COMMITTEE CERTIFICATION OF APPROVED VERSION
The committee for Jason Alan Wicker certifies that this is the approved version of the
following dissertation:
MUTATIONAL ANALYSIS OF THE WEST NILE VIRUS NS4B
PROTEIN
Committee:
Alan D.T. Barrett, Ph.D. Supervisor
James C. Lee, Ph.D.
Richard M. Kinney, Ph.D.
Stephen Higgs, Ph.D.
Norbert Roberts, M.D.
______________________________
Dean, Graduate School
i
MUTATIONAL ANALYSIS OF THE WEST NILE VIRUS
NS4B PROTEIN
By
Jason Alan Wicker, B.S., B.A.
Dissertation
Presented to the Faculty of
The University of Texas Graduate School of
Biomedical Sciences at Galveston
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
Approved by the Supervisory Committee
Alan D.T. Barrett, Ph.D.
James C. Lee, Ph.D.
Richard M. Kinney, Ph.D.
Stephen Higgs, Ph.D.
Norbert Roberts, M.D.
January, 2007
Galveston, Texas
Key words: phenotypic variation, membrane protein, molecular engineering, attenuation
of mouse neuroinvasiveness/neurovirulence
ii
There is something fascinating about science. One gets such wholesale returns of
conjecture out of such a trifling investment of fact.
- Mark Twain, Life on the Mississippi (1883)
iii
ACKNOWLEDGEMENTS
First of all, I would like to thank my mentor, Dr. Alan Barrett, for his guidance
and advice during my dissertation work. His countless hours reviewing manuscripts,
listening to my presentations, and giving scientific input facilitated both the design of a
successful project and my development as a researcher. While constantly busy, he
always had time to set up a time to meet and discuss current findings and future
experiments. In addition, he kept me on the straight and narrow and leaned on me when
necessary to maximize my productivity and mold me into a better scientist. Next I would
like to thank my fellow graduate student and colleague, Melissa Whiteman. She trained
me in virtually every aspect of virology utilized in this project and had the magic touch
when it came to the infectious clone. I would like to think that at least some of her
technical expertise and voodoo rubbed off on me as I learned everything from plaque
assays to animal work from her. I would also like to thank all other members of the
Barrett laboratory with whom I’ve worked including Dr. Todd Davis, Dr. David Beasley,
Dr. Mike Holbrook, Dr. Juliet Bryant, Dr. Amber Engel, Dr. Jana von Lindern, Dr.
Sareen Galbraith, Dr. Fiona May, Dr. Monica McArthur, Greg Gromowski, Li Li, Shuliu
Zhang, and Sandra Rivas. Specifically, Todd and David were instrumental in carrying
out the initial animal experiments, Mike kindly provided the murine dendritic cell line,
and Jana helped with phylogenetic analysis. Li and Shuliu kept the lab running smoothly
and were always willing to help out. Amber, Greg, and I spent countless hours in either
serious or not serious deep philosophical conversations related to virtually any possible
subject either in lab or at “coffee time,” thereby making my time spent in the Barrett lab a
truly memorable experience. In addition to being my colleagues, these people were also
my friends and participated in activities including a night at Molly’s, fierce tennis
matches, wild parties (at least for science nerds), and various epic camping, fishing, or
kayaking trips. The many good friends I’ve met here at UTMB have made studying and
working in Galveston (affectionately known as Galvetraz) a lot of fun.
iv
I would like to thank the members of my dissertation committee; Dr. Stephen
Higgs, Dr. Norbert Roberts, Dr. Jim Lee, and Dr. Rich Kinney for helping me develop a
successful project and guiding me through the process of completing my research and
dissertation. In addition, Rich Kinney provided the West Nile infectious clone that
served as an integral component of my project, and Jim Lee assisted with the
interpretation of the NS4B amino acid substitutions. I would like to thank Dr. Kley
Hughes and Martha Lewis in the Microbiology and Immunology department for always
being available to guide me through the graduate school process. In addition, I would
like to thank Dr. Steve Weinman and Lyska Morrison in the M.D./Ph.D. combined
degree program for continued support during my time at UTMB. Finally, a number of
scientists at UTMB gave me helpful advice and suggestions along the way including Dr.
Robert Shope, Dr. Robert Tesh, Dr. Vivian Braciale, Dr. Shinji Makino, and Dr. Peter
Mason. I want to thank Brad Schneider and Charlie McGee for helping with quantitative
real-time RT-PCR, Dr. Gavin Bowick for helping with densitometry analysis, and Dr.
Kui Li for providing the human hepatocyte cell lines. Finally, I would like to thank my
parents, Tom and Lou Ann Wicker, for their continued love and support during my time
down in Texas and my feline roommate, Donner, who always knew just when to take a
nap on an important piece of paper or take a leisurely stroll across my laptop computer
keyboard while I was diligently working on this dissertation. The funding for this
research was provided in part by the State of Texas Advanced Research Program, the
NIH T32 Training Grant for research of Emerging Infectious Diseases (NIH T32AI
7526), and the Clayton Foundation for Research.
v
MUTATIONAL ANALYSIS OF THE WEST NILE VIRUS
NS4B PROTEIN
Publication No._____________
Jason Alan Wicker, B.S., B.A.
The University of Texas Graduate School of Biomedical Sciences at Galveston, 2007
Supervisor: Alan D.T. Barrett, Ph.D.
West Nile virus (WNV) is a member of the genus Flavivirus in the family
Flaviviridae. The WNV genome is a positive-sense RNA molecule approximately 11kb
in length encoding a single polyprotein that is cleaved by a combination of viral and host
proteases to produce three structural and seven nonstructural proteins. The NS4B protein
is a small hydrophobic protein approximately 27kD in size that is hypothesized to
participate both in the viral replication complex and evasion of host innate immune
defenses. The objective of this dissertation was to investigate the role of the NS4B
protein in viral cell multiplication and mouse virulence phenotypes by studying
recombinant mutant viruses encoding amino acid substitutions of selected residues within
the NS4B protein. The first aim of this project used protein modeling and phylogenetic
analysis of the NY99 WNV NS4B protein in comparison to NS4B proteins from other
flavivirus and WNV strains to identify amino acid residues with a theoretical probability
of contributing to the function of NS4B. The second aim utilized site-directed
mutagenesis of a WNV NY99 infectious clone to introduce amino acid substitutions into
the NS4B protein primarily targeting a highly conserved N-terminal domain, the variable
central hydrophobic region, and the four cysteine residues. Out of fourteen recombinant
viruses encoding engineered substitutions, two highly attenuated mutant viruses were
identified (C102S and P38G/T116I viruses) that exhibited temperature-sensitive and
mouse attenuation (greater than 10,000,000-fold compared to wild-type) phenotypes.
The third aim investigated the putative underlying molecular mechanisms responsible for
the attenuation of the C102S and P38G/T116I viruses. Both NS4B mutants exhibited
vi
reduced multiplication kinetics both in mice and in murine macrophage and dendritic cell
types critical for mediating the antiviral immune response. In addition, preliminary data
identified a series of genes by DNA microarray analysis that exhibited differential
expression in wild-type WNV-infected cells compared to C102S mutant-infected cells
that may be involved in viral manipulation of cellular processes. This study has for the
first time demonstrated the role of the NS4B protein as mediator of WNV temperaturesensitive and mouse attenuation phenotypes and has led to the identification of putative
molecular mechanisms that may be involved.
vii
TABLE OF CONTENTS
Acknowledgements
iv
Mutational Analysis of the West Nile virus NS4B protein
vi
Table of Contents
viii
List of Tables
xiv
List of Figures
xvi
List of Abbreviations
xx
CHAPTER 1
1
INTRODUCTION
1
1.1 Overview……………………………………………………………………………...1
1.2 Isolation and Classification………………………………………………………….1
1.3 Phylogeny of WNV…………………………………………………………………...2
1.4 Biology and Ecology………………………………………………………………….3
1.5 Epidemiology…………………………………………………………………………3
1.6 Introduction to the Americas………………………………………………………..5
1.7 Clinical manifestations of WNV…………………………………………………….6
1.8 Pathogenesis………………………………………………………………………….7
1.9 Molecular Virology…………………………………………………………………..8
1.9.1 Virion structure…………………………………………………………….8
1.9.2 Genomic organization……………………………………………………...9
1.9.3 WNV structural proteins…………………………………………………10
1.9.4 WNV nonstructural proteins…………………………………………….14
1.9.5 WNV untranslated regions………………………………………………23
1.9.6 Virus life cycle…………………………………………………………….24
viii
1.10 Immunology………………………………………………………………………..26
1.10.1 Immune response to WNV……………………...………………………26
1.10.2 Immune evasion by WNV……………………...……………………….28
1.10.3 Vaccine designs…………………………………………………………..29
1.11 Specific aims………………………………………...……………………………..30
1.11.1 Specific aim 1…………………………...………………………………..31
1.11.2 Specific aim 2(a)…………………...…………………………………….32
1.11.3 Specific aim 2(b)………………...……………………………………….32
1.11.4 Specific aim 2(c)……..…………………………………………………..33
1.11.5 Specific aim 3……..……………………………………………………...33
CHAPTER 2
46
MATERIALS AND METHODS
46
2.1 Buffers and solutions……………………………………………………………….46
2.2 Cell culture media recipes………………………………………………………….47
2.3 Protein modeling of NS4B………………………………………………………….49
2.4 Amino acid alignments and phylogenetic analysis of flaviviral NS4B proteins...50
2.5 Cell culture techniques……………………………………………………………..50
2.6 The WNV two-plasmid infectious clone…………………………………………...51
2.7 Site-directed mutagenesis…………………………………………………………..51
2.8 Miniprep of plasmid DNA………………………………………………………….52
2.9 Maxiprep of plasmid DNA…………………………………………………………52
2.10 Construction and rescue of recombinant viruses……………………………….53
2.11 Viral RNA extraction……………………………………………………………...54
2.12 Genomic sequencing of recombinant viruses……………………………………54
2.13 Plaque assays………………………………………………………………………56
ix
2.14 Temperature-sensitivity assay……………………………………………………56
2.15 Growth curves……………………………………………………………………..56
2.16 Mouse virulence studies………………………………………………………….57
2.17 Virus multiplication in mice……………………………………………………..58
2.18 Quantitative real-time RT-PCR assays…………………………………………58
2.19 PAGE and Western blots………………………………………………………...59
2.20 Cloning and quasispecies analysis……………………………………………….59
2.21 Cytokine array membranes………………………………………………………60
2.22 Gene expression studies…………………………………………………………..60
CHAPTER 3
66
AMINO ACID SEQUENCE ANALYSIS AND PROTEIN MODELING
OF THE WEST NILE VIRUS NS4B PROTEIN……………………………………..66
3.1 Abstract……………………………………………………………………………...66
3.2 Introduction…………………………………………………………………………67
3.3 Results……………………………………………………………………………….68
3.3.1 Phylogenetic analysis of WNV NS4B compared to NS4B
proteins from other flaviviruses…………………………………………68
3.3.2 Phylogenetic analysis of WNV 382-99 NS4B protein
compared to other WNV strains…………………………………………69
3.3.3 Topological modeling of the WNV NS4B protein………………………70
3.4 Discussion…………………………………………………………………………...71
x
CHAPTER 4
84
DISRUPTION OF RESIDUES IN THE CENTRAL
HYDROPHOBIC REGION OF THE WNV NS4B PROTEIN
84
4.1 Abstract……………………………………………………………………………...84
4.2 Introduction…………………………………………………………………………85
4.3 Results……………………………………………………………………………….86
4.3.1 Rescue of recombinant viruses…………………………………………..86
4.3.2 Temperature sensitivity assay……………………………………………87
4.3.3 Multiplication kinetics in cell culture……………………………………87
4.3.4 Mouse neuroinvasive phenotype…………………………………………87
4.4 Discussion…………………………………………………………………………...87
CHAPTER 5
96
DISRUPTION OF THE CYSTEINE RESIDUES
IN THE WNV NS4B PROTEIN
96
5.1 Abstract……………………………………………………………………………...96
5.2 Introduction…………………………………………………………………………97
5.3 Results……………………………………………………………………………….98
5.3.1 Rescue of recombinant viruses…………………………………………..98
5.3.2 Temperature sensitivity assay……………………………………………98
5.3.3 Multiplication kinetics in cell culture……………………………………99
5.3.4 C102S mutant is attenuated for neuroinvasiveness
and neurovirulence in mice………………………………………………99
5.3.5 Reversion of the C102S mutation………………………………………100
xi
5.3.6 C102A substitution is associated with a virulent phenotype in mice...100
5.3.7 RNA and protein levels in wild-type and C102S
virus-infected Vero cell culture…………………………………………100
5.4 Discussion………………………………………………………………………….101
CHAPTER 6
114
DISRUPTION OF A CONSERVED N-TERMINAL MOTIF
IN THE WNV NS4B PROTEIN
114
6.1 Abstract…………………………………………………………………………….114
6.2 Introduction………………………………………………………………………..115
6.3 Results……………………………………………………………………………...116
6.3.1 Design of amino acid substitutions……………………………………..116
6.3.2 Rescue of recombinant viruses…………………………………………116
6.3.3 Recombinant P38G/T116I virus is temperature sensitive
and exhibits a small-plaque phenotype………………………………...117
6.3.4 Multiplication kinetics of recombinant viruses in cell culture………..117
6.3.5 P38G/T116I virus is attenuated for neuroinvasiveness but
not neurovirulence in mice……………………………………………...118
6.3.6 Isolation of P38G/T116I derivatives encoding
compensatory mutations………………………………………………..118
6.3.7 Analysis of compensatory mutants……………………………………..119
6.3.8 Mutation rate of the P38G/T116I virus when passaged
in Vero cells at 37°C or 41°C…………………………………………...120
6.3.9 RNA and protein levels in wild-type and P38G/T116I
virus-infected Vero cell culture…………………………………………122
6.4 Discussion………………………………………………………………………….122
xii
CHAPTER 7
138
ANALYSIS OF IMMUNE MECHANISMS POTENTIALLY CONTRIBUTING
TO ATTENUATION OF THE NS4B MUTANTS
138
7.1 Abstract…………………………………………………………………………….138
7.2 Introduction………………………………………………………………………..139
7.3 Results……………………………………………………………………………...141
7.3.1 Multiplication of attenuated NS4B mutants in mice………………….141
7.3.2 Multiplication kinetics in relevant murine cell types…………………142
7.3.3 Comparison of the neuroinvasive phenotype
in both inbred and outbred mice……………………………………….142
7.3.4 Multiplication kinetics in human hepatocyte-derived cell lines
expressing varying RIG-1 and TLR3 phenotypes…………………….143
7.3.5 Cytokine responses in West Nile virus-infected cells……………….....144
7.3.6 Gene expression in P388.D1 cells infected with either wild-type
or NS4B C102S mutant West Nile virus……………………………….144
7.4 Discussion………………………………………………………………………….145
CHAPTER 8
164
DISCUSSION
164
REFERENCES
176
APPENDIX 1
218
xiii
LIST OF TABLES
Table 2-1. GenBank accession numbers of flavivirus NS4B amino acid sequences
used in alignments …………...……………………………………………….61
Table 2-2. Origins and citations for cell lines used in these experiments………………..62
Table 2-3. Primers used for site-directed mutagenesis of the WNV infectious clone…...63
Table 2-4. Primers used for WNV genomic amplification and sequencing……………..64
Table 3-1. GenBank accession numbers of flavivirus NS4B amino acid sequences
used in alignments…………………………………………………………….75
Table 3-2. NS4B amino acid identity rates between West Nile virus strain NY99
382-99 and other WN and flavivirus strains………………………………….76
Table 4-1. Temperature sensitive and mouse virulence phenotypes of central
hydrophobic mutants…………………………………………………………91
Table 5-1. Temperature sensitive phenotypes of recombinant wild-type WNV
and the five cysteine mutants……………………………………………….105
Table 5-2. Mouse virulence phenotypes of recombinant wild-type WNV
and the five cysteine mutants……………………………………………….106
Table 6-1. Temperature sensitive phenotypes of recombinant wild-type WNV
and the N-terminal mutants…………………………………………………127
Table 6-2. Mouse virulence phenotypes of recombinant wild-type WNV
and the N-terminal mutants…………………………………………………128
Table 6-3. Temperature sensitive and mouse virulence phenotypes of P38G/T116Iderived viruses encoding compensatory substitutions……………………...129
Table 6-4. Analysis of the mutation rate of the P38G/T116I virus when passaged
in Vero cells at either 37°C or 41°C compared to variability in the
parental stock…………………………………………………………..……130
xiv
Table 7-1. Viremia and brain titers from mice inoculated with 100 pfu of either
wild-type WNV, the C102S mutant, or the P38G/T116I mutant
virus via the intraperitoneal route…………………………………………..154
Table 7-2. Virulence phenotypes of wild-type WNV and the attenuated NS4B
mutants in different mouse strains………………………………………….155
Table 7-3. Differentially expressed genes in mouse macrophage P388.D1 cells
infected with either wild-type NY99 West Nile virus or the attenuated
NS4B C102S mutant virus at 12 hours post-infection (moi-5)……………..156
xv
LIST OF FIGURES
Figure 1-1. Diagram showing the main components of the West Nile virus
transmission cycle (adapted from CDC, Arbonet)………………………….35
Figure 1-2. Worldwide distribution of West Nile virus in 2006
(adapted from CDC, Arbonet)………………………………………………36
Figure 1-3. Phylogram showing relationships of different flaviviruses based on the
NS5 polymerase domain (adapted from Kuno and Chang, 2005)…………...37
Figure 1-4. Phylogram showing relationships of different WNV isolates
utilizing complete genomic nucleotide sequences
(adapted from Bakonyi et al., 2006)………………………………………...38
Figure 1-5. Reported incidence of neuroinvasive West Nile virus disease by county,
United States, 1999-2004. Reported to Centers for Disease Control and
Prevention through April 21, 2005 (adapted from Hayes et al., 2005)……..39
Figure 1-6. Distribution of human WNV cases by year…………………………………40
Figure 1-7. West Nile virus genome organization showing co- and posttranslational processing reactions…………………………………………...41
Figure 1-8. Diagram of the DEN E protein showing linear amino acid sequence (Panel A)
and ribbon structure (Panel B). Domain I is shown in red, domain II in
yellow, and domain III in blue. (adapted from Modis et al., 2003)………….42
Figure 1-9. West Nile virus life cycle (adapted from Solomon and Barrett in Nash and
Burger (Eds) Clinical Neurovirology, Marcel Decker 2003)……………….43
Figure 1-10. Conformational changes of the virion during maturation (Panel A)
and fusion with host cell membranes (Panel B)
(adapted from Mukhopadhyay et al., 2005)………………………………..44
Figure 1-11. Molecular processes that signal the host response to HCV infection
(adapted from Gale Jr. and Foy, 2005)…………………………………….45
Figure 2-1. Diagram of the WN-NY99 two-plasmid infectious clone…………………..65
xvi
Figure 3-1. Flaviviral NS4B complete amino acid alignment. Residues exhibiting
complete conservation (yellow), high-identity (blue), high-homology
(green), or high-variability (white) are denoted……………………………..77
Figure 3-2. Phylogenetic tree generated by the neighbor-joining analysis (PAUP) of
aligned amino acid sequences of the NS4B proteins from different
flaviviruses with bootstrap resampling analysis (500 replicates). Cell
fusing agent virus (CFAV) and Kamiti river virus (KRV) together were
used as the outgroup………………………………………………………...78
Figure 3-3. Phylogenetic tree generated by neighbor-joining analysis (PAUP) of aligned
NS4B amino acid sequences of 115 West Nile virus strains and 1 Japanese
encephalitis virus (used as the outgroup). Branches are drawn to scale……79
Figure 3-4. Predicted secondary structure of the West Nile virus NS4B protein generated
using the SOSUI hydrophobicity plotting program (Hirokawa et al., 2003)..80
Figure 3-5. Predicted topology of the West Nile virus NS4B protein generated using the
ConPredII hydrophobicity plotting program (Arai et al., 2004)……………81
Figure 4-1. A model for the NS4B protein was produced based on hydrophobicity
plots highlighting the central hydrophobic region………………………….92
Figure 4-2. Amino acid substitutions in the central hydrophobic region are highlighted
on a flaviviral NS4B amino acid alignment (panel A) and compared to the
corresponding residues in the WNV sequence (panel B)…………………..93
Figure 4-3. Multiplication kinetics of recombinant wild-type and mutant viruses in
monkey kidney Vero cells at 37°C (panel A) and 41°C (panel B)………...94
Figure 4-4. Multiplication kinetics of recombinant wild-type and mutant viruses in
mouse Neuro2A (panel A) and mosquito C6/36 cells (panel B)…………...95
Figure 5-1. A model for the NS4B protein developed using hydrophobicity plots that
highlights the cysteine residues……………………………………………107
Figure 5-2. Complete amino acid alignments including both tick-borne and mosquitoborne flaviviruses showing conservation of the WNV C102 residue within
the DEN and JE genetic groups. This residue is not found in the tick-borne
flaviviruses or yellow fever virus. In contrast, the WNV C120 and C237
residues are only found in WNV and Kunjin virus while C227 is found
throughout the JE genetic group…………………………………………..108
xvii
Figure 5-3. Amino acid substitutions in the central hydrophobic region are show on a
flaviviral NS4B amino acid alignment highlighting the C102 residue
(panel A) and compared to the corresponding residues in the WNV
sequence (panel B)…………………………………………………...…….109
Figure 5-4. Multiplication kinetics of recombinant wild-type and cysteine mutant
viruses in monkey kidney Vero cells (MOI = 0.01) at 37°C (panel A)
and 41°C (panel B)………………………………………………………...110
Figure 5-5. Multiplication kinetics of recombinant wild-type and cysteine mutant
viruses in mouse Neuro2A (panel A) and mosquito C6/36 cells (panel B)
(MOI = 0.01)……………………………………………………………….111
Figure 5-6. C102S and wild-type viral RNA levels were assayed in Vero cells at 37°C
(panel A) and 41°C (panel B)……………………………………………...112
Figure 5-7. C102S and wild-type viral envelope protein levels were assayed in Vero
cells at 37°C and 41°C……………………………………………………..113
Figure 6-1. A model for the NS4B protein developed using hydrophobicity plots that
highlights the mutated N-terminal residues……………..…………………107
Figure 6-2. NS4B amino acid alignments showing conservation of the target Nterminal residues within both mosquito- and tick-borne flaviviruses……...108
Figure 6-3. Multiplication kinetics of recombinant wild-type and N-terminal mutant
viruses in monkey kidney Vero cells (MOI = 0.01) at 37°C (panel A) and
41°C (panel B)……………………………………………………………..109
Figure 6-4. Multiplication kinetics of recombinant wild-type and N-terminal mutant
viruses in mouse Neuro2A (panel A) and mosquito C6/36 cells at an
MOI of 0.01………………………………………………………………..110
Figure 6-5. Chromatogram data of sequenced PCR products of NS4B regions
amplified from either parental P38G/T116I virus or P38G/T116I virus
incubated for 96 hours at 41°C…………………………………………….111
Figure 6-6. P38G/T116I mutant and wild-type viral RNA levels were assayed in
Vero cells at 37°C and 41°C……………………………………………….112
Figure 6-7. P38G/T116I and wild-type viral protein levels were assayed in Vero
cells at 37°C and 41°C……………………………………………………..113
xviii
Figure 7-1. Multiplication kinetics of recombinant wild-type and mutant viruses in
mouse neuronal Neuro2A cells (panel A) or macrophage P388.D1 cells
at an MOI of 0.1 (panel B)…………………………………………………158
Figure 7-2. Multiplication kinetics of recombinant wild-type and mutant viruses in
Mouse dendritic cell-derived DC2.4 cells an an moi of 0.1 (panel A) or 5
(panel B)……………………………………………………………………159
Figure 7-3. Multiplication kinetics of recombinant wild-type and mutant viruses in
human hepatocyte Huh7 (panel A) and Huh7 FT3.7 (panel B) infected at
an moi of 5. Huh7 cells are RIG-1+/TLR3- while the Huh7 FT3.7 cells
are RIG-1+/TLR3+………………………………………………………...160
Figure 7-4. Multiplication kinetics of recombinant wild-type and mutant viruses in
human hepatocyte Huh7.5 (panel A) and Huh7.5iTLR3.16 (panel B) cells
infected at an moi of 5. Huh7.5 cells are RIG-1-/TLR3- while
Huh7.5iTLR3.16 cells are RIG-1-/TLR3+………………………………...161
Figure 7-5. Multiplication kinetics of recombinant wild-type and mutant viruses in
T-antigen immortalized non-neoplastic PH5CH8 hepatocytes infected at
an moi of 5. PH5CH8 cells are RIG-1+/TLR3+………………………….162
Figure 7-6. Raybiotech cytokine arrays were used to assay cytokine expression in
Wild-type WNV-infected (moi = 5) or uninfected mouse neuronal
Neuro2A, macrophage P388, or dendritic DC2.4 cell lines……..………...163
xix
LIST OF ABBREVIATIONS
Å
μL
Ala (A)
ALF
ALKV
APOIV
Arg (R)
Asn (N)
Asp (D)
AST
BAGV
BBB
BGS
BSQV
C protein
C6/36
CDC
cDNA
CFAV
CNF
CPE
CS
Cx
Cys (C)
DC2.4
DENV
DNA
dsRNA
DTV
E protein
EDTA
eGFP
EIF
ENTV
ER
FBS
Gln (Q)
Glu (E)
Gly (G)
angstrom
microliters
alanine
Alfuy virus
Alkhurma virus
Apoi virus
arginine
asparagines
aspartic acid
average survival time
Bagaza virus
blood-brain barrier
bovine growth serum
Bussuquara virus
capsid protein
mosquito (Aedes albopictus) cell line
Centers for Disease Control
complementary DNA
cell fusing agent virus
central nervous system
cytopathic effect
cyclization sequence
Culex
cysteine
murine dendritic cell line
dengue virus
deoxyribonucleic acid
double-stranded RNA
deer tick virus
envelope protein
di-sodium ethylenediamine tetra-acetic acid
enhanced green fluorescent protein
eukaryotic initiation factor
Entebbe bat virus
endoplasmic reticulum
fetal bovine serum
glutamine
glutamic acid
glycine
xx
HCV
His (H)
Huh7
Ile (I)
ic
IGUV
IL
ILHV
ip
IFN
IRF
ISG
JAK
JEV
kb
kD
KEDV
KOKV
KRV
KUNV
LB
LD50
Leu (L)
LGTV
LIV
Lys (K)
M protein
MEM
Met (M)
MMLV
MODV
moi
mL
mRNA
MTase
MVEV
NAMRU
NEAA
NS
nt
NTPase
NY99
OAS
hepatitis C virus
histidine
human hepatocyte cell line
isoleucine
intracerebral
Iguape virus
interleukin
Ilheus virus
intraperitoneal
interferon
interferon regulated factor
interferon stimulated gene
Janus kinase
Japanese encephalitis virus
kilobase
kilodalton
Kedougou virus
Kokobera virus
Kamiti River virus
Kunjin virus
Luria-Bertani
lethal dose 50
leucine
langat virus
louping ill virus
lysine
membrane protein
modified eagle medium
methionine
Montana myotis leukoencephalitis virus
Modoc virus
multiplicity of infection
milliliters
messenger RNA
methyltransferase
Murray Valley encephalitis virus
Naval Medical Research Unit
non-essential amino acids
nonstructural
nucleotide
nucleoside triphosphatase
New York 1999
2’,5’-oligoadenylate synthase
xxi
OHFV
ORF
P388.D1
PAGE
PBS
PCR
PD50
PFU
Phe (F)
PKR
POWV
prM
Pro (P)
RabV
RBV
RdRp
RC
RF
RI
RIG-1
RNA
RNase
ROCV
RT
SD
SDS
SEPV
Ser (S)
SL
SLE
STAT
SVP
TBE
Thr (T)
TLR
TMD
TNF
TPB
Trp (W)
Tyr (Y)
USUV
UTMB
UTR
Omsk hemorrhagic fever virus
open reading frame
murine macrophage cell line
polyacrylamide gel electrophoresis
phosphate buffered saline
polymerase chain reaction
protective dose 50
plaque forming unit
phenylalanine
dsRNA-dependent protein kinase
Powassan virus
premembrane protein
proline
Rabensburg virus
Rio Bravo virus
RNA-dependent RNA polymerase
replication complex
replicative form
replicative intermediate
retinoic acid-inducible gene
ribonucleic acid
ribonuclease
Rocio virus
reverse transcription
standard deviation
sodium dodecyl sulfate
Sepik virus
serine
stem loop
St. Louis encephalitis virus
signal transducer and activator of transcription
subviral particle
tick-borne encephalitis virus
threonine
Toll-like receptor
transmembrane domain
tumor necrosis factor
tryptose phosphate buffer
tryptophan
tyrosine
Usutu virus
University of Texas Medical Branch
untranslated region
xxii
Val (V)
Vero
WNV
wt
YFV
YOKV
ZIKV
valine
African green monkey kidney cells
West Nile virus
wild-type
yellow fever virus
Yokose virus
Zika virus
xxiii
CHAPTER 1
INTRODUCTION
1.1 Overview
First isolated in 1937, West Nile virus (WNV) is a mosquito-borne flavivirus that
is maintained in nature between avian amplifying hosts and a mosquito vector, primarily
members of the Culex genus (Fig. 1-1). Other vertebrates can become infected, however
viremias are not thought to reach sufficient levels to infect feeding mosquitoes resulting
in a designation as an incidental host. Human infection with WNV is usually either
asymptomatic or associated with a non-specific febrile illness. Nonetheless, sporadic
outbreaks have been reported in Africa, the Middle East, Europe, and Asia. Between
1996 and 1999, the emergence of an especially virulent strain of WNV caused major
epidemics associated with relatively high rates of meningitis, encephalitis, and death in
Romania, the Volga delta in southern Russia, and in the northeastern United States. The
subsequent spread of WNV throughout the United States marked one of the most closely
studied emergence events of a novel virus interacting with naïve host and vector species.
West Nile virus also became the most widely distributed member of the Flavivirus genus
as it continued its migration north into Canada and south into Mexico, the Caribbean, and
Central and South America (Fig. 1-2).
1.2 Isolation and Classification
West Nile virus was first isolated in December 1937 at Omogo in the West Nile
district of Uganda from the blood of a febrile woman (Smithburn et al., 1940). Serum
from her blood was inoculated into mice via the intracerebral route, and nine out of ten
inoculated mice succumbed to infection (Smithburn et al., 1940). The agent was
1
described as filterable and was capable of causing disease in both mice and monkeys
following intracerebral inoculation (Smithburn et al., 1940). Subsequent crossneutralization studies established that WNV was serologically related to, but distinct from
St. Louis and Japanese B encephalitis (SLE and JE) viruses leading to its classification as
a group B arbovirus (Smithburn, 1942). Additional cross-neutralization studies later
placed WNV in an antigenic group composed of SLE, JE, Murray Valley encephalitis
(MVE), Kunjin (KUN), Usutu (USU), Kokobera (KOK), Stratford, and Alfuy (ALF)
viruses (deMadrid and Porterfield, 1974).
Currently, the Flavivirus genus in the family Flaviviridae consists of more than 70
viruses. The majority are considered arboviruses (arthropod-borne) because they are
transmitted by mosquitoes, ticks, or mites. Historically, members of the Flavivirus genus
were classified into subgroups based on antigenic relationships. The advent of
convenient nucleic acid sequencing has allowed for improved classification of
flaviviruses based on genetic relationships. The members of the Flavivirus genus can
also be subdivided by vector into mosquito-, tick-, and non-vector-borne viruses, and
these groupings correlate loosely with genetic and antigenic relationships (Kuno and
Chang, 2005; Billoir et al., 2000; Fig. 1-3).
1.3 Phylogeny of WNV
Phylogenetic analyses have led to the identification of 2 distinct lineages of WNV
strains that can be further divided into subclades (Berthet et al., 1997; Charrel et al.,
2003; Lanciotti et al., 1999, 2002). Lineage I WNV strains have been isolated from
Africa, the Middle East, Europe, North America, and Australia while lineage II strains
come from Africa and Madagascar (Murgue et al., 2002). Following phylogenetic
analysis, the Australian KUN virus has been classified as a subclade of lineage I WNV
(Fig. 1-4). Certain WNV isolates from India also are classified as a subclade of lineage I
WNV and display only weak antigenic cross-reactivity with other lineage I viruses
(Lanciotti et al., 2002). Recently, viruses have been isolated from the Czech republic
2
(Rabensburg virus) and the Caucasus region of Russia (LEIV-Krnd88-190 isolate) that
either represent new lineages of WNV or could be classified as novel flavivirus species
(Bakonyi et al., 2005, 2006; Fig. 1-2).
1.4 Biology and Ecology
The primary WNV transmission cycle occurs between mosquito vectors of the
genus Culex and a wide variety of avian hosts including Passeriformes (song birds),
Charadriiformes (shorebirds), Strigiformes (owls), and Falcinoformes (hawks) (Hayes et
al., 2005a). Occasionally WNV is isolated from ticks, however the role of ticks in the
WNV transmission cycle remains unclear. The amplifying avian host allows the
production of sufficiently high viremias to allow transmission to feeding mosquitoes. An
alternative method of nonviremic transmission has been observed with the simultaneous
co-feeding of mosquitoes where infected mosquitoes were able to transmit WNV to
uninfected mosquitoes feeding in close proximity (Higgs et al., 2005). In this situation,
the host does not develop a viremia before transmission to the vector occurs. The
biological significance of this transmission pathway remains unclear. In temperate
regions, WNV transmission occurs from May through October while in warmer climates
transmission can occur year-round (Savage et al., 1999; Tesh et al., 2004). Various
vertebrate hosts including certain birds, humans, and equines serve as incidental hosts
that are not capable of supporting sufficient viremias to efficiently transmit WNV to
feeding mosquitoes.
1.5 Epidemiology
The first reported outbreak of WNV in Israel occurred from 1951-1952 which led
to a four-year epidemiological survey studying WNV prevalence conducted by the
United States Naval Medical Research Unit (NAMRU) in the Upper Nile delta of Egypt.
In humans, exposure to WNV was prevalent with greater than 60% of the population
3
possessing antibodies to WNV (Taylor et al., 1956). The virus was found to be capable
of infecting a variety of domestic livestock with the exception of goats, as determined by
seroconversion (Taylor et al., 1956). Experimental infections of horses by infected
mosquitoes showed that neutralizing antibodies were induced, but only a small proportion
of animals developed detectable viremias (Taylor et al., 1956). Experiments focusing on
arthropods found that Culex antennatus, Cx univittatus, and Cx pipiens were capable of
transmitting WNV while soft ticks of the genus Ornithodoros could become infected with
WNV but were unable to transmit it (Taylor 1956). Neutralizing antibodies to WNV
were found in a high proportion of crows (65%) and sparrows (42%) (Taylor et al.,
1956).
Following the initial outbreak, subsequent human and veterinary outbreaks have
been reported in Israel (1957, 1962, and 1998-2000), France (1962 and 2000), South
Africa (1974 and 1983-1984), Algeria (1994), Romania (1996), Tunisia (1997),
Democratic Republic of Congo (1998), Italy (1998), Russia (1999), and the United States
(1999-present) (Murgue et al., 2002). Prior to the introduction of WNV into North
America, lineage 2 strains were found in areas with an endemic transmission cycle and
high levels of anti-WNV antibodies in the human population (Morvan et al., 1990).
Lineage I strains were associated with short-term epidemics in regions lacking an
endemic transmission cycle where the majority of the population was naïve to WNV
infection, such as within Europe (Scherret et al., 2001). In addition, WNV was thought to
be incapable of establishing an endemic transmission cycle in Central and Eastern Europe
because of the cold climate and harsh winters (Hayes et al., 2001). In recent years,
endemic transmission cycles have been identified within Central Europe for both lineage
I and II strains plus the putative lineage IV Rabensburg virus (Bakonyi et al., 2006).
Persistently infected birds or mosquitoes may allow overwintering of WNV even in
regions where cold winters lead to a temporary interruption of the transmission cycle
(Komar, 2002; Tesh et al., 2004). In humans, transmission can also occur through blood
transfusions or organ donations (Pealer et al., 2006; Iwamoto et al., 2006).
4
1.6 Introduction to the Americas
In late August of 1999, an outbreak of encephalitis was detected in Queens, New
York, USA. At the same time, increased mortality was observed in certain avian species,
including wild crows and captive exotic birds (Briese et al., 1999). Serological analysis
of serum samples from infected patients revealed the presence of a “SLE-like” virus.
Since SLEV does not normally cause avian mortality, additional analyses were conducted
that utilized degenerate primer sets to identify WNV as the culprit in the outbreak (Briese
et al., 1999). The prototype WNV NY99 strain, designated WNV 382-99, was isolated
from a dead Chilean flamingo (Phoenicopterus chilensis) in 1999 and was found to be
most closely related to a lineage I WNV strain isolated from a dead goose in Israel in
1998 (Lanciotti et al., 1999). The initial outbreak involved 62 human cases including 7
deaths, and 25 equine cases with 9 deaths. In 1999 WNV was reported in 4 states,
however WNV was detected in 8 states in 2000 and 16 states in 2001 (Figs. 1-5 and 1-6).
In 2002, WNV underwent a drastic expansion across the United States and was detected
in 46 states causing 4,156 human cases with 284 deaths and over 14,000 equine cases. In
2003, there were 9,862 human cases with 264 deaths and over 4,000 equine cases (Hayes
and Gubler, 2006). The drop in equine cases coincided with the implementation of a
veterinary vaccination program. In the Gulf Coast region of Texas and Louisiana, WNV
transmission has been found to occur year-round (Tesh et al., 2004).
Phylogenetic analysis of North American WNV strains detected limited evolution
of WNV during its spread across the continent (Beasley et al., 2003; Davis et al., 2003).
In 2002, a dominant North American genotype was detected that displaced the original
NY99 genotype (Davis et al., 2005). In the spring of 2003, the first WNV isolates were
detected in Mexico (Beasley et al., 2004; Deardorff et al., 2006). The lower than
expected number of human cases in Mexico may be due to poor surveillance, or the
circulation of endemic flaviviruses may provide cross-immunity to WNV infection (Tesh
et al., 2002; Elizondo-Quiroga et al., 2005). The predominant North American WNV
vector species include Culex pipiens in the Northeast, Culex quinquefasciatus in the
5
South, and Culex tarsalis west of the Mississippi River (Turell et al., 2005). Important
North American avian hosts include passerines such as common grackles, corvids (crows,
jays, magpies), house finches, and house sparrows (Komar et al., 2003; Hayes et al.,
2005a). At least 29 species of mammals, including cats, dogs, sheep, wolves, and
skunks, are capable of being infected by WNV (McLean et al., 2001, 2002). Direct
alligator-to-alligator transmission has been found to occur in captive but not wild
populations, and persistent WNV infection of alligators could provide a mechanism of
overwintering (Klenk et al., 2004). The NY99-derived WNV strains exhibit much
greater virulence for American crows than certain Old World isolates (Kinney et al.,
2006; Brault et al., 2004).
1.7 Clinical Manifestations of WNV
WNV infection in humans is usually asymptomatic or presents as a nonspecific
febrile illness (Hayes et al., 2005b). Induced infection of terminally ill patients with the
Egypt 101 WNV strain found that 89% of patients lacked any clinical symptoms other
than fever while 11% of patients exhibited clinical signs of diffuse encephalitis between 6
and 13 days post-inoculation (Southam and Moore, 1954). Over 95% of patients
exhibited detectable viremias at 24 hours while 87% of patients had viremias at 72 hours
post-infection (Southam and Moore, 1954). Intracerebral inoculation of monkeys with
Egypt 101 WNV resulted in a clinical pattern were fever developed at 7-8 days postinfection, ataxia, convulsions, and weakness occurred at 10-13 days post-infection, and
death could occur at day 12-19 post-infection (Pogodina et al., 1983).
In humans, the first naturally occurring case of encephalitis was not described
until 1957 when 12 residents of a nursing home in Israel were affected (Solomon and
Vaughn, 2002). Neurological manifestations of WNV infection include aseptic
meningitis, encephalitis, or myelitis (Solomon and Vaughn, 2002). In days 1-7 following
infection, patients may exhibit myalgia, fever, headache, weakness, gastrointestinal
symptoms, drowsiness, and confusion (Hayes et al., 2005b). Some infected individuals
6
exhibit asymmetric weakness or paralysis in addition to dyskinesias such as
parkinsonism, myoclonus, and intention tremor (Granwehr et al., 2004). Ocular
symptoms of WNV infection include neuritis, chorioretinitis, retinal hemorrhages,
nystagmus, and optic edema (Granwehr et al., 2004). Approximately 15% of WNVinfected individuals develop symptoms of WN fever while less that 1% develop
neuroinvasive disease. The fatality rate for patients exhibiting CNS symptoms of WNV
infection is between 5-10% (Solomon and Vaughn, 2002).
1.8 Pathology
Pathological findings associated with human WNV infection include perivascular
cuffing of the brain parenchyma along with infiltrates of microglia and
polymorphonuclear leukocytes which are prominent in the brainstem and spinal cord but
can also be identified in the thalamus, cortex, and cerebellum (Sampson et al., 2000).
Lesions in response to WNV infection occur in both white and gray matter (Solomon and
Vaughn, 2002). Neuronal injury can also be present and is characterized by acute
neuronal necrosis and neuronophagia (Granwehr et al., 2004). Inflammation can also be
detected that is more prominent in the deep nuclei and Purkinje-cell layer of the
cerebellum as compared to cortical neurons. Severe loss of anterior horn neurons has
also been detected in the spinal cord in conjunction with axonal degeneration (Granwehr
et al., 2004). Patients infected with WNV have been found to exhibit changes in the
basal ganglia as visualized by magnetic resonance images which are characteristic of
other flaviviral encephalitic infections (Solomon et al., 2003).
Pathological analysis has also been conducted on other avian and mammalian
hosts susceptible to WNV infection in addition to the mosquito vector. Birds infected
with WNV exhibited pathologic changes in a variety of organs including brains, spleens,
and kidneys (Steele et al., 2000). Brain hemorrhage, splenomegaly, meningoencephalitis,
and myocarditis were the most prominent lesions, and cellular targets included neurons,
glial cells, peripheral ganglia, Purkinje cells, renal tubular epithelium, and myocardial
fibers (Steele et al., 2000). Hamsters infected with WNV exhibited splenic necrosis in
7
addition to neurological pathology. Beginning on day 5 post-infection hamsters exhibited
pathologic changes in Purkinje cells of the cerebellar cortex, subcortical gray matter, and
cerebral cortex (Xiao et al., 2001). On day 6, these changes become more severe,
especially the pathology of the cerebellar Purkinje cells (Xiao et al., 2001). On day 10,
most pathologic abnormalities localized to the brain stem, which exhibited focal neuronal
degeneration surrounded by microglial infiltration (Xiao et al., 2001). Long-term WNV
infection of Culex pipiens quinquefasciatus was found to result in cellular degeneration
and apoptotic-like cell death in the salivary glands (Girard et al., 2005). Thus WNV
infection is capable of inducing pathological changes in humans, birds, and mosquitoes.
1.9 Molecular Virology
1.9.1
Virion structure
Like other flaviviruses, West Nile virus (WNV) virions are approximately 500Å
in diameter and contain a nucleocapsid core surrounded by a host-derived lipid bilayer
envelope approximately 35 to 40Å thick embedded with viral membrane (M) and
envelope (E) proteins (Fig. 1-7) (Mukhopadhyay et al., 2003). The virus is roughly
spherical and exhibits icosahedral symmetry with a highly ordered scaffold of 90
glycoprotein E dimers and a relatively smooth surface without the distinct spikes or
surface projections characteristic of other enveloped viruses such as influenza and
measles viruses (Kuhn et al., 2002). Virions contain the electron-dense nucleocapsid
core composed of the positive-sense, single-stranded RNA genome in association with
the viral capsid (C) protein. Cross-sectional analysis of virions shows concentric layers
of varying electron density suggesting a multilayer organization (Mukhopadhyay et al.,
2003). Mature virions are composed of 6% RNA, 66% protein, 9% carbohydrate, and
17% lipid and sediment between 170 and 210S upon centrifugation (Lindenbach & Rice,
2001). During the process of maturation, flaviviral virions undergo a pH-induced
conformational change involving the premembrane (prM) and envelope (E) proteins
8
where 60 trimers of prM-E heterodimers dissociate to form 90 E homodimers giving the
virion a relatively smooth surface (Mukhopadhyay et al., 2005). Additional
conformational changes occur when the virus undergoes fusion with a host cell in which
the E homodimers dissociate into monomers which then rearrange into homotrimers
(Allison et al., 1995). Subviral particles (SVPs) are frequently observed in flavivirusinfected cell cultures and have been found to be composed of viral M and E proteins in
association with host-derived lipids without the nucleocapsid core (Allison et al., 2003).
These particles undergo similar post-translational modifications as infectious virions but
tend to be smaller in size with an average diameter of 315Å (Allison et al., 2003). Such
SVPs exhibit similar cell fusion characteristics to infectious virions and can be used to
induce protective immunity against certain flavivirus infections (Konishi et al., 1992).
1.9.2
Genomic organization
The WNV genome is a single-stranded, positive-sense RNA genome
approximately 11 kilobases (kb) in length that contains a type I methylated 5’ cap
(m7GpppAmpN2) and lacks a 3’ polyadenylated tail (Wengler et al., 1978). The genomic
RNA codes for a single open reading frame (ORF) translated into a single polyprotein
that is co- and post-translationally cleaved by a combination of the viral protease and host
endopeptidases to produce three structural and seven nonstructural proteins (Fig. 1-8)
(Coia et al., 1988). The virus-encoded protease composed of the NS2B and NS3 proteins
cleaves at the amino termini of NS2B, NS3, NS4A, and NS5 proteins in addition to
mediating internal cleavage of the C, NS2A, and NS3 proteins (Castle et al., 1985;
Speight et al., 1988; Wengler et al., 1990). An unidentified host signal peptidase cleaves
the amino termini of prM, E, NS1, and NS4B proteins while the host furin protease is
responsible for removing the “pr” portion of prM to form the mature M protein. The
protease responsible for mediating NS1/NS2A junction cleavage remains unknown but is
provided by the host (Lindenbach and Rice, 2003). Flavivirus genomic RNA serves as
the messenger RNA and is therefore infectious upon transfection into host cells (Peleg,
1969).
9
1.9.3
WNV structural proteins
Capsid (C) protein
The C protein is a small, highly basic protein approximately 11kD in size that is
involved in forming the viral nucleocapsid in association with genomic RNA. The
mature C protein contains 107 amino acid residues and includes two basic RNA binding
domains at the N- (residues 3 to 32) and C- (residues 84-107) termini (Khromykh and
Westaway, 1996). In mature virions, the C protein exists as tetrameric dimers and each
subunit consists of four α-helices (Ma et al., 2004). An internal hydrophobic region
including residues 29 to 69 is known to be essential for dimerization as well as mediating
interactions with cellular membranes (Bhuvanakantham and Ng, 2005). The C-terminal
domain of the C protein contains a signal sequence that is cleaved by the NS2B/NS3 viral
protease and is involved in translocating the prM protein into the ER lumen (Amberg and
Rice, 1999). The C protein associates with the ER membrane and is primarily found in
the cytoplasm although it has sometimes been detected in the nucleus (Wang et al.,
2002). The nuclear localization signal in the C protein is composed of residues 85-10 in
addition to the highly conserved residues 42 and 43 (Mori et al., 2005). The C gene
region also encodes the 5’ cyclization nucleotide sequence that is required for viral RNA
replication (Khromykh and Westaway, 1997). Kunjin virus (KUNV) C protein has also
been shown to translocate into the nucleus following interactions with perinuclear
membranes of infected Vero cells (Westaway et al., 1997).
The pre-membrane/membrane (prM/M) protein
The pre-membrane (prM) protein is a small glycoprotein approximately 26 kD in
size that is known to be involved in the maturation of flaviviral virions. The immature
prM protein is translocated into the ER lumen by the C-terminal signal sequence of the C
protein. Coordinated processing of the prM protein occurs and is delayed until the capsid
signal sequence is removed (Amberg et al., 1994). The prM signalase cleavage site is
known to be suboptimal, and the introduction of an optimized cleavage site into yellow
fever virus (YFV) was found to be lethal for virus production, although progeny viruses
10
were obtained that encoded additional substitutions in the signal sequence (Lee et al.,
2000). The N-terminal region of the prM protein contains between one and three Nlinked glycosylation sites (Chambers et al., 1990) and six conserved cysteine residues
that mediate the formation of three disulfide bonds (Nowak et al., 1987). All cysteines
are removed during processing into the mature M form of the protein. The C-terminal
region of the prM protein is composed of two transmembrane regions that are involved in
anchoring to the membrane along with the E protein signal sequence (Markoff et al.,
2004). Immature prM protein forms a heterodimer with E protein, and the conserved
histidine residue at position 99 is required for this function (Lin and Wu, 2005). These
heterodimers form spikes on the surface of the immature virion, and the prM protein is
located at the tip of the spikes (Heinz et al., 1994). The prM protein is thought to serve as
a chaperone for the E protein and prevents premature pH-dependent conformational
changes within the low pH secretory pathway (Guirakhoo et al., 1992). Cleavage of the
prM protein by either furin or a furin-like peptidase occurs just after the virion leaves the
trans-Golgi network and results in the production of mature M protein and the secreted pr
fragment (Stadler et al., 1997; Murray et al., 1993). The timing of this cleavage step is
critical to produce properly formed mature virions as the E protein requires a specific pH
to form the necessary homodimers. Mutations in the furin recognition site were found to
prevent prM processing and resulted in the production of immature viral particles
(Elshuber et al., 2003). Dengue virus (DENV) M protein has been found to contain a
proapoptotic ectodomain comprised of the C-terminal residues 32-40, which is also
present in other mosquito-borne flaviviruses, including WNV, although the mechanism of
action remains unclear (Catteau et al., 2003).
Envelope (E) protein
The E protein is approximately 53 kD in size and is the major structural protein
on the surface of virions. The mature virion is coated with 90 head-to-tail homodimers of
the E protein rich in β-sheets that lie parallel with the virus envelope (Fig. 1-8). Crystal
structures have been determined for the E proteins of tick borne encephalitis (TBE) virus
11
(Rey et al., 1995), dengue-2 (DEN2) virus (Modis et al., 2003), and WNV (Nybakken et
al., 2006; Kanai et al., 2006). The E protein is a class II fusion protein where E
homodimers convert into a trimeric fusogenic complex upon exposure to low pH in host
cells (Heinz and Allison, 2001). Each E protein monomer is composed of an ectodomain
containing the N-terminal 400 amino acids that consists of three domains (I, II, and III)
where domain I serves as the hinge region, domain II serves as the dimerization domain,
and domain III contains the receptor-binding region (Mandl et al., 2000). The E protein
also serves as the primary target of neutralizing antibodies (Roehrig et al., 1989). The E
protein contains twelve conserved cysteine residues that form six disulfide bonds critical
for stabilizing the structure of the protein (Nowak and Wengler, 1987). The C-terminus
of the E protein is a stem region that contains two α-helices that are involved in
stabilizing the structure of the fusion trimer (Ferlenghi et al., 2001).
Domain I forms the central domain and is composed of residues 1-51, 137-189,
and 285-302 in the TBEV E protein. This domain is involved in stabilizing the protein
and many flaviviruses contain a conserved N-linked glycosylation site that is thought to
contribute to this function. However, various naturally occurring flaviviruses are known
to lack this glycosylation site. Studies of glycosylated and non-glycosylated strains have
been mixed in linking the glycosylation phenotype to attenuation. In some cases, E
glycosylation was associated with decreased virulence (Chambers et al., 1998; Halevy et
al., 1994) while in others there was no change in virulence (Chambers et al., 1998;
Scherret et al., 2001). Studies of glycosylated and non-glycosylated WNV strains have
shown that the presence of the glycosylation motif increased the mouse neuroinvasive
phenotype (Beasley et al., 2005; Shirato et al., 2004) although a comparison of naturally
occurring lineage I and II WNV strains found no distinct correlation between
glycosylation and neuroinvasion (Beasley et al., 2004).
Domain II forms the dimerisation domain and is comprised of residues 52-136
and 190-284 in TBEV E protein (Rey et al., 1995). This domain contains the fusion
protein and hinge region which are critical for mediating the conformational changes
during fusion to host cells. When the E protein is exposed to low pH in host endosomes,
12
the hinge region exhibits significant flexibility allowing for exposure of the fusion region
and interactions with host membranes (Zhang et al., 2004). This rearrangement is a twostep process comprised of a reversible dissociation of E homodimers followed by the
irreversible formation of thermodynamically favored trimers (Allison et al., 1995).
Introduced mutations predicted to decrease flexibility have also been shown to decrease
the efficiency of fusion (Hurrelbrink and McMinn, 2001). In addition, amino acid
substitutions localizing to the hinge region have been found in a variety of different
attenuated flaviviruses including JE (Cecilia and Gould, 1991; Arroyo et al., 2001;
Monath et al., 2002; Hasegawa et al., 1992), DEN2 (Gualano et al., 1998), and LGT
viruses (Campbell and Pletnev, 2000).
Domain III is composed of residues 303-395 in TBEV E protein (Rey et al.,
1995) and contains the putative receptor-binding region (Crill and Roehrig, 2001).
Neutralizing antibodies against flaviviruses have frequently been shown to bind to
domain III. The domain III protein has an Arg-Gly-Asp (RGD) integrin-like motif and
immunoglobulin-constant domain-like structure consisting of a β-barrel containing six
antiparallel β-sheets (Rey et al., 1995). Langat virus (LGTV) E protein domain III has
been found to compete with infectious virions for receptor binding (Bhardwaj et al.,
2001), and the receptor-binding site is found in an exposed loop known as the FG loop
(Zhang et al., 2004). Various mutations observed in attenuated flavivirus strains localize
to this region including those in hamster-passaged Asibi YFV (McArthur et al., 2005),
chimeric DEN2/DEN4 viruses (Hiramatsu et al., 1996), and MVEV and JEV strains that
exhibit altered binding to glycosaminoglycans (Lee and Lobigs, 2004). For WNV, a
specific interaction between domain III and the putative receptor, αvβ3 integrin, was found
to induce phosphorylation of a kinase that could mediate ligand-receptor internalization
of virus into cells (Chu and Ng, 2004; Lee et al., 2006).
13
1.9.4
WNV nonstructural proteins
NS1 protein
The NS1 protein is approximately 46 kD in size and can either be intracellular,
associated with the host cellular surface, or secreted (Smith and Wright, 1985; Post et al.,
1991). Secreted NS1 is hexameric and consists of three sets of homodimers (Crooks et
al., 1994). Interestingly, NS1 has been found to be secreted by mammalian cells but not
by insect cells (Smith and Wright, 1985). Secreted NS1 has been found to localize to the
liver when injected into mice and has found to be capable of inducing a protective
immune response (Alcon-LePoder et al., 2005; Flamand et al., 1999). Intracellular NS1
is a hydrophobic membrane-associated dimer that contains 12 conserved cysteines that
form 6 disulfide bonds (Winkler et al., 1988; Blitvich et al., 2001). The NS1 protein is
cleaved from E by a host signalase after translocation into the ER lumen that requires a
hydrophobic signal sequence at the end of the E protein (Coia et al., 1988; Chambers et
al., 1990a; Falgout et al., 1989).
The NS1 protein is required for RNA replication and co-localizes with other
members of the viral replication complex (RC) along with dsRNA (Mackenzie et al.,
1996). Flaviviral NS1 proteins also contain multiple N-glycosylation sites, and ablation
of these sites have resulted in impaired RNA accumulation, delayed virus production, and
decreased neurovirulence in YF and WN viruses (Muylaert et al., 1996; Whiteman et al.,
submitted for publication). Screening of 28 YFV NS1 charged-amino-acid-to-alanine
mutants resulted in the identification of a temperature-sensitive mutant virus encoding a
R299A amino acid substitution that exhibited decreased RNA accumulation at 39°C
compared to 32°C (Muylaert et al., 1997). A set of lethal substitutions was also
identified that clustered in the N-terminal portion of NS1 while those impairing
replication relative to wild-type were distributed throughout the protein (Muylaert et al.,
1997). The NS1 protein can be successfully trans-complemented (Lindenbach and Rice,
1997) and is thought to interact with the RC via the NS4A protein (Khromykh et al.,
1999). The interaction between the NS1 and NS4A protein has been shown to be critical
for viral replication, and disruption of this interaction has been found to result in a lack of
14
minus-strand RNA accumulation (Lindenbach and Rice, 1999). While transcomplemented wild-type DEN NS1 was incapable of allowing for YF viral replication,
DEN NS1 proteins encoding suppressor mutations were found interact with YF NS4A
thereby allowing for successful viral replication (Lindenbach and Rice, 1999). A role for
the NS1 protein in viral packaging or assembly has not been determined although NS1 is
not required in cis (Jones et al., 2005).
NS2A protein
The NS2A protein is a small, hydrophobic protein approximately 22 kD in size
that localizes to the ER membrane. The N-terminus of NS2A resides within the ER
lumen while the C-terminus localizes to the cytoplasm (Preugschat et al., 1990). The
NS2A protein localizes to the viral RC and has been shown to bind NS3, NS5, and the
3’UTR of viral RNA (Mackenzie et al., 1998). The N-terminus of NS2A is cleaved by an
unknown host protease in the ER (Falgout and Markoff, 1995) while the C-terminus is
cleaved by the viral protease (Preugschat et al., 1990). The NS2A protein also contains
an internal viral protease cleavage site which results in the formation of NS2Aα and NS1’
products (Kummerer and Rice, 2002). Ablation of the internal viral protease cleavage
site was found to prevent production of infectious virus although a spontaneous mutation
in the helicase region NS3 was found to suppress this phenotype allowing for virus
production (Kummerer and Rice, 2002).
The NS2A protein has been found to be critical for KUNV assembly at the level
of RNA packaging, and a single I59N substitution was found to completely block virus
production (Liu et al., 2003). Amino acid substitutions in the DEN4 NS2A protein were
found to confer a temperature-sensitive phenotype in cell culture and attenuation of viral
replication in mouse brain (Blaney Jr. et al., 2003b). NS2A has also been shown to
inhibit IFN-β promoter-driven transcription, and a single A30P substitution in KUNV
NS2A was found to reduce this inhibition and allowed more efficient persistent
replication in hamster BHK21 and human HEK 293 cell lines (Liu et al., 2004). Further
analysis of the KUNV NS2A A30P mutant showed that this virus was defective in its
15
ability to inhibit α/β-interferon induction and was highly attenuated for neuroinvasiveness
and neurovirulence in mice (Liu et al., 2006). The NS2A A30P mutant was found to
exhibit decreased multiplication kinetics compared to wild-type virus in interferonproducing A549 cells while no decrease in multiplication was observed in interferondeficient BHK cells (Liu et al., 2006). Thus, the flaviviral NS2A protein seems to be
critical in both viral assembly and modulation of the host immune response.
NS2B protein
The NS2B protein is a small, hydrophobic protein approximately 15 kD in size
that serves as a cofactor with NS3 to form the active viral protease (Arias et al., 1993;
Preugschat et al., 1990; Chambers et al., 1989). The NS2B protein interacts with NS3 via
a conserved hydrophilic domain, and substitutions that destabilize this interaction have
been found to abolish protease activity (Chambers et al., 1993; Leung et al., 2001).
Cleavage at the NS2B/NS3 junction is required for viral replication, and NS2B can be
trans-complemented (Chambers et al., 1995; Wu et al., 2003). The NS2B protein also
has been found to regulated cleavage of the C protein (Amberg and Rice, 1999).
Expression of the NS2B protein in an inducible Escherichia coli system leads to
increased membrane permeability rendering the bacteria susceptible to hygromycin B
(Chang et al., 1999).
NS3 protein
The NS3 protein is approximately 69 kD in size and serves as a multifunctional
protein with protease, helicase, and NTPase activities. The NS3 protein is associated
with ER membranes when in complex with NS2B, although it localizes to the viral RC on
its own suggesting that NS2B is required for protease activity but not the helicase or
NTPase activities (Westaway et al., 1997). The N-terminal third of NS3 serves as the
trypsin-like serine protease that contains a catalytic triad composed of His-53, Asp-77,
and Ser-138 residues in YFV (Chambers et al., 1990b). Mutations within the catalytic
triad, especially Ser-138, have been found to decrease or prevent efficient cleavage
16
(Chambers et al., 1990b; Pugachev et al., 1993). The crystal structure has been solved for
the DEN2V NS3 protease that allows for improved modeling and identification of critical
residues in various flaviviruses (Murthy et al., 1999). The NS3 protease in conjunction
with the required NS2B cofactor is responsible for various viral polyprotein processing
events not mediated by host proteases. These include the NS2A-NS2B, NS2B-NS3,
NS3-NS4A, NS4A-2kNS4B, and NS4B-NS5 junctions (Cahour et al., 1992; Preugschat
and Strauss, 1991; Lin et al., 1993; Chambers et al., 1990). In addition, the NS2B/NS3
protease is involved in regulating the removal of the prM signal sequence from C
(Amberg et al., 1994; Lobigs, 1993). The consensus viral protease recognition sequence
is composed of a motif containing K-R or R-R followed by G, S, or A (Preugschat et al.,
1990; Billoir et al., 2000).
The C-terminal two-thirds of NS3 encode overlapping helicase, NTPase, and
RTPase activities (Li et al., 1999; Wengler & Wengler, 1991). The helicase activity is
required to unwind RNA secondary structure in the 3’UTR thus allowing efficient RNA
replication (Lindenbach and Rice, 2003). The NS4B protein has been found to modulate
the NS3 helicase activity by dissociating NS3 from single-stranded RNA in DENV
(Umareddy et al., 2006). In addition, coimmunoprecipitation demonstrated that DEN2
NS3 protein interacts directly with the NS5 polymerase protein and induces differential
phosphorylation of NS5 (Kapoor et al., 1995). The NTPase activity utilizes energy from
ATP hydrolysis to unwind base-paired regions of RNA to allow synthesis of new strands
(Li et al., 1999). The RTPase activity cleaves the terminal γ-phosphate from the 5’
triphosphate end of the RNA allowing the addition of the cap structure (Wengler and
Wengler, 1993). Amino acid substitutions within the DEN4 NS3 protein have been
found to confer a temperature-sensitive phenotype in cell culture and decreased
replication in mouse brain (Blaney Jr. et al., 2003b). Other amino acid substitutions
within the NTPase and helicase regions have also been shown to decrease infectivity in
cell culture (Liu et al., 2003; Kinney et al, 1997; Blaney Jr. et al., 2002). The structure of
the C-terminal NTPase/helicase region of NS3 has been determined and shows a tunnel
large enough to accommodate single-stranded RNA (Xu et al., 2005; Wu et al., 2005).
17
Three domains were identified, and two of these were found to bind nucleotides in a
triphosphate pocket for NTPase and RTPase while the third domain was involved in
RNA and protein recognition (Wu et al., 2005). Expression of NS3 alone has also been
implicated in the induction of host cell apoptosis through the recruitment of caspase-8
(Ramanathan et al., 2006).
NS4A protein
The NS4A protein is a small hydrophobic protein approximately 16 kD in size
that is thought to participate in viral RNA replication by serving as an anchor for other
members of the viral RC (Mackenzie et al., 1998). An interaction between the NS1 and
NS4A proteins has been found to be critical for successful viral replication by allowing
minus-strand RNA accumulation (Lindenbach and Rice, 1999). The N-terminus of the
NS4A protein is cleaved by the viral protease (Chambers et al., 1989; Preugschat and
Strauss, 1991). The C-terminus of the NS4A protein at the NS4A/2kNS4B junction is
also cleaved by the viral protease, and this reaction must occur before downstream
cleavage of the 2k peptide from the NS4B protein by a host endopeptidase (Lin et al.,
1993; Cahour et al., 1992; Preugschat and Strauss, 1991). The NS4A-2k-NS4B cleavage
events are highly regulated, and immature NS4A/B polyprotein has been detected in
mammalian but not mosquito cells suggesting that this product may provide a unique
function in viral replication (Preugschat and Strauss, 1991). Cleavage of the flaviviral
NS4A/B polyprotein by the viral protease is thought to be a key event in initiating the
induction of membrane proliferation (Roosendaal et al., 2006). In addition, removal of
the 2k peptide is hypothesized to allow targeting of mature NS4A to the Golgi
membranes were viral RCs occur (Mackenzie et al., 1999). Expression of NS4A in
bacteria has been found to alter membrane permeability, rendering them susceptible to
hygromycin B inhibition (Chang et al., 1999).
18
NS4B protein
The NS4B protein is a small, hydrophobic protein approximately 27 kD in size
that is thought to participate both in the viral RC and in evasion of the host immune
response. The NS4B protein is preceded by a 2k signal peptide that targets the NS4B
protein to the ER membrane although this peptide is not required for membrane insertion
since NS4B is an integral membrane protein (Miller et al., 2006). The mature NS4B
protein is produced following removal of the 2k peptide by a host signalase. This
reaction is preceded by the cleavage of the 2k/NS4B protein from the NS4A protein by
the viral NS2B/NS3 protease (Chambers et al., 1989; Lin et al., 1993; Cahour et al.,
1992; Preugschat and Strauss, 1991). Among mosquito-borne flaviviruses, the NS4B
protein exhibits approximately 35% identity. While there is little amino acid similarity
with other members of the family Flaviviridae, such as Hepatitis C virus (HCV),
topologies and functions are thought to be conserved (Lundin et al., 2003). Both HCV
and DEN2 NS4B proteins have been found to localize to discrete cytoplasmic foci within
the ER that are thought to represent viral RCs (Miller et al., 2006; Lundin et al., 2003).
Expression of HCV NS4B in cell culture also leads to ER-derived membrane
proliferation resulting in ultrastructural alterations known as “membranous webs” while
expression of NS4A/B does not (Egger et al., 2002; Konan et al., 2003). While primarily
distributed within the ER (Kim et al., 2004; Khromykh et al., 1996), KUNV NS4B
protein has also been shown to translocate into the nucleus following interactions with
reticular perinuclear membranes of infected Vero cells (Westaway et al., 1997).
Topologically, both DEN2V and HCV NS4B proteins have been shown to
primarily localize the N-terminus to the ER lumen while the C-terminus was in the
cytoplasm (Lundin et al., 2003; Miller et al., 2006). Hydrophobicity models predict that
flaviviral NS4B proteins contain between three and five transmembrane domains (TMDs)
(Lundin et al., 2003; Miller et al., 2006). Experimentally, DENV NS4B has been shown
to contain three TMDs that are capable of independently targeting a marker protein to
intracellular membranes (Miller et al., 2006). The HCV NS4B protein has been found to
be palmitoylated at two cysteine residues, C257 and C261, and that palmitoylation of
19
C261 is critical for modulating the formation of high-order NS4B multimers (Yu et al.,
2006). In addition, the HCV NS4B protein contains an N-terminal amphipathic helix and
nucleotide-binding motif that have been found to modulate RNA replication (Elazar et
al., 2004; Einav et al., 2004). Amino acid substitutions predicted to localize on both
lumenal and cytoplasmic sides of the ER membrane have also been found to affect the
establishment of HCV replicon colonies (Lindstrom et al., 2006). The NS4B protein has
also been found to modulate NS5 polymerase and NS3 helicase activities in the viral
replication complex of HC and DEN2 viruses (Piccininni et al., 2002; Umareddy et al.,
2006).
The HCV NS4B protein has been shown to be capable of transforming cells in
cooperation with the Ha-ras oncogene suggesting a role in the onset of malignant
transformation (Park et al., 2000) and has been shown to modulate an unfolded protein
response (Zheng et al., 2005). In addition, HCV NS4B has been found to specifically
interact with the CREB-RP/ATF6 heat shock protein (Tong et al., 2002). Expression of
KUNV NS4B protein in bacteria has been shown to increase membrane permeability
rendering them susceptible to hygromycin B inhibition (Chang et al., 1999) while bovine
viral diarrhea virus (BVDV) NS4B has been shown to play a role in cytopathogenicity
(Qu et al., 2001).
Various attenuated or passage-adapted flavivirus strains have exhibited
substitutions in a central hydrophobic region. A single DEN4 NS4B P101L substitution
was found to confer a small-plaque phenotype in mosquito C6/36 cells while increasing
plaque size in monkey Vero and human Huh7 cells (Hanley et al., 2003). Substitutions
have also been observed in this region with chimeric WN/DEN4 virus (Pletnev et al.,
2002), chimeric DEN2/4 virus (Blaney Jr., 2003a), Vero cell-passaged DEN4 virus
(Blaney Jr. et al., 2003a), JEV vaccine strains (Ni et al., 1995), YFV vaccine strains
(Hahn et al., 1987; Wang et al., 1995), and hamster-passaged Asibi-derived YFV
(McArthur et al., 2003). Except for the DEN4 P101L mutant, each of the described
viruses encodes additional amino acid substitutions scattered throughout the genome that
make determination of the significance of the NS4B mutations difficult. The P101L
20
substitution has also been found to prevent oligomerization of the DEN4 NS4B protein
(Miller et al., 2006).
Recently, DEN2, YF, and WN virus NS4B proteins have been shown to inhibit
interferon signaling at the level of signal transducer and activator of transcription
(STAT1) phosphorylation (Munoz-Jordan et al., 2003; Munoz-Jordan et al., 2005).
Deletion analyses identified the first 125 amino acid residues as being critical for this
function. In addition, the mature NS4B protein could inhibit interferon signaling but not
the NS4A/B precursor protein (Munoz-Jordan et al., 2005). Amino acid substitutions
within the NS4B protein have also been identified in HCV replicons exhibiting an
interferon-resistant phenotype (Namba et al., 2004). Thus, the role of the NS4B protein
in both viral replication and immune evasion may be conserved among members of the
family Flaviviridae.
NS5 protein
The NS5 protein is the largest flaviviral protein at 103 kD and serves as both a Sadenosyl-methionine (SAM)-dependent methyltransferase (MTase) and the viral RNAdependent RNA polymerase (RdRp). The NS5 protein can be trans-complemented, and
the minimal region for efficient complementation is the first 316 amino acids (Liu et al.,
2002). The NS5 protein of YFV plus the NS5A proteins of HCV and BVDV have been
shown to be phosphorylated by serine/threonine kinases suggesting that this represents a
common regulatory mechanism throughout the family Flaviviridae (Reed et al., 1998). In
DEN2V, the interaction between NS3 and NS5 has been shown to result in differential
phosphorylation of NS5 (Kapoor et al., 1995). The phosphorylation occurs at multiple
serine residues and is altered in the nuclear form of NS5 as compared to the cytoplasmic
form (Kapoor et al., 1995). The NS5 protein localizes to the nucleus by a signal
sequence and is modulated by a cellular nuclear import receptor (Johansson et al., 2001).
Approximately 20% of the RNA-dependent RNA polymerase activity from cells infected
with WNV, JEV, or DENV is resident within the nucleus (Uchil et al., 2006).
21
The N-terminal MTase portion of NS5 is responsible for RNA capping and also
encodes a GTPase activity. The crystal structure of the DEN1V MTase has been
determined that showed a structurally novel way of promoting specific binding of GTP
(Egloff et al., 2002). The C-terminus of NS5 encodes the RdRp activity and contains a
highly conserved GDD motif (Rice et al., 1985; Tan et al., 1996). Mutations within or
near this critical GDD motif have been found to ablate the polymerase activity (Guyatt et
al., 2001). The RdRp activity is dependent on interactions between NS5 and conserved
stem-loop, cyclization motifs, and pseudoknot elements in the 3’ UTR (Ackermann and
Padmanabhan, 2001). Incubation temperature was found to modulate the initiation but
not elongation phase of RNA synthesis (Ackermann and Padmanabhan, 2001). While
RNA viruses are generally regarded as having high mutation rates, the YFV NS5
polymerase was found to exhibit high fidelity with error rates as low as 1.9 x 10-7 to 2.3 x
10-7 per copied nucleotide (Pugachev et al., 2004). This resulting genetic stability is
thought to be at least partially in response for YFV to alternate between a mosquito
vector and vertebrate host. Amino acid mutation hotspots were observed in the Nterminal portion of prM, the central portion of E, and in NS4B suggesting that relatively
frequent substitutions in these proteins may be beneficial for virus assembly (Pugachev et
al., 2004).
The crystal structure for the HCV NS5A polymerase has been determined and
exhibits an RNA-binding groove running between the finger and thumb domains (Ago et
al., 1999). Structural determinations of flaviviral NS5 MTase and RdRp domains should
facilitate the development of antiviral agents targeting these regions. The NS5 protein
may also be involved in inhibiting interferon signaling pathways at the level of JAKSTAT phosphorylation for certain flaviviruses, possibly by activating protein tyrosine
phosphatases (Best et al., 2005; Lin et al., 2006). In addition, paired charge-to-alanine
mutagenesis of DEN4 NS5 generated mutant viruses that exhibited a temperaturesensitive phenotype in cell culture and attenuation of replication in mouse brain (Hanley
et al., 2002).
22
1.9.5
WNV untranslated regions
The 5’ UTR
The 5’ UTR is approximately 100 nucleotides in length and contains several
critical elements that are required for viral RNA replication. A highly conserved
cyclization sequence (5’CS) (UCAAUAUG) is found just downstream of the 5’UTR in
the capsid protein gene, spanning KUNV nucleotides +137 to +144, and is thought to
interact with a complememtary sequence in the 3’ UTR. The 5’CS is located within the
ORF approximately 34 to 40 nucleotides downstream of the start codon (Markoff, 2003).
Structurally, the flaviviral 5’ UTR is thought to consist of a stem with a small top loop
and a larger side loop. The 3’ portion of the 5’UTR is thought to contain a short stem
that encompasses the start codon for the ORF (Markoff, 2003). This structure is highly
conserved in various members of the Flavivirus genus, and mutations in the doublestranded stem region were either lethal for the virus or resulted in decreased plaque size
(Cahour et al., 1995).
The 3’ UTR
The 3’ UTR is variable in length among different flaviviruses but is
approximately 600 nucleotides in length. There is a highly conserved large 3’ stem-loop
(SL1) followed by a smaller stem-loop (SL2) in the 3’ terminus of the RNA although the
region immediately downstream of the stop codon is highly variable (Brinton et al.,
1986). A pseudoknot structure formed by the interaction of an unstable region of the 5’
side of SL1 with a conserved loop in SL2 has also been detected (Shi et al., 1996). The
3’ UTR contains three cyclization sequences (C1, C2, C3) that interact with elements in
the 5’ portion of the genome to allow cyclization and RNA replication to occur.
Substitutions targeting the cyclization sequences or the top loop of SL1 have been shown
to reduce RNA replication (Khromykh et al., 2003; Lo et al., 2003; Elghonemy et al.,
2005). A 30 nucleotide deletion in the 3’ UTR of DEN4 virus has also been found to
decrease replication in rhesus monkeys while maintaining the induction of a protective
immune response and has been used to develop candidate live attenuated DEN vaccine
23
strains (Hanley et al., 2004). Intriguingly, under certain cellular conditions the RNA capdependent mechanism is not required for initiation of DENV translation, however the
interaction of the 5’ and 3’ UTRs with other host components is necessary to allow for
translation (Edgil et al., 2006).
1.9.6
Virus life cycle
The virus life cycle of WNV begins with the interaction of the E protein domain
III receptor binding domain with the receptor, currently thought to be αVβ3 integrin (Chu
and Ng, 2004; Lee et al., 2006). The virus undergoes receptor-mediated endocytosis, and
a decrease in pH of the host endosome induces conformational changes that allow the
dissociation of E protein dimers and association of E protein trimers (Mukhopadhyay et
al., 2005) (Figs. 1-9 and 1-10). The virion disassembles and releases the nucleocapsid
into the cytoplasm in close proximity to the ER membrane. The positive-sense RNA
genome dissociates from the nucleocapsid and is then available to undergo translation.
Translation occurs utilizing host ribosomal components primarily via a capdependent mechanism mediated by the eukaryotic initiation factor 4E (EIF4E) capbinding protein. Under certain conditions, a cap-independent mechanism can be utilized
where the direct interactions of the viral 5’ and 3’ UTRs with host components initiates
translation (Edgil et al., 2006). Following translation of the single ORF, a combination of
host signalases and the viral NS2B/NS3 protease leads to production of the mature viral
proteins. The viral products such as the NS4B protein induce ER membrane proliferation
and reorganization that facilitates the formation of the viral RC in double-layered
membrane vesicles (Uchil and Satchidanandam, 2003; Mackenzie et al., 2005). The
NS4A protein is known to link the intracellular form of NS1 to the replication complex
(Lindenbach and Rice, 1999) while NS2A is capable of binding the NS3 protein that
provides helicase and NTPase activities, the NS5 protein encoding RdRp and MTase
activities, and the 3’ UTR that is required for initiation of replication (Mackenzie et al.,
24
1998). The NS4B protein has also been found to localize to the viral RC and is involved
in modulating the NS3 helicase activity (Umareddy et al., 2006).
RNA replication then occurs within the viral RC where the intermediate RNA
species are protected against proteolytic degradation (Uchil and Satchidanandam, 2003).
Cyclization of the 3’ and 5’ UTRs is required to initiate viral RNA replication. Three
forms of viral RNA are present during replication including the single-stranded, positivesense genomic RNA, a double-stranded RNA product termed the replicative form (RF),
and a partially double-stranded species termed the replicative intermediate (Chu and
Westaway, 2003). Approximately 10- to 100-fold more positive-sense RNA is produced
than negative-sense RNA (Muylaert et al., 1996), and these positive strands accumulate
to await packaging into virions. Negative-sense RNA is only found in double-stranded
RNA species in complex with newly formed positive-sense RNA (Chu and Westaway,
1985). Positive-sense RNA is then subjected to capping by the N-terminal portion of the
NS5 protein that encodes the MTase and GTPase functions (Egloff et al., 2002).
Synthesized viral genomic RNA is then coated with the membrane-associated
capsid protein to form the nucleocapsid before budding into the ER lumen where the
nucleocapsid interacts with prM-E heterodimers to form the immature virion
(Mukhopadhyay et al., 2005). The immature virion is covered with 60 irregular trimeric
surface spikes composed of three prM-E heterodimers. Further post-translational
modifications such as glycosylation occur as the immature virion proceeds through the
ER. Within the Golgi apparatus, a pH-induced conformational change causes
dissociation of the trimeric spikes and formation of 90 E homodimers that lie flat on the
surface of the virion (Mukhopadhyay et al., 2005). A 30° shift in the E hinge region
allows for furin cleavage of the prM protein, which facilitates the maturation of the
virions (Guirakhoo et al., 1992). Virions then collect in Golgi-derived vesicles before
being released at the cell membrane to await fusion with a naïve host cell.
25
1.10 Immunology
1.10.1 Immune response to WNV
Dissemination of WNV infection
Following peripheral inoculation, the initial round of WNV replication is thought
to occur in Langerhans dendritic cells within the skin (Diamond et al., 2003b). The virusinfected dendritic cells are thought to migrate to draining lymph nodes where
dissemination into the bloodstream occurs, leading to a primary viremia (Kwan et al.,
2005). WNV then infects various peripheral organs and may cross the blood-brain
barrier (BBB) through direct hematogenous seeding, alterations in BBB integrity, or by
transport in virus-infected immune cells that travel to the CNS (Samuel et al., 2006b).
Following introduction into the CNS, WNV can cause serious neurological disease
including paralysis, meningitis, encephalitis, and death.
The interferon response
The host has evolved a variety of immune mechanisms to counteract WNV
infection. Interferon (IFN) is one of the first parts of the antiviral response and works by
inducing an antiviral state (Fig. 1-11). There are two types of IFN: type I IFN includes
IFN-α and IFN-β while type II IFN is denoted as IFN-γ. IFN-α/β is produced in direct
response to virus infection usually after detection of double-stranded (ds) RNA while
IFN-γ is primarily produced in response to detection of virus-infected cells by activated T
lymphocytes and natural killer (NK) cells. IFN-α/β leads to the upregulation of genes
with antiviral functions and is involved in stimulating dendritic cell maturation (AsselinPaturel et al., 2004). IFN-α/β has also been found to restrict viral tropism, decrease viral
burden, and prevent neuronal death in WNV-infected mice (Samuel et al., 2005). IFN- γ
has been shown to be critical in preventing viral dissemination to the CNS by activating
γδ T lymphocytes and by preventing WNV production in activated dendritic cells
(Shrestha et al., 2006b).
26
Intracellular viral sensing components
Cells infected with WNV are able to respond directly to virus dsRNA products
with nucleic acid sensors such as Toll-like receptor 3 (TLR3) and the retinoic acidinducible gene I (RIG-I). While TLR3 is a critical component of the antiviral response, it
has also been shown that TLR3-dependent inflammatory responses can mediate WNV
entry into the brain causing lethal encephalitis by tumor necrosis factor-α (TNFα)induced permeablization of the BBB (Wang et al., 2004). Interestingly, TLR3 knockout
mice exhibited increased viral loads in the periphery but were more resistant to lethal
WNV infection than wild-type mice (Wang et al., 2004). The transcription factor IFN
regulatory factor 3 (IRF3) is induced by WNV infection and is involved in constraining
WNV and limiting cell-to-cell spread (Fredericksen et al., 2004). The absence of IRF3
was found to significantly increase WNV plaque size and increase the production of
infectious virions (Fredericksen et al., 2004). The IFN- α/β-induced protein dsRNAdependent protein kinase (PKR) and 2’,5’-oligoadenylate sythetase (OAS) proteins are
also involved in modulating the antiviral response by targeting components of the
eukaryotic translation complex such as eukaryotic translation initiation factor 2 (eIF2) for
phosphorylation (Samuel et al., 2006a). The OAS enzymes are also known to bind and
activate RNase L, an enzyme that cleaves viral RNA. Mice lacking PKR and RNase L
were highly susceptible to WNV infection and exhibited 90% mortality compared to
wild-type mice that exhibited 30% mortality (Samuel et al., 2006a).
Cellular-mediated and humoral immunity
Macrophages are known to clear WNV infection through direct viral clearance,
antigen presentation, and induction of cytokine and chemokine responses (Shirato et al.,
2006; Coccia et al., 2002). Natural killer cells are also involved in clearing virus-infected
cells (Vargin et al., 1986). B lymphocytes and antibody production have been shown to
be involved in limiting WNV dissemination as mice lacking these proteins (μMT mice)
exhibited increased viral burdens and increased vulnerability to lethal infection (Diamond
et al., 2003c). Passive antibody transfer from immune mice was found to protect the
27
μMT mice against lethal infection (Diamond et al., 2003c). CD8+ T lymphocytes have
been found to be critical in resolving WNV infection by using perforin to clear WNV
from infected neurons (Shrestha et al., 2006a). Studies have also shown the critical role
of neutralizing antibodies in protective immunity against WNV infection (Beasley and
Barrett, 2002).
1.10.2 Immune evasion by WNV
Flaviviruses have developed a variety of mechanisms to counteract the host
immune response (Katze et al., 2002). Various nonstructural proteins have been
implicated in inhibiting IFN signaling by blocking STAT1 and STAT2 phosphorylation
(Guo et al., 2005). The KUNV NS2A, NS2B, NS3, NS4A, and NS4B proteins were
found to prevent the translocation of STAT2 into the cell nucleus (Liu et al., 2005). The
NS4B protein from DEN2, WNV, and YF viruses has also been found to block STAT1
phosphorylation, and the N-terminal 125 amino acids were found to be critical for this
function (Munoz-Jordan et al., 2003; Munoz-Jordan et al., 2005). Others have shown that
the NS5 protein rather than the NS4B protein is primarily responsible for blocking JAKSTAT signaling (Best et al., 2005; Lin et al., 2006). Resistance to IFNα/β has been
shown to be a critical determinant of WNV fitness and virulence, and strains defective in
this function tend to be attenuated in mice and certain cell types (Liu et al., 2006; Keller
et al., 2006). Flaviviruses are also known to utilize antigenic variability to counteract the
humoral and cellular components of the host immune response (reviewed by Diamond,
2003a).
Flaviviruses have developed various methods to counteract intacellular virusresponse components. WNV is known to delay IRF3 activation through both RIG-Idependent and -independent pathways thereby circumventing the host immune response
until the infection is well underway (Fredericksen et al., 2006). WNV also interferes
with poly(I:C)-induced interferon gene transcription as demonstrated in replicon-bearing
HeLa cells (Scholle and Mason, 2005). The DENV2 NS5 protein has been shown to
induce interleukin-8 (IL-8) transcription in HEK293 cells suggesting that this chemokine
28
may be involved in the inflammatory response to DENV infection (Medin et al., 2005).
Neurovirulent WNV strains have been found to induce different gene expression patterns
in infected cells and mice as compared to infection by attenuated WNV strains (Shirato et
al., 2004; Venter et al., 2005).
1.10.3 Vaccine designs
Two veterinary vaccines are currently available for use in equines, however a
human vaccine has not yet been developed. An inactivated WNV vaccine strain based on
the NY99 strain has been shown to be both safe and effective in equines, and immunity is
known to last for at least one year (Ng et al., 2003). A recombinant canarypox-based
vaccine that expresses the WNV prM and E protein antigens has been shown to be safe
and effective in multiple animal models and has recently been licensed for use in equines
(Minke et al., 2004). Alternative vaccine designs include chimeric WN/YF 17D and
WN/DEN4 constructs as candidate live attenuated vaccines that exhibit reductions in
virulence compared to wild-type NY99 WNV without compromising the induction of a
protective immune response (Monath et al., 2001; Arroyo et al., 2004; Pletnev et al.,
2002). This strategy has been successful with Chimerivax-JE which utilizes the safe YF
17D backbone to express WNV prM and E proteins and has successfully undergone
phase I and II clinical trials (Monath, 2006). In addition, the chimeric WN/DEN4 virus
has been found to successfully protect rhesus monkeys against WNV infection (Pletnev et
al., 2003). Alternatively, the attenuated lineage II prototype Uganda37 WNV strain could
be utilized to induce protective immunity against the more virulent NY99 strain
(Yamshchikov et al., 2004). Chimeric viruses encoding NY99 structural proteins and
attenuated Uganda37 nonstructural proteins resulted in equal attenuation and superior
immunogenicity compared to the parental Uganda37 virus (Borisevich et al., 2006).
Due to the theoretical possibility of flavivirus vaccine recombination and the
known risk of disease in immunocompromised individuals associated with live vaccines,
some have suggested that the development of non-live flavivirus vaccines should be
encouraged (Seligman and Gould, 2004). A trans-packaging system has been developed
29
to produce WN virus-like particles (VLPs) in which WN C, prM, and E proteins are
expressed using Sindbis virus-derived RNAs (Scholle et al., 2004). Such VLPs could
likely be used as a non-live method of vaccination. Direct inoculation of WNV E domain
III has also been shown to protect mice against WNV infection (Chu et al., 2006).
There are currently no clinically licensed antiviral agents for use in response to
WNV infection. A humanized monoclonal antibody against the WNV E protein (hE16)
administered after neuronal infection was recently found to protect hamsters against
lethal encephalitis. Hamsters that received an intraperitoneal injection of hE16 antibody
five days following viral injection exhibited 80-90% survival compared to 37% survival
in placebo-injected hamsters (Morrey et al., 2006). The recent advances in understanding
the flaviviral life cycle and the structure and function of viral components should allow
for the development of new antiviral agents that can be utilized during the course of
clinical infection.
1.11 Specific aims
Although the flaviviral nonstructural NS4B protein is known to be essential for
viral replication, its function is poorly understood. Various functions have been proposed
for the NS4B protein, its precise role in the viral life cycle remains unclear. The NS4B
protein is extremely hydrophobic which makes direct manipulation and purification
difficult. While NS4B amino acid sequence homologies are negligible among members
from the different genera of the family Flaviviridae, protein topologies and functions are
thought to be conserved.
Amino acid substitutions have been found to localize within a central
hydrophobic region of the NS4B protein in a variety of attenuated or passage adapted
flaviviruses. Most of these mutations occur in addition to other substitutions scattered
throughout the genome, therefore the contribution of the NS4B mutations to observed
alterations in phenotype remain unknown. A single DEN2V NS4B P101L substitution
30
was found to confer a decrease in plaque size in mosquito cells while increasing plaque
size in monkey Vero and human Huh7 cells (Hanley et al., 2003). This situation is
unique because the P101L substitution was the only mutation in the entire genome
allowing the conclusion that this mutation was directly responsible for the observed
phenotype.
Utilizing a WNV infectious clone, this dissertation proposes to identify critical
residues within NS4B by introducing amino acid substitutions targeting distinct regions
of the protein by site-directed mutagenesis. It is likely that some of the introduced amino
acid substitutions will lead to the production of recombinant mutant viruses exhibiting
altered multiplication kinetics in cell culture and attenuation of neuroinvasiveness in
mice. Modeling of the NS4B protein will be conducted utilizing hydrophobicity plotting
programs to assist with the identification of residues to be targeted by mutagenesis.
Following the identification of attenuated WNV mutant recombinant viruses, additional
analyses will be conducted to elucidate the molecular mechanism of attenuation focusing
on aspects of the immune system in addition to gene expression profiles. Therefore, the
following specific aims are proposed:
1.11.1 Specific aim 1
Compare amino acid sequences of NS4B proteins from different flaviviruses and WNV
isolates and produce a topological model of the WNV NS4B protein utilizing
hydrophobicity plotting programs.
Hypothesis: Analyzing conserved and variable regions within the WNV NS4B protein in
conjunction with predicted topological data will allow for the identification of amino acid
residues that may be critical for NS4B structure and function.
Rationale: Amino acid sequence alignments will be conducted comparing the WNV
NY99 NS4B protein to other published WNV NS4B amino acid sequences and other
flavivirus NS4B proteins. These data should allow for the identification of conserved and
variable regions of the protein. Hydrophobicity plotting programs will also be utilized to
obtain a working topological model of the NS4B protein as structural data is not
31
available. The combination of alignment and hydrophobicity data should allow for the
identification of amino acid residues within the WNV NS4B protein that may be critical
for NS4B function and will be targeted for mutagenesis.
1.11.2 Specific aim 2(a)
Conduct site-directed mutagenesis targeting the central hydrophobic region where
mutations have been found to localize in various attenuated or passage-adapted flavivirus
strains.
Hypothesis: Recombinant mutant viruses encoding amino acid substitutions targeting the
central hydrophobic domain will exhibit altered multiplication in cell culture and
attenuation in mice.
Rationale: Based on the alignments and models from specific aim 1 in conjunction with
the literature, a series of amino acid substitutions targeting the central hydrophobic
domain has been designed. Various attenuated or passage-adapted flavivirus strains have
been found to encode amino acid substitutions within a variable central hydrophobic
region. Engineered amino acid substitutions will be introduced into the WNV infectious
clone, and produced recombinant mutant viruses will be assayed for alterations in
multiplication in cell culture as well as attenuation of neuroinvasiveness in mice as
compared to wild-type WNV.
1.11.3 Specific aim 2(b)
Conduct site-directed mutagenesis targeting the four cysteine residues as cysteines are
known to be involved in catalytic reactions, disulfide bonds, or other protein-protein
interactions.
Hypothesis: Recombinant mutant viruses encoding amino acid substitutions targeting the
four cysteine residues will exhibit altered multiplication in cell culture and attenuation in
mice.
Rationale: The WNV NS4B protein encodes four cysteine residues (C102, C120, C227,
and C237). Since cysteine residues are often critical for the structure or function of
32
proteins, each cysteine will be mutated to serine using site-directed mutagenesis of the
WNV infectious clone. Resulting recombinant mutant viruses will be assayed for
alterations of multiplication kinetics in cell culture as well as attenuation of
neuroinvasiveness and neurovirulence in mice compared to wild-type WNV. Viruses
found to be attenuated will be subjected to further analysis to elucidate the molecular
mechanism responsible for observed alterations in phenotype.
1.11.4 Specific aim 2(c)
Conduct site-directed mutagenesis targeting a highly conserved N-terminal motif that is
found throughout mosquito- and tick-borne flaviviruses.
Hypothesis: Recombinant mutant viruses encoding amino acid substitutions targeting the
conserved N-terminal motif will exhibit altered multiplication in cell culture and
attenuation in mice.
Rationale: The WNV NS4B protein contains a N-terminal motif that is highly conserved
among various mosquito- and tick-borne flaviviruses. It is possible that the high degree
of conservation is due to these residues serving critical roles pertaining to NS4B structure
and function. Site-directed mutagenesis will be utilized to introduce amino acid
substitutions targeting the N-terminal motif into the WNV infectious clone. Recombinant
mutant viruses will be assayed for alterations of multiplication kinetics in cell culture and
attenuation of neuroinvasiveness in mice as compared to wild-type WNV.
1.11.5 Specific aim 3
Recombinant mutant viruses exhibiting attenuation of neuroinvasiveness in mice will be
subjected to further analysis in order to determine the molecular mechanism of
attenuation focusing on specific immune system components.
Hypthesis: Recombinant mutant viruses encoding NS4B amino acid substitutions that
exhibit attenuation of the mouse neuroinvasive phenotype will also show differences in
modulating components of the immune response as compared to wild-type WNV.
33
Rationale: Attenuated recombinant mutant viruses encoding NS4B amino acid
substitutions likely display a reduced ability to modulate the immune response.
Flaviviral NS4B has been shown to disrupt IFN signaling, and disruption of this function
would be expected to attenuate the virus. Attenuated viruses encoding NS4B mutations
may be unable to successfully modulate the host immune response which would explain
the observed alterations in phenotype. Multiplication kinetics will be assayed in mice as
well as in cell lines known to be involved in the host immune response. In addition,
differences in gene expression profiles will be assayed in cells infected with either
virulent wild-type WNV or attenuated NS4B mutant WNV strains.
34
Figure 1-1. Diagram showing the main components of the West Nile virus
transmission cycle (adapted from CDC, Arbonet). The primary transmission cycle
consists of an amplifying avian host and a mosquito vector. A wide variety of
vertebrates including humans and horses can be infected with WNV but serve as
incidental dead-end hosts.
35
Figure 1-2. Worldwide distribution of West Nile virus in 2006 is shown in blue
(Adapted from CDC, Arbonet). The closely related Kunjin virus in Australia
appears in periwinkle. West Nile virus was first detected in North America in 1999
and has since moved north into Canada and south into the Caribbean, Central, and
South America.
36
Figure 1-3. Phylogram showing relationships of different flaviviruses based on the
NS5 polymerase domain (Adapted from Kuno and Chang, 2005). West Nile virus
groups most closely with JEV, USUV, and MVEV.
37
Figure 1-4. Phylogram showing relationships of different WNV isolates utilizing
complete genomic nucleotide sequences (Adapted from Bakonyi et al., 2006).
Shading highlights the co-circulation of both Lineage 1 and Lineage 2 WNV strains
in Hungary.
38
Figure 1-5. Reported incidence of neuroinvasive West Nile virus disease by county,
United States, 1999-2004. Reported to Centers for Disease Control and Prevention
through April 21, 2005 (Adapted from Hayes et al., 2005)
Incidence per million
0-9.99
10-99.99
>100
Any WNV Activity
39
Figure 1-6. Distribution of human cases by year. Numbers of detected human
WNV cases are shown in red while deaths attributed to WNV infection are shown in
green. Increased ratios of detected cases to deaths in 2003 (9858:262) as compared
to 2002 (4161:277) were likely the result of increased surveillance.
10000
9858
9000
8000
7000
6000
5000
4161
4052
4000
3000
3000
2000 62
7
1000
1999
Cases
Deaths
2448
19 2
48 5
2000
2001
277
2002
262
2003
40
87
2004
119
2005
146
2006
Figure 1-7. West Nile virus genome organization showing co- and post-translational
processing reactions (Provided by A.D.T. Barrett). The positive-sense RNA genome
encodes a single open reading frame that is translated into a polyprotein which is coand post-translationally cleaved into three structural and seven nonstructural
proteins by a combination of the viral NS3 protease and host endopeptidases.
5’NCR Structural proteins
NS1 NS2A NS2B
3’NCR
NS3 NS4A 2KNS4B NS5





C prM E
Non-structural proteins
RNA
Polyprotein

cap
Co- and Post-translational Processing
C
prM
pr
E
NS1
NS2A NS2B
M

Signal peptidase site
Unique site
NS2B-NS3 protease site
NS3
NS3’
NS4A 2K
NS4B
NS5
NS3”
NS3 Protease, helicase, NTPase
NS5 Methyltransferase, RNA polymerase
41
Figure 1-8. Diagram of the DEN E protein showing linear amino acid sequence
(Panel A) and ribbon structure (Panel B). Domain I is shown in red, domain II in
yellow, and domain III in blue. (adapted from Modis et al., 2003).
42
Figure 1-9. West Nile virus life cycle (Adapted from Solomon and Barrett in Nash
and Burger (Eds) Clinical Neurovirology Marcel Decker 2003). West Nile virus
enters the host cell by receptor mediated endocytosis and undergoes low pHdependent membrane fusion to uncoat the RNA genome. The translated
polyprotein is cleaved and processed, RNA replication occurs, and immature virions
undergo processing in intracellular vesicles. Virions undergo additional maturation
in the Golgi apparatus and are transported in vesicles to thte cell surface, where the
mature virions are released to infect new cells.
43
Figure 1-10. Conformational changes of the virion during maturation (Panel a) and
fusion with host cell membranes (Panel b). (Adapted from Mukhopadhyay et al.,
2005). During maturation (a), 60 trimers of prM-E heterodimers rearrange to form
90 E homodimers. When the virus undergoes fusion with the host cell (b), the E
homodimers dissociate into monomers before reassociating into homotrimers.
44
Figure 1-11. Molecular processes that signal the host response to HCV infection
(Adapted from Gale Jr. and Foy, 2005). Following infection, viral RNA binds to
RIG-1 or TLR3 and initiates IRF-3 activation (a). Signal transduction pathways
induced by IRF3 result in IFN-β production (b), and binding of IFN-β to its receptor
leads to activation of the Tyk2 and Jak1 kinases that phosphorylate STAT1-STAT2
heterodimers and direct the formation of the trimeric ISGF3 complex (c). The
ISGF3 complex translocates into the nucleus and initiates ISG expression, thereby
inducing an antiviral state within the cell.
45
CHAPTER 2
MATERIALS AND METHODS
2.1 Buffers and solutions
GTE cell suspension buffer
50 mM Glucose (Sigma)
25 mM Tris-Cl (Sigma)
10 mM EDTA (Sigma)
Alkaline lysis buffer
0.2 N NaOH (Sigma)
1% SDS (Sigma)
PAGE loading buffer (2X)
100 mM Tris-Cl pH = 6.8 (Sigma)
4% Sodium dodecyl sulfate (SDS) (Sigma)
0.2% Bromophenol blue (Sigma)
20% Glycerol (Sigma)
200 mM β-mercaptoethanol (Sigma)
PAGE Laemmli running buffer (10X)
25 mM Tris-Cl (Sigma)
250 mM Glycine (Sigma)
0.1% SDS (Sigma)
46
Western blot transfer buffer (10X)
500 mM Trizma base (Sigma)
4 M Glycine (Sigma)
Mix 100mL 10X transfer buffer, 200mL methanol (Sigma), and 700mL ddH20 for 1L of
1X Western transfer buffer
Phosphate Buffered Saline (PBS)/Tween buffer)
1X PBS
0.1% Tween 20 (Sigma)
PBS/milk blocking buffer
1X PBS
5% Milk (Carnation)
Luria-Betani (LB) broth and agar (per liter)
20g LB powder (Difco)
Add ddH20 to 1L. For LB agar, add 15g Bacto agar (Difco) per liter before dilution.
Tryptose phosphate buffer (TPB) (2X)
29.5g TPB powder (Sigma)
500 mL ddH20
2.2 Cell culture media recipes
Monkey kidney Vero cell media
1X MEM supplemented with Earle’s salts and L-glutamine (Gibco)
8% bovine growth serum (BGS) (Hyclone)
100 units/mL penicillin (Gibco)
100 μg/mL streptomycin (Gibco)
47
1X Non-essential amino acids (NEAA) (Gibco)
1X L-glutamine (Gibco)
Mouse neuronal Neuro2A cell media
1X MEM supplemented with Earle’s salts and L-glutamine (Gibco)
8% BGS (Hyclone)
100 units/mL penicillin (Gibco)
100 μg/mL streptomycin (Gibco)
0.075% sodium bicarbonate (Sigma)
1 mM sodium pyruvate (Sigma)
1X NEAA (Gibco)
Macrophage P388.D1 cell media
1X RPMI 1640 with L-glutamine (Gibco)
4% BGS (Hyclone)
1X NEAA (Gibco)
Dendritic DC2.4 cell media
1X RPMI 1640 with L-glutamine (Gibco)
8% BGS (Hyclone)
0.075% sodium pyruvate (Sigma)
1X NEAA (Gibco)
1X L-glutamine (Gibco)
100 units/mL penicillin (Gibco)
100 μg/mL streptomycin (Gibco)
Mosquito C6/36 cell media
1X MEM with Earles’s salts and L-glutamine (Gibco)
10% Fetal bovine serum (FBS) (Hyclone)
48
0.075% TPB
100 units/mL penicillin (Sigma)
100 μg/mL streptomycin (Sigma)
NEAA (Gibco)
Human hepatocyte (Huh) cell media
1X MEM with Earle’s salts and L-glutamine (Gibco)
8% FBS (Hyclone)
100 μg/mL streptomycin (Gibco)
100 units/mL penicillin (Sigma)
For Huh FT3.7 and Huh 7.5-iTLR3.16 cells, supplement with 2 μg/mL blasticidin
(Sigma).
Plaque assay overlay media
2X MEM Dilute 10X MEM (Sigma) in H20
4% BGS (Hyclone)
200 units/mL penicillin (Sigma)
200 μg/mL streptomycin (Sigma)
5% sodium bicarbonate (Sigma)
1X NEAA (Gibco)
Before overlay, mix 50:50 with 2% agar (Sigma)
2.3 Protein modeling of NS4B
The amino acid sequence of the NS4B protein from the prototypical North
American West Nile virus strain, NY99 (382-99), was entered into the SOSUI
(http://bp.nuap.nagoya-u.ac.jp/sosui/) and Consensus prediction (ConPredII)
(http://bioinfo.si.hirosaki-u.ac.jp/~ConPred2/) hydrophobicity plotting programs. The
49
complete genome of the WNV NY99 (382-99) strain was previously sequenced by
Lanciotti et al. (1999) and stored in GenBank (accession number AY196835). The amino
acid sequence (255 aa residues) of the utilized WNV NS4B protein is shown below. The
hydrophobicity predictions allowed the development of a topological model of the WNV
NS4B protein incorporating elements of both SOSUI and ConPred II programs.
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDY
INTSLTSINVQASALFTLARGFPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPG
WQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQKKVGQIMLILVSLAAVVVNPSV
KTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
2.4 Amino acid alignments of and phylogenetic analyis of flaviviral NS4B proteins
Flaviviral NS4B protein amino acid sequences from 35 different mosquito-borne,
tick-borne, insect-borne, and non-vector flaviviruses (Table 2-1) for which data were
available in GenBank were subjected to alignment using the AlignX program of the
Vector NTI Suite software package (Informax). A phylogenetic tree was constructed
from the aligned flaviviral NS4B proteins using the neighbor-joining algorithm of the
PAUP program with 500 bootstrap replicates (Version 4.0b11, Sinauer Associates).
Similarly, amino acid sequences from 115 different WNV complete genome sequences
available in GenBank were subjected to alignment by using the AlignX program
(Appendix 1). A phylogenetic tree was constructed from the aligned WNV NS4B
proteins using the neighbor-joining algorithm of the PAUP program (Version 4.0b11,
Sinauer Associates).
2.5 Cell culture techniques
Cell lines used for virus experiments were maintained in appropriate media
(Section 2.2) and were incubated at 37°C in the presence of 5% CO2 with the exception
of mosquito C6/36 cells which were incubated at 28°C in the absence of CO2. Origins
50
and citations for the cell lines used in these experiments are shown in Table 2-2. All cells
were passaged by dissociating the cell monolayer from the flask with trypsin/EDTA
(Gibco) with the exception of C6/36 cells which were disrupted by jarring of the flask.
2.6 The WNV two-plasmid infectious clone
The two-plasmid WNV NY99 virus infectious clone was constructed utilizing a
derivative of plasmid pBRUC-139S (Fig. 2-1). The 5’ plasmid, pWN-AB, contained
WNV nucleotides 1 to 2495 and a promoter for T7 polymerase. The 3’ plasmid, pWNCG, contained nucleotides 2495 to 11029 and an introduced 3’ XbaI restriction site used
to linearize the plasmid (Fig. 2-1). Nucleotide sequencing of the NY99 infectious clonederived virus identified seven nucleotide differences from the published WNV NY99
nucleotide sequence (GenBank AF196835) including two that encoded amino acid
substitutions: C1428U, U1855C, C3880U (NS2A H118Y), A4922G (NS3 K104R),
G7029U, U8811C, and A10851G.
2.7 Site-directed mutagenesis
The 3’ plasmid of the WNV infectious clone WN/IC P991 served as the template
for introduction of mutations (Beasley et al., 2005). Mutagenesis was conducted using
the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) following the protocol
accompanying the kit. Sets of primers were designed for each engineered mutation
(D35E, P38G, P38A, W42F, Y45F, C102S, C102A, C120S, C227S, C237S, L97M,
A100V, V106I, L108P, T116I) including sufficiently long flanking regions to obtain a
predicted melting temperature of at least 78˚C (Table 2-3). Mutagenesis reactions were
carried out in a thermocycler following specific cycling parameters listed below.
51
95°C for 1 minute
1X
95°C for 50 seconds
60°C for 50 seconds
34X
68°C for 12 minutes
72°C for 7 minutes 1X
Products were then digested with Dpn I to remove parental DNA and transformed into
XL-10 Gold ultracompetent cells that were plated on LB/ampicillin plates. Four colonies
from each mutagenesis reaction were picked and miniprepped, and sequencing was
conducted to confirm the presence of the desired mutation and absence of additional
mutations in the NS4B gene. Appropriate plasmids were grown in 200mL cultures to
obtain concentrated DNA for further manipulation.
2.8 Miniprep of plasmid DNA
For minipreps, bacterial cells were grown overnight in 3mL LB broth
supplemented with ampicillin (100 μg/mL) at 37°C. The cultures were subjected to
centrifugation, and supernatants were decanted to obtain the cell pellet. Plasmid DNA
was then purified from the bacterial pellet using the GenElute Plasmid Miniprep kit
(Sigma) according to manufacturers’ instructions.
2.9 Maxiprep of plasmid DNA
For maxipreps, bacterial cells were grown overnight in 200mL LB broth
supplemented with ampicillin (100 μg/mL) at 37°C. The cultures were centrifuged for 5
minutes at 6,000 rpm at 4°C. The supernatant was decanted while the cell pellet was
52
resuspended in 4.5mL of cold GTE buffer. Bacterial cellular membranes were disrupted
with the addition of 9mL alkaline lysis buffer. Cellular debris was precipitated with 9mL
cold 3M potassium acetate (pH = 4.8) and centrifuged for 10min at 10,000 rpm.
Supernatant was transferred to another tube containing 16mL isopropanol (Fisher), mixed
by inversion, and centrifuged for 15min at 10,000 rpm. Each pellet was dissolved in
400μL TE buffer (Sigma) and incubated with 50μL of RNase A stock solution
(10mg/mL) for 60min at 37°C. The plasmid DNA preparation was extracted once with
500μL phenol/chloroform/isoamyl alcohol (Invitrogen) and once with 500μL chloroform
(Sigma). The aqueous fraction was then subjected to ethanol precipitation and
resuspended in an appropriate volume of TE buffer representing a high salt crude extract.
To decrease the concentration of salt in the preparation, aliquots of crude extract were
desalted using the Qiaquick PCR purification kit (Qiagen). Alternatively, some plasmids
were purified using the Qiagen plasmid Maxi kit (Qiagen) following the protocol
supplied by the manufacturer.
2.10
Construction and rescue of recombinant viruses
The WNV NY-99 infectious clone was constructed in two plasmids as described
by Beasley et al. (2005). Three μg each of 5’ pWN-AB and 3’ pWN-CG infectious clone
plasmids were digested simultaneously with NgoMIV and XbaI restriction enzymes.
Appropriate DNA fragments were visualized on an agarose gel and purified using a gel
extraction kit (Qiagen). Fragments were ligated overnight at room temperature using T4
DNA ligase. DNA was linearized by digesting with XbaI, treated with Proteinase K
(Roche), and was extracted twice with phenol/chloroform/isoamyl alcohol (Invitrogen)
and once with chloroform (Sigma). DNA was ethanol precipitated, and the pellet was
resuspended in TE buffer. The resulting product served as the template for transcription
using a T7 ampliscribe kit (Epicentre) and A-cap analog (NEB). Following 3 hours
incubation at 37˚C, the transcription reaction was added to 1.5x107 Vero cells suspended
53
in 500uL PBS, and transfection was accomplished by using electroporation. The cells
were placed in a cuvette (0.2cm electrode gap) on ice, pulsed twice at 1.5kV, infinite
Ohms, and 25uF, incubated at room temperature for ten minutes, transferred to 75-cm2
flasks containing MEM with 8% FBS, and incubated at 37˚C and 5% CO2. Viruses were
harvested on day 5 or 6 post-transfection, when CPE was evident. Culture medium
containing virus was clarified by centrifugation for 5 minutes at 12000 rpm, and 1-mL
aliquots were stored at -80˚C. Viral RNA was extracted using the Viral RNA Mini-Spin
kit (Qiagen). The presence of the engineered NS4B mutation was confirmed by
amplifying the NS4B region using the Titan One-Step RT-PCR kit (Roche) and DNA
sequencing. Full-length genomic sequencing was conducted on recombinant C102S and
P38G/T116I mutant viruses to check for additional mutations. None were identified.
2.11
Viral RNA extraction
Viral RNA was extracted from 140 μL of infected Vero cell culture supernatants
using the QiaAMP viral RNA extraction kit according to the protocol provided by the
manufacturer (Qiagen).
2.12
Genomic sequencing of recombinant viruses
Each generated recombinant mutant virus was subjected to reverse transcriptionpolymerase chain reaction (RT-PCR) of the NS4B gene region followed by direct
nucleotide sequencing of the product to confirm the presence of the engineered amino
acid substitution and absence of any additional mutations. Selected viruses including the
attenuated C102S and P38G/T116I mutants, the S102C revertant, and several
P38G/T116I-derived viruses encoding additional putative compensatory substitutions
were subjected to full-length genome sequencing. Seven sets of primers were utilized for
54
both genome amplification via RT-PCR and sequencing (Table 2-4). For example, the
NS4B region was amplified using the WN 6640 and complementary (c)WN 8155 primers
using the touchdown (TD) RT-PCR reaction show below. The use of a touchdown
protocol, where annealing temperatures were decreased each cycle, allowed for the
simultaneous amplification of the entire genome in one set of seven reactions.
94°C for 2 minutes
1X
94°C for 30 seconds
55°C for 1 minute
30X
-0.5°C/cycle
72°C for 3 minutes
94°C for 30 seconds
40°C for 1 minute
10X
72°C for 3 minutes
72°C for 7 minutes
1X
Sequencing reactions were performed in the UTMB Biomolecular Resource
Facility’s DNA sequencing laboratory using the ABI PRISM Big Dye Teminator v3.7
cycle sequencing kits (Applied Biosystems) according to the manufacturers’ protocol and
analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Analysis and
assembly of sequencing data were performed using the ContigExpress program in the
Vector NTI Suite software package (Informax).
55
2.13
Plaque assays
Infectivity titers of recombinant viruses were determined by plaque titration in
Vero cells at 37˚C. Vero cells were allowed to grow to approximately 90% confluency in
six-well plates. Growth medium, MEM with Earle’s salts and L-glutamine (Gibco)
supplemented with 8% bovine growth serum (BGS) (Hyclone), 100units/mL penicillin
(Gibco), and 100 μg/mL streptomycin (Gibco), was removed, and cells were rinsed with
PBS. Serial dilutions of each virus were adsorbed, 200 μL per well, for 30 minutes at
room temperature before overlaying the cell monolayer with 4mL of a 50:50 mixture of
2% agar (Sigma) and 2X MEM (Gibco) containing 4% BGS. Two days following the
first agar overlay, 2mL of a mixture of 2% agar and 4% BGS 2X MEM containing 2%
neutral red (Gibco) was added to each well. Plaques were visualized and counted the
following day, and viral titers were calculated.
2.14 Temperature-sensitivity assay
Each rescued recombinant mutant virus was assayed by plaque titration in Vero
cells at both 37°C and 41°C to determine if the virus exhibited a temperature-sensitive
phenotype. Temperature-sensitive viruses were defined as those which exhibited a > 2.0
log10 pfu/mL decrease in infectivity titers at 41°C compared to the permissive 37°C
temperature. Viruses found to be temperature-sensitive were also assayed by plaque
titration at 39.5°C to determine if this temperature was permissive.
2.15 Growth curves
Growth curves were conducted for wild-type WNV as well as the recombinant
mutant viruses to determine viral multiplication kinetics in a variety of cell types at
56
specific temperatures including monkey kidney Vero cells (both 37°C and 41°C), mouse
neuronal Neuro2A cells (37°C), mouse macrophage P388.D1 cells (37°C), mouse
dendritic DC2.4 cells (37°C), mosquito C6/36 cells (28°C), and a series of human
hepatocyte (Huh)-derived cell lines (37°C). Cells were grown in 6-well plates in
appropriate media (Section 2.2) to approximately 90% confluency and were infected with
200 μL virus diluted in PBS to obtain a specific multiplicity of infection (MOI).
Following adsorption of virus for 30 minutes, the cells were washed three times with
PBS, and 4 mL of the appropriate culture medium was added. Aliquots were removed at
0, 12, 24, 48, 72, and 96 hours following infection and frozen at -80°C. Harvested
aliquots were then subjected to Vero cell plaque titration to determine viral multiplication
kinetics. Each growth curve was performed in triplicate, and each plaque assay was
undertaken in duplicate.
2.16 Mouse virulence studies
Recombinant viruses were diluted in PBS, and 10-fold serial doses ranging from
103 pfu to 10-1 pfu were injected by the intraperitoneal (ip) route in a volume of 100 μL
into groups of five 3-4 week old female outbred NIH Swiss mice (Harlan). Clinical signs
of infection such as ruffled fur or hunched posture were noted during the following 14
days, and the LD50 value was calculated for each virus. Three weeks following
inoculation, surviving mice were challenged ip with a uniformly lethal dose (100 LD50)
of wild-type NY-99 WNV to determine if they had developed a protective immune
response. If a virus was found to be attenuated via the ip route, it was administered by
the intracerebral (ic) route to investigate the mouse neurovirulence phenotype. Inbred
BALB/c and C57BL/6 mice (Harlan) were also used for some determinations of
neuroinvasiveness with the attenuated NS4B mutant viruses. All procedures with
animals were carried out in Biosafety Level 3 facilities according to guidelines of the
57
Committee on Care and Use of Laboratory Animals under an animal-care protocol
approved by the University of Texas Medical Branch.
2.17 Virus multiplication in mice
NIH Swiss mice were inoculated with 100 pfu of either wild-type WNV or
appropriate recombinant mutant virus via the ip route. Three mice were sacrificed for
each virus per day for six days. Brains were harvested, and blood was collected by
cardiac puncture. Brains were homogenized, and homogenates were subjected to plaque
assays to determine infectivity titers in the brain. The limit of detection was 2.7 log10
pfu/brain. Viral RNA was also isolated from brains (Qiagen) and was subjected to RTPCR utilizing the Titan kit (Roche) to detect low levels of virus in the brain below the
level of detection for plaque assays. Blood from cardiac puncture was centrifuged at
1200 rpm, and serum was separated and frozen. Serum samples were plaqued to
determine viral titers, and the limit of detection was 1.7 log10 pfu/mL.
2.18 Quantitative real-time RT-PCR assays
Virus-infected Vero cell monolayers (moi of 0.01) were rinsed three times with
PBS, and total viral and cellular RNA was isolated from cellular lysates at 0, 12, 24, and
48 hours post-infection by using the Qiagen RNeasy Mini kit. A ~100-bp fragment of the
3’ noncoding region was amplified using TaqMan one-step RT-PCR as described by
Vanlandingham et al. (2004). These primers were first described by Lanciotti et al.
(2000). RNA levels were quantified by comparison to a standard curve generated from
serial 10-fold dilutions of WNV RNA.
58
2.19 PAGE and Western blots
To determine WNV envelope (E) protein levels, cell lysates were generated by
solubilizing virus- or mock-infected Vero monolayers in 0.5 mL of RIPA buffer
containing 1% SDS. Equal volumes (8 μL) of each lysate were run in 12.5% SDS-PAGE
gels (BioRad) at 50mAmp and transferred to Sequi-Blot PVDF membranes (BioRad) at
100V in duplicate. One membrane was probed with rabbit polyclonal anti-WNV
envelope domain III antibody to determine viral E protein levels, while the other
membrane was probed with mouse anti-β-actin antibody (Sigma) to assay cellular protein
levels. Visualization of membranes was accomplished using BCIP/NBT (5-Bromo-4chloro-3-indolyl phosphate/Nitro blue tetrazolium) development following conjugation
with alkaline phosphatase (AP)-conjugated anti-rabbit and anti-mouse secondary
antibodies (Sigma).
2.20 Cloning and quasispecies analysis
Viral RNA was isolated from the attenuated parental P38G/T116I viral stock in
addition to virus samples from the 96-hour timepoint in Vero cells incubated at 37°C and
41°C utilizing the Viral RNA isolation kit (Qiagen). RT-PCR was conducted on the 96
hour timepoint samples utilizing the Titan RT-PCR kit (Roche) and NS4B-specific
primers, and PCR products were cloned into the T-easy vector (Promega). Resulting
plasmids were transformed into E. coli DH5α cells and selected on LB/ampicillin plates.
Twenty-one to 23 colonies each were picked from the parental sample used to infect Vero
cell cultures, 96-hour 37°C sample, and 96-hour 41°C sample. The NS4B region was
amplified by RT-PCR and sequenced for each cDNA clone, and frequencies of
nucleotide and encoded amino acid substitutions for the cDNA clones from each of the
three samples were determined.
59
2.21 Cytokine array membranes
Mouse neuronal Neuro2A, macrophage P388.D1, and dendritic DC2.4 cells were
utilized to assay alterations of cytokine expression in response to infection by WNV.
Cells were allowed to grow to 90% confluency in six-well plates and were either infected
with 200μL wild-type WNV at an MOI of 5 or were mock-infected with 200μL growth
media. Following adsorption for 30 minutes, cells were rinsed with PBS, and appropriate
growth media was added to each cell type. Aliquots were removed at 24 hours postinfection and were then used to assay supernatant cytokine expression with RayBio
Mouse Cytokine Antibody Array III membranes (Raybiotech) according to the protocol
supplied with the kit. Membranes were scanned and subjected to densitometry analysis
to account for variations in background intensity.
2.22 Gene expression studies
Mouse macrophage P388.D1 cells were allowed to grow to 100% confluency in
six-well plates and were infected with 200μL of either wild-type WNV or the attenuated
NS4B C102S mutant virus at an MOI of 5 in triplicate. At 12 hours post-infection cells
were harvested, and cellular RNA was isolated using the RNA aqueous Midi kit
(Ambion). Purified RNA samples were submitted to the UTMB Recombinant DNA
Laboratory and analyzed using Affymetrix DNA microarrays to identify differentially
regulated genes in wild-type versus C102S mutant virus-infected cells. Gene expression
patterns were further analyzed by the UTMB Bioinformatics Program using Ingenuity
Pathways Analysis (version 3.0). Genes that exhibited ≥ 2-fold difference in expression
and a p-value of less than 0.001 by one-way Anova were defined as significant.
60
Table 2-1. GenBank accession numbers of flavivirus NS4B amino acid sequences
used in alignments
Virus
Abbreviation
Alfuy virus
Murray Valley encephalitis virus
Japanese encephalitis virus
Usutu virus
West Nile virus
Bagaza virus
Ilheus virus
Rocio virus
St. Louis encephalitis virus
Bussuquara virus
Iguape virus
Kedougou virus
Zika virus
Dengue 1 virus
Dengue 2 virus
Dengue 3 virus
Dengue 4 virus
Kokobera virus
Entebbe bat virus
Yokose virus
Sepik virus
Yellow fever virus
Alkhurma virus
Deer tick virus
Powassan virus
Langat virus
Louping ill virus
Tick-borne encephalitis virus
Omsk hemorrhagic fever virus
Apoi virus
Modoc virus
Montana myotis leukoencephalitis virus
Rio Bravo virus
Kamiti River virus
Cell fusing agent virus
ALFV
MVEV
JEV
USUV
WNV
BAGV
ILHV
ROCV
SLEV
BSQV
IGUV
KEDV
ZIKV
DEN1V
DEN2V
DEN3V
DEN4V
KOKV
ENTV
YOKV
SEPV
YFV
ALKV
DTV
POWV
LGTV
LIV
TBEV
OHFV
APOIV
MODV
MMLV
RBV
KRV
CFAV
61
GenBank accession
number
AY898809
NC000943
NC001437
NC006551
AF196835
AY632545
AY632539
AY632542
AY632544
AY632536
AY632538
AY632540
AY632535
NC001477
AF169686
NC001475
AY776330
AY632541
AY632537
AB114858
AY632543
AY640589
NC004355
NC003218
L06436
NC003690
Y07863
U27495
AY193805
AF160193
AJ242984
NC004191
AF144692
NC005064
M91671
Table 2-2. Origins and citations for cell lines used in these experiments.
Cell line
ATCC number
Origin
Citation
Vero
CCL-81
Epithelial cells from Cercopithecus
aethiops (African green monkey)
Nippon Rinsho 1963
21, 1209
Neuro2A
CCL-131
Neuroblasts from the brains of
albino strain A Mus musculus
(mouse)
Klebe et al., 1969
P388.D1
CCL-46
Lymphoblasts from strain BALB/c
Mus musculus (mouse)
Am. J. Pathol 1957
33, 603
C6/36
CRL-1660
Cells from the larvae of Aedes
albopictus (Asian tiger mosquito)
Igarashi, 1978
DC2.4
NA
Dendritic cells from the bone
marrow of strain C57BL/6 Mus
musculus (mouse)
Shen et al., 1997
Huh7
NA
Hepatocellular carcinoma cells from Nakabayaski et al.,
Homo sapiens (human)
1982
PH5CH8
NA
Non-neoplastic hepatocytes
transformed with large T antigen
from Homo sapiens (human)
Huh7 FT3.7
Huh7.5
Huh7.5-iTLR3.16
Li et al., 2005
Kindly provided by K. Li and S.M. Lemon
62
Table 2-3. Primers used for site-directed mutagenesis of the WNV infectious clone.
Orientation is noted as forward (F) or reverse (R), and introduced nucleotide
mutations are denoted in red.
Primer
Name
D35E-F
D35E-R
P38G-F
P38G-R
P38A-F
P38A-R
W42F-F
W42F-R
Y45F-F
Y45F-R
L97M-F
L97M-R
A100V-F
A100V-R
V106I-F
V106I-R
L108P-F
L108P-R
T116I-F
T116I-R
C102S-F
C102S-R
C102A-F
C102A-R
C120S-F
C120S-R
C227S-F
C227S-R
C237S-F
C237S-R
Sequence (5’ → 3’)
GGGAGAGTTTCTTTTGGAGTTGAGGCCGGCAAC
GTTGCCGGCCTCAACTCCAAAAGAAACTCTCCC
GAGTTTCTTTTGGACTTGAGGGGTGCAACAGCCTGGTCACTG
CAGTGACCAGGCTGTTGCACCCCTCAAGTCCAAAAGAAACTC
GAGTTTCTTTTGGACTTGAGGGCTGCAACAGCCTGGTCACTG
CAGTGACCAGGCTGTTGCAGCCCTCAAGTCCAAAAGAAACTC
GGCCGGCAACAGCCTTCTCACTGTACGCTGTGAC
GTCACAGCGTACAGTGAGAAGGCTGTTGCCGGCC
GCCTGGTCACTGTTCGCTGTGACAACAGCG
CGCTGTTGTCACAGCGAACAGTGACCAGGC
GGAGTGTCGGCTCTCATGCTAGCAGCCGGATGC
GCATCCGGCTGCTAGCATGAGAGCCGACACTCC
GGCTCTCCTGCTAGCAGTCGGATGCTGGGGACAAG
CTTGTCCCCAGCATCCGACTGCTAGCAGGAGAGCC
GGATGCTGGGGACAAATCACCCTCACCGTTACG
CGTAACGGTGAGGGTGATTTGTCCCCAGCATCC
GGGGACAAGTCACCCCCACCGTTACGGTAAC
GTTACCGTAACGGTGGGGGTGACTTGTCCCC
GGTAACAGCGGCAACACTCCTTTTTTGCC
GGCAAAAAAGGAGTGTTGCCGCTGTTACC
CCTGCTAGCAGCCGGATCCTGGGGACAAGTCACCC
GGGTGACTTGTCCCCAGGATCCGGCTGCTAGCAGG
CCTGCTAGCAGCCGGAGCATGGGGACAAGTCACCC
GGGTGACTTGTCCCCATGCTCCGGCTGCTAGCAGG
GCGGCAACACTCCTTTTTTCCCACTATGCCTACATGG
CCATGTAGGCATAGTGGGAAAAAAGGAGTGTTGCCGC
CTGCCATCGGACTCTCCCACATCATGCGTGGGG
CCCCACGCATGATGTGGGAGAGTCCGATGGCAG
CGTGGGGGTTGGTTGTCATCTCTATCCATAACATGGAC
GTCCATGTTATGGATAGAGATGACAACCAACCCCCACG
63
Table 2-4. Primers used for WNV genomic amplification and sequencing. Forward
primers are denoted with a (+) while reverse primers are denoted with a (-).
Primer name
WN 1+
cWN 1346WN 1272+
cWN 2500WN 2495+
cWN 3795WN 3739+
cWN 5248WN 5199+
cWN 6701WN 6640+
cWN 8155WN 8086+
cWN 9592WN 9511+
cWN 11029-
Primer sequence (5’→ 3’)
AGTAGTTCGCCTGTGTGA
CCTATTGCCTTGGTAGAGCAGGC
CAACGGCTGCGGACTATTTGG
GCCGGCTGATGTCTATGGCAC
GCCGGCAAGAGCTGAGATGTG
CGCTTTGAGAAACGATGCCACC
GGAGACGTGGTACACTTGCGC
GGCCTCTTTGATGATCTGTGGCAG
CGGCGCCGGTAAAACAAGG
CCAATGCCCTTCCGCTGC
GCCTTATTGAGTGTGATGACCATGG
GCTCTTCAACCTCAGCACTTGACG
CCTTCTGAGTGTTGTGACACCCTCC
CCACATCATCTGGGCCAATCACC
GCCCTAAACACTTTCACCAACCTGG
AGATCCTGTGTTCTCGCACCACC
64
Figure 2-1. Diagram of the WN-NY99 two plasmid infectious clone
West Nile virus genome
5’UTR
C
prM
NS2A
NS1
E
(structurals)
NS2B
NS3
NS4A
NS4B
3’UTR
NS5
(nonstructurals)
T7 promotor
pBr322
NgoMIV
pBr322
Mutagenesis
Xba1 NgoMIV
XbaI
Cut with NgoMIV and XbaI
Ligate with T4 DNA ligase
Linearize with XbaI
pBr322
T7
5’UTR
C
prM
E
NS1
NS2A
NS2B
NS3
NS4A
Transcription and transfection
Vero
65
NS4B
NS5
3’UTR
CHAPTER 3
AMINO ACID SEQUENCE ANALYSIS AND PROTEIN MODELING
OF THE WEST NILE VIRUS NS4B PROTEIN
3.1 Abstract
West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein
approximately 27 kilodaltons (kD) in size that is hypothesized to participate in the viral
replication complex. The precise function of the NS4B protein remains poorly defined
due to the unique challenges in isolation and purification presented by its extreme
hydrophobicity. The NS4B protein is thought to be involved in altering host cellular
membrane arrangements and is known to co-localize with other viral components of the
replication complex. To gain a better understanding of this protein, NS4B amino acid
sequence alignments were generated for WNV and other flaviviruses for which the NS4B
gene has been sequences. Phylogenetic analyses were conducted to determine the degree
of conservation of the NS4B protein in relation to that of more commonly studied
proteins such as the envelope (E) and RNA-dependent RNA polymerase (NS5) proteins.
Topological models of the WNV NS4B protein were generated using a combination of
hydrophobicity plotting programs to determine which regions were likely exposed and
which served as transmembrane domains. Taken together, these studies allowed for the
identification of distinct regions that may be critical in mediating the function of the
NS4B protein in the viral replication complex.
66
3.2 Introduction
The NS4B is a small hydrophobic protein approximately 27kD in size that colocalizes with other components of the membrane-bound viral replication complex.
Although NS4B is known to be required for viral replication, its precise function remains
unclear. The NS4B protein is known to localize to the endoplasmic reticulum (ER) and
induces alterations in ER-membrane organization resulting in a reticular staining pattern
with discreet cytoplasmic foci (Lundin et al., 2003). Mature NS4B is cleaved from an
NS4A-2k-NS4B precursor by the NS2B-NS3 viral protease (Preugschat et al., 1991).
The 2k-peptide is an ER-targeting signal peptide that is not required for insertion of the
NS4B protein into membranes (Miller et al., 2006). Cleavage of the NS4A-2k-NS4B
results in mature NS4B as well as the NS4A-2k intermediate that can also induce
membrane rearrangements (Roosendaal et al., 2006).
While Hepatitis C virus (HCV) NS4B, a member of the genus Hepacivirus of the
family Flaviviridae, displays little amino acid homology with NS4B proteins from the
genus Flavivirus, topologies and functions are thought to be conserved. HCV NS4B has
been found to contain a nucleotide binding motif and is known to hydrolyze GTP (Einav
et al., 2004). HCV NS4B also forms multimers and is capable of regulating the activity
of the NS5B polymerase (Yu et al, 2006; Piccininni et al., 2002). Recently, DEN4 NS4B
has been found to interact with the NS3 protein and enhance its helicase activity by
dissociating NS3 from single-stranded RNA (Umareddy et al., 2006). Evidence was also
presented that indicates DEN4 NS4B is capable of forming multimers further suggesting
that protein function is conserved throughout the family Flaviviridae.
To better elucidate the role of the NS4B protein in flaviviral replication, WNV
will be used as a model system to test the effects of engineered amino acid substitutions
targeting critical regions of the NS4B protein on viral phenotypes. In this study, NS4B
amino acid alignments and phylogenetic analysis will be utilized to determine the
location of both conserved and variable domains encoded by WNV and other
67
flaviviruses. Hydrophobicity plotting programs will also be utilized to generate models
of WNV NS4B to identify hydrophobic and hydrophilic sequments of the protein. Taken
together, amino acid sequence analysis combined with topological modeling of the
protein product should allow for identification of certain critical regions likely to be
responsible for facilitating viral replication.
3.3 Results
3.3.1 Phylogenetic analysis of WNV NS4B compared to NS4B proteins from other
flaviviruses
Amino acid sequences from the WNV NS4B protein were compared to those
obtained from GenBank for a wide variety of mosquito-borne, tick-borne, non-vector,
and insect flaviviruses (Table 3-1). Mosquito-borne viruses utilized included Alfuy virus
(ALFV), Murray valley encephalitis virus (MVEV), Japanese encephalitis virus (JEV),
Usutu virus (USUV), Bagaza virus (BAGV), Ilheus virus (ILHV), Rocio virus (ROCV),
St. Louis encephalitis virus (SLEV), Bussuquara virus (BSQV), Iguabe virus (IGUV),
Kedougou virus (KEDV), Zika virus (ZIKV), dengue serotypes 1-4 (DENV1-4),
kokobera virus (KOKV), Entebbe bat virus (ENTV), Yokose virus (YOKV), Sepik virus
(SEPV), and yellow fever virus (YFV). The vectors for the bat-isolated YOK and ENT
viruses have not yet been specifically identified, however mosquitoes are thought to be
involved in transmission (Tajima et al., 2005). Tick-borne viruses selected for
comparison were Alkhurma virus (ALKV), deer tick virus (DTV), Powassan virus
(POWV), Langat virus (LGTV), louping ill virus (LIV), Central European tick-borne
encephalitis virus (TBEV), and Omsk hemorrhagic fever virus (OHF). Non-vector-borne
viruses included Apoi virus (APOIV), Modoc virus (MODV), Montana myotis
leukoencephalitis virus (MMLV), and Rio Bravo virus (RBV). The insect flaviviruses
used for this comparison were cell fusing agent virus (CFAV) and Kamiti river virus
(KRV). Alignments of NS4B protein amino acid sequences were generated using the
AlignX program (Fig. 3-1).
68
A phylogenetic tree of the different NS4B amino acid sequences was produced
using parsimony analysis (PAUP) with bootstrap resampling analysis (500 replicates).
The insect viruses CFAV and KRV were used together as the outgroup. As expected,
groups of flaviviral NS4B proteins correlated closely with classification of mosquitoborne, tick-borne, or non-vector-borne viruses (Fig. 3-2). WNV NS4B was found to
group most closely with NS4B proteins from ALF, MVEV, JE, and USU viruses. The
next most closely related NS4B amino acid sequences came from BAG, ILH, ROC, and
SLE viruses. Based on amino acid identity, the JEV NS4B protein was most closely
related to WNV NS4B with 65% identity (Table 3-1). MVEV and USUV NS4B amino
acid sequences were both 64% identical to that of WNV. In contrast, BAGV and ILHV
NS4B proteins only displayed 53% identity to WNV NS4B.
3.3.2 Phylogenetic analysis of WNV 382-99 NS4B protein compared to other WNV
strains
NS4B protein amino acid sequences from the 115 WNV complete genomes
(denoted by GenBank accession number) currently available in GenBank were aligned
using the AlignX program (Appendix 1). These sequences were also analyzed using
maximum parsimony and neighbor-joining programs in the PAUP software package.
JEV NS4B was used as the outgroup. The resulting phylogenetic tree exhibited four
lineages (Fig. 3-3). Lineage 1 was further subdivided into lineage 1a encompassing both
Old and New world WNV strains and lineage 1b comprised of the Australian Kunjin
virus (KUNV). Lineage 2 included the original WNV Ug37 isolate, the laboratory
Sarafend strain, and the Hung2004 strain among others. The WNV Rus98 virus strain
isolated from a tick in the Caucasus in 1998 is the only member of lineage 3 while
Rabensburg virus is (RabV) the only member of lineage 4 according to this analysis.
NS4B amino acid sequences from lineage 1a WNV strains all exhibited 99% or
greater identity to NS4B from the WNV 382-99 strain (Table 3-2). Lineage 1b KUNV
NS4B was 97% identical while the lineage 2 strains were 91-93% identical to WNV 38299 NS4B. Lineage 3 Rus98 and lineage 4 RabV NS4B proteins were 89% and 88%
69
identical to WNV382-99 NS4B respectively. The next most closely related flaviviral
NS4B amino acid sequence was JEV at 65% identity. All lineage 2 strains contained an
insertion at position 27, making the complete NS4B protein 256 amino acids in length as
opposed to the 255 amino acids encoded by the WNV 382-99 strain (Appendix 1).
Except for the WNV Mad98 strain which encoded a glycine (G) at NS4B 27, all of the
lineage 2 viruses encoded a threonine (T) residue at this position. In fact, lineage 2, 3,
and 4 strains all exhibit a great deal of heterogeneity within the N-terminal region
encompassing NS4B amino acid residues 11-32. However, the lineage 3 and 4 viruses
did not encode the insertion at position NS4B 27.
3.3.3
Topological modeling of the WNV NS4B protein
Two hydrophobicity plotting programs were utilized to generate topological
models of the WNV 382-99 NS4B protein. The model generated using the SOSUI
program predicted four transmembrane domains (Hirokawa et al., 2003) (Fig. 3-4).
Transmembrane domain 3 (TMD3) comprised of amino acid residues 103-127 was the
most hydrophobic segment and was predicted to serve as the primary helix. The
Consensus Prediction (ConPredII) program predicted the existence of five TMDs (Arai et
al., 2004) (Fig. 3-5). This program was also capable of predicting cytoplasmic and
lumenal ectodomains of the NS4B protein resulting in additional information compared
to that obtained using the SOSUI program. The 3 N-terminal predicted TMDs were
highly conserved between the two programs. However, TMD4 differed by
approximately 10 amino acids between the two programs while TMD5 was only
predicted by the ConPredII program. Both programs predicted the N-terminus of the
NS4B protein to localize in the ER-lumen while the C-terminus was predicted to reside in
the cytoplasm.
70
3.4
Discussion
In this study, the WNV NS4B protein amino acid sequence was subjected to
alignment, phylogenetic analysis, and hydrophobicity plotting to elucidate potentially
important regions within the NS4B protein. Phylogenetic analysis of the WNV NS4B
382-99 protein amino acid sequence in comparison to other those from other flaviviruses
resulted in the identification of the most closely related proteins to WNV NS4B (Fig. 31). The resulting phylogram was quite similar to one previously developed utilizing the
polymerase domain of the flaviviral NS5 protein (Kuno et al., 2005). The WNV NS4B
protein was found to group most closely to NS4B proteins from ALF, MVE, JE, and
USU viruses. The WNV NS5 polymerase domain and NS3 gene also grouped most
closely with MVE, USU, and JE viruses (Kuno et al., 2005; Cook and Holmes, 2006).
JEV NS4B was found to exhibit 65% identity to WNV NS4B while MVEV and USUV
NS4B proteins were both 64% identical to WNV NS4B (Table 3-2). ROCV and SLEV
NS4B proteins were more distantly related with 56% identity while ILHV and BAGV
proteins exhibited 53% identity to WNV NS4B. These data indicate that flaviviral
phylogenetic relationships are highly conserved when utilizing either NS4B or NS5
polymerase regions.
The WNV 382-99 NS4B protein amino acid sequence was also subjected to
alignment and phylogenetic analysis with NS4B protein sequences from other WNV
strains (Appendix 1). Every WN NS4B sequence available (115 complete WNV
sequences in GenBank) was utilized to obtain maximum coverage. Four distinct lineages
of WN viruses were exhibited utilizing neighbor-joining analysis (Fig. 3-3). The
phylogram generated in this study is rather similar to one previously published based on
complete genomic nucleotide sequences of selected WNV strains, which also denoted
four lineages (Bakonyi et al., 2006). The exact classifications of the lineage 3 Rus98 and
lineage 4 Rab viruses remain undetermined as these may either be classified as new
lineages of WNV or as completely novel flaviviruses. Based on amino acid comparisons
of the NS4B protein, WNV 382-99 is much more closely related to Rus98 and Rab
71
viruses (89% and 88% NS4B amino acid identity respectively) than the next most closely
related flavivirus JEV (65% NS4B identity). This strongly suggests that Rus98 and Rab
viruses are new lineages of WNV rather than representing novel flaviviruses. This is also
supported by the fact that the lineage 2 strains are not much more closely related to WNV
382-99 than Rus98 and Rab viruses with NS4B amino acid identities ranging from 9193%. WNV 382-99 NS4B amino acid identities with proteins from JEV, USUV, and
MVEV exhibit a significant decrease to 64-65%.
In contrast, NS4B amino acid sequences from all lineage 1a strains are nearly
identical with identities of 99% or greater. It is intriguing that this lineage of viruses
includes isolates from both the New and Old World over a period greater than fifty years.
The degree of NS4B amino acid conservation is interesting given that lineage 1a NS4B is
optimized for a wide variety of hosts and vectors as is evident with WNV being the most
geographically widespread member of the JEV complex. The most divergent lineage 1a
NS4B amino acid sequences differ only at three amino acid positions from that of WNV
382-99. Lineage 1b KUNV is slightly more divergent with only 97% identity. When
comparing the phylogram with the alignment, it was noted that each member of lineage 2
encoded an insertion at position NS4B 27 resulting in a 256 amino acid NS4B protein as
compared to a 255 amino acid NS4B protein. This insertion was not observed in any
member of lineages 1, 3, or 4. It thus appears that the insertion at NS4B position 27 is
the defining characteristic of lineage 2 with respect to this segment of the genome. In
summary, the NS4B amino acid sequences from lineage 1a were highly conserved both
spatially and temporally. Also, a novel insertion was identified in each lineage 2 strain at
NS4B position 27. Finally, NS4B amino acid identities suggest that Rus98 and Rab
viruses represent new lineages of WNV rather than novel flaviviruses.
Topological models were generated of the WNV 382-99 NS4B protein utilizing
SOSUI and ConPredII hydrophobicity plotting programs. The SOSUI program was
designed to distinguish between membrane and soluble proteins in addition to predicting
transmembrane helices for proteins determined to reside in membranes (Hirokawa et al.,
2003). The ConPredII program is a consensus program that utilizes a variety of other
72
protein modeling algorithms including SOSUI for increased accuracy and greater
predictive capabilities (Arai et al., 2004). The model generated from the SOSUI program
predicted four transmembrane domains (TMDs) with TMD3 including amino residues
104-126 thought to represent the primary helix. ConPredII predicted five TMDs as an
additional C-terminal TMD5 (amino acid residues 226-246) was present on this model
that was not depicted by SOSUI. The N-terminal three TMDs were highly conserved
with both SOSUI and ConPredII programs. TMD4 was slightly more variable as the
SOSUI program predicted this helix to be comprised of residues 182-204 while
ConPredII predicted residues 172-192 to form TMD4.
Miller et al. (2006) utilized a series of hydrophobicity plotting programs to
produce models of the DENV4 NS4B protein. SOSUI and ConPredII analysis of the
DENV4 NS4B protein correlated closely with the models generated for the WNV NS4B
protein when homologous amino acids from the two proteins were aligned. SOSUI
predicted four TMDs while ConPredII predicted five TMDs for both DEN2 and WN
NS4B proteins. Miller et al. (2006) constructed a panel of NS4B fragments fused to
eGFP and tested their ability to localize to the ER-membrane. It was found that predicted
TMDs 3, 4, and 5 were each sufficient on their own to localize to the ER-membrane
resulting in a reticular staining pattern. In contrast, predicted TMDs 1 and 2 were not
capable of transporting eGFP on there own while a fragment encompassing predicted
TMDs 1-3 was capable of localizing to the ER-membrane. It thus seems that the
existence of TMDs 3, 4, and 5 was confirmed while TMDs 1 and 2 may not be
biologically significant. It seems likely though that within the context of the complete
protein adjacent to TMD3, the TMDs 1 and 2 would still occur. The SOSUI program
predicted that WNV NS4B TMD3 served as the primary transmembrane helix. In
addition, Umareddy et al. (2006) determined that DEN2 NS4B protein was capable of
forming multimers and that amino acid residues 91-136 were critical in mediating this
process. Upon alignment, these amino acids would correspond to WNV NS4B residues
98-142. This overlaps closely with the predicted primary TMD3 comprised of residues
106-126 and raises the possibility that a critical function of predicted TMD3 is to mediate
73
the formation of high-order multimers. Engineering amino acid substitutions into this
region could disrupt the ability of the NS4B protein to form multimers and lead to
attenuation of the virus.
In summary, phylogenetic analyses targeting the NS4B region of different WNV
and other flavivirus strains exhibited similarities to previously published phylograms
suggesting the conservation of genetic relationships throughout the flavivirus genome.
WNV NS4B was highly conserved throughout lineage 1a while lineage 2 isolates each
displayed an amino acid insertion at position 27. Based on NS4B amino acid identity,
Rus98 and Rab viruses likely comprise new lineages of WN instead of representing novel
flavivirus strains. Finally, hydrophobicity plotting of the WNV NS4B amino acid
sequence led to the generation of models that should facilitate the engineering and
interpretation of disruptive amino acid substitutions.
74
Table 3-1. GenBank accession numbers of flavivirus NS4B amino acid sequences
used in alignments
Virus
Abbreviation
Alfuy virus
Murray Valley encephalitis virus
Japanese encephalitis virus
Usutu virus
West Nile virus
Bagaza virus
Ilheus virus
Rocio virus
St. Louis encephalitis virus
Bussuquara virus
Iguape virus
Kedougou virus
Zika virus
Dengue 1 virus
Dengue 2 virus
Dengue 3 virus
Dengue 4 virus
Kokobera virus
Entebbe bat virus
Yokose virus
Sepik virus
Yellow fever virus
Alkhurma virus
Deer tick virus
Powassan virus
Langat virus
Louping ill virus
Tick-borne encephalitis virus
Omsk hemorrhagic fever virus
Apoi virus
Modoc virus
Montana myotis leukoencephalitis virus
Rio Bravo virus
Kamiti River virus
Cell fusing agent virus
ALFV
MVEV
JEV
USUV
WNV
BAGV
ILHV
ROCV
SLEV
BSQV
IGUV
KEDV
ZIKV
DEN1V
DEN2V
DEN3V
DEN4V
KOKV
ENTV
YOKV
SEPV
YFV
ALKV
DTV
POWV
LGTV
LIV
TBEV
OHFV
APOIV
MODV
MMLV
RBV
KRV
CFAV
75
GenBank accession
number
AY898809
NC000943
NC001437
NC006551
AF196835
AY632545
AY632539
AY632542
NC007580
AY632536
AY632538
AY632540
AY632535
NC001477
AF169686
NC001475
AY776330
AY632541
AY632537
AB114858
AY632543
AY640589
NC004355
NC003218
L06436
NC003690
Y07863
U27495
AY193805
AF160193
AJ242984
NC004191
AF144692
NC005064
M91671
Table 3-2. NS4B amino acid identity rates between West Nile virus strain NY99 382-99 and other WN and
flavivirus strains.
Virus name
Code
GenBank accession
number
Year, location isolated
Host
WNV lineage,
clade
NS4B amino acid
identity to
WNV382-99 (%)
WNV 382-99
NY99
AF196835
1999, New York
Flamingo
1a
100
WNV Israel98
Is98
AF481864
1998, Israel
Stork
1a
100
WNV VLG4
Rus99
AF317203
1999, Volgograd
Human
1a
>99
WNV TX2002
TX2002
DQ164198
2002, Texas
Human
1a`
>99
WNV CO2003
CO2003
DQ164203
2003, Colorado
Magpie
1a
>99
WNV Eg101
Eg51
AF260968
1951, Egypt
Human
1a
>99
76
WNV RO9750
Ro96
AF260969
1996, Romania
Culex pipiens
1a
99
WNV LA2002
LA2002
DQ080062
2002, Louisiana
Mosquito
1a
99
WNV Kunjin
KUNV
D00246
1960, Australia
Culex annulirostris
1b
97
WNV Hung2004
Hu04
DQ116961
2004, Hungary
Goshawk
2
93
WNV B956
Ug37
AY532665
1937, Uganda
Human
2
92
WNV Sarafend
Sarafend
AY688948
Unknown
Laboratory strain
2
91
WNV Mad78
Mad78
DQ176636
1978, Madagascar
Parrot
2
91
WNV LEIV-Krnd88-190
Rus98
AY277251
1998, Caucasus
Tick
Undetermined (3?)
89
Rabensburg virus
RabV
AY765264
1997, Czech Republic
Culex pipiens
Undetermined (4?)
88
Japanese encephalitis virus
JEV
NC001437
65
Usutu virus
USUV
AY453411
64
Murray valley encephalitis virus
MVEV
NC000943
64
Alfuy virus
ALFV
AF013360
62
St. Louis encephalitis virus
SLEV
NC007580
56
Rocio virus
ROCV
AY632542
56
Bagaza virus
BAGV
AY632545
53
Ilheus virus
ILHV
AY632539
53
76
Figure 3-1. Flaviviral NS4B complete amino acid alignment. Residues exhibiting complete conservation
(yellow), high-identity (blue), high-homology (green), or high-variability (white) are denoted.
77
ALFV
MVEV
JEV
USUV
W NV
BAGV
ILHV
ROCV
SLEV
BSQV
IGUV
KEDV
ZIKV
DENV1
DENV2
DENV3
DENV4
KOKV
ENT V
Y OKV
SEPV
Y FV
ALKV
DT V
POW V
LGT V
LIV
T BEV
OHFV
APOIV
MODV
MMLV
RBV
CFAV
KRV
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
1
10
20
30
40
50
60
70
86
NEFGMLEKTKEDLRHLFVGSKP-ANEAI--SMTTSMFTLDLRPATAWALYGGSTVVFTPMIKHIVTSQYVTTSLASISAQAGTLFT
NEYGMLERTKTDIRNLFGKSLIEENEVH--IPPFDFFTLDLKPATAWALYGGSTVVLTPLIKHLVTSQYVTTSLASINAQAGSLFT
NEYGMLEKTKADLKSMFVGKTQASGLTG--LPSM---ALDLRPATAWALYGGSTVVLTPLLKHLITSEYVTTSLASINSQAGSLFV
NEYGMLERTKSDLGKIFSSTRQPQSALP--LPSMNALALDLRPATAWALYGGSTVVLTPLIKHLVTSEYITTSLASISAQAGSLFN
NEMGWLDKTKSDISSLFGQRIEVKENFS--MGEF---LLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFT
NEMGWLEQTKKDVASLFGR----AHHQE--PSRWEMPWPDLRPATAWAAYAGATTFLTPLLKHLIVTEYVNFSLMAVTAQAGALFG
NEMGWLETTKKDIGKLFRS---SGDTQE--QSTWQSWAPEVRAATAWAGYAGLTVFLTPLFRHLITTQYVSFSLTAITAQASALFG
NEMGWLDTTKRDLGKLFSGP--SAVTTS--RWEPLKLALALKPATAWAGYAGMTMLLTPLFRHLITTQYISFSLTAITSQASALFG
NEMGLLEKTKSDIAKLFGSQPGSVGFAI--RTTPWDISLDIKPATAWALYAAATMVMTPLIKHLITTQYVNFSLTAIASQAGVLLG
NEMGMLERTKQDLAGVFHK---TERKS---TEFTLLTPPDLRPATAWSIYAIGTTLITPLIHHMITTHYANFSLMAMANQAGSLFG
NEMGYLEKTKNDIISLWGR---SREQN---STLQEWFIMDIKPATAWTLYAVTTTILTPFIQHHITTHYANVSLSAIAAQAGNLFM
NEAGLLERTKADIRGLLKK---EEVNEP--GWSLPRLELDLKPATTWTLYAVITIILSPFVQHSIITTYNNFSLTAIGNQAGILFG
NELGWLERTKNDIAHLMGR---REEG----ATMGFSMDIDLRPASAWAIYAALTTLITPAVQHAVTTSYNNYSLMAMATQAGVLFG
NEMGLLETTKKDLGIGHVAAEN--------QHHATMLDVDLRPASAWTLYAVATTVITPMMRHTIENTTANISLTAIANQAAILMG
NEMGFLEKTKKDLGLG-NIATQ--------QPESNILDIDLRPASAWTLYAVATTFITPMLRHSIENSSVNVSLTAIANQATVLMG
NEMGLLETTKRDLGMS-KEPGV--------VSPTSYLDVDLHPASAWTLYAVATTVITPMLRHTIENSTANVSLAAIANQAVVLMG
NEMGLIEKTKTDFGFY-QVKT-----------ETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMG
NEMGWLEKTKADLSWVVRG------R-S--STTTPVVELDMKPATAWTLYALATTLLTPLFQHLIVTKYANISLMAIASQAGTLFS
NENGYLEKTKADIFGHKQMRTMPVNG--------SWMSFDLRPGSAWAVYAFVVGIFSPLYHHAESINYGAISLQGITQSAAAFFQ
NENGYLEKTKEDLFGRRALNSSNVYAN---LPVEKWLSLDLQPATSWTLYAVIVGVLSPLYHHIEHVNYGAISLQGISQGAAALFQ
NEMGMLEKTKRDIFG--TTVVEEGKK-------WTFPELDLHPGAAWTVYVGLVTLVTPMLHHWIKVDYGNISLSGITQNAQVLGL
NELGMLEKTKEDLFGKKNLIPSSASP-------WSWPDLDLKPGAAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSF
NEMGMLDKTKADLAGLMWHGEQRHPA------WEEWTNVDIQPARSWGTYVLIVSLFTPYMLHQLQTKIQQLVNSSVASGAQAMRD
NELGYLERTKADIAGLFRYDTQGDRV------WDTWTNIDIQPARSWGTYVFIVSLFTPYMLHQLQTKIQRLVNSSVAAGTQAMKD
NELGYLEQTKTDISGLFRREDQGGMV------WDAWTNIDIQPARSWGTYVLIVSLFTPYMLHQLQTKIQRLVNSSVAAGTQAMRD
NEMGLLEKTKADLAALFARDQGETVR------WGEWTNLDIQPARSWGTYVLVVSLFTPYMLHQLQTRIQQLVNSAVASGAQAMRD
NEMGFLEKTKADLSAMLWSGHEEHRQ------WSEWTNVDIQPARSWGTYVLVVSLFTPYIIHQLQTKIQQLVNSAVASGAQAMRD
NEMGFLEKTKADLSTALWSEREEPRP------WSEWTNVDIQPARSWGTYVLVVSLFTPYIIHQLQTKIQQLVNSAVASGAQAMRD
NEMGFLEKTKADLSAVLWSEREEPRV------WSEWTNIDIQPAKSWGTYVLVVSLFTPYIIHQLQTRIQQLVNSAVASGAQAMRD
NEMGFLERTKKDFREFFRKEVNMDGEP----TQWRIFDLDICPMVSWSLYVLLVTGLRPVCLHGLQMMTQRVVTGAISGRSDLLGQ
NELRWLENTKEDIKQLFGEKIHMGIS----SGGDFWKYIDLKPLSIWGTYATLVTFMRPQMLHNLRMFTQRIVAGSVSGKLDTLNG
NEMRWLENTKKDLFGQPQTSPSVNTG----GIIQDLLQLDIRPMNVWGTYVALVTVARPQALHNLKMFTKKIVSGVVAGKESAMER
NEMRLLENTKRDIMDLFKRDTTVNESPVFHYTWESLMEWDIRPLTIWATYVVFVTLARPQALHNLKMFTQRVITGTVAGKHDMVNL
WEMRMFPNIRSDLMELVKAVKEPEEVVNSGPSFPSWEIAQ---GKGATMLDSLQVFFFITVLSTKFLYWFQENWTARMYAMKHPEM
WEMRLFPNIRGDIMEMASAMKEPQETQSQASTISGSFFTSRVRGERATMLDSLQVFFFVTVLMNEFIIWVQENWIAQMYVMKHPEM
NEMGWLEKTK DL
L
LDLRPATAWALYAVLTTLLTPLI H I T Y NLSLSAIA QA AL
77
Figure 3-1 (continued). Flaviviral NS4B complete amino acid alignment. Residues exhibiting complete
conservation (yellow), high-identity (blue), high-homology (green), or high-variability (white) are denoted.
78
ALFV
MVEV
JEV
USUV
W NV
BAGV
ILHV
ROCV
SLEV
BSQV
IGUV
KEDV
ZIKV
DENV1
DENV2
DENV3
DENV4
KOKV
ENT V
Y OKV
SEPV
Y FV
ALKV
DT V
POW V
LGT V
LIV
T BEV
OHFV
APOIV
MODV
MMLV
RBV
CFAV
KRV
Consensus
(87)
(84)
(85)
(82)
(85)
(82)
(81)
(82)
(83)
(85)
(81)
(81)
(82)
(80)
(79)
(78)
(78)
(75)
(78)
(79)
(84)
(78)
(80)
(81)
(81)
(81)
(81)
(81)
(81)
(81)
(83)
(83)
(83)
(87)
(84)
(87)
(87)
87
100
110
120
130
140
150
160
172
LPKGIPFSNIDMTVALVFLGCWGQITLTTLLTAVVLGVVHYGYLLPGWQAEALRAAQKRTAAGIMKNAVVDGIVATDVPELERTTP
LPKGIPFTDFDLSVALVFLGCWGQVTLTTLIMATILVTLHYGYLLPGWQAEALRAAQKRTAAGIMKNAVVDGIVATDVPELERTTP
LPRGVPFTDLDLTVGLVFLGCWGQITLTTFLTAMVLATLHYGYMLPGWQAEALRAAQRRTAAGIMKNAVVDGMVATDVPELERTTP
LPRGLPFTELDFTVVLVFLGCWGQVSLTTLITAAALATLHYGYMLPGWQAEALRAAQRRTAAGIMKNAVVDGLVATDVPELERTTP
LARGFPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTP
LGKGMPFVAIDLSTPLLLLGCWGQFTMTTTLTTIMLLIIHYAFLIPGWQAEAMRSAQRRTAAGVMKNPVVDGIVATDVPDLEASTP
LSAGYPFVGIDLAVGFLLLGCYGQYNLPTAVATGLLLLAHYGYMIPGWQAEAMRAAQKRTAAGVMKNAVVDGIVATDIPEVDTATP
LNSGYPFVGVDLSVVFLLVGCYGQYNLPTTMATIGLLVGHYAFMIPGWQAEAMRAAQRRTAAGVMKNAVVDGIVATDIPEMDTATP
LTNGMPFTAMDLSVPLLVLGCWNQMTLPSLAVAVMLLAIHYAFMIPGWQAEAMRAAQRRTAAGIMKNAVVDGIVATDIPDLSPATP
MQTGAPFSKMDWAVPAIVVGCWQQLTPATLMTALVLLAVHYIYMIPGWQAGAARAAQRRTAAGIMKNPVVDGLVVTDIPTLEEVDP
MKNGHPFTQLDWAVPLLALGCWSTMTPLALVAATLLLLVHYAYMIPGWQAMGARSAQARTAAGIMKNPVVDGVTVTDIPELEVPDP
MGTGVPFYKWDWGVPLLLLGCATQITPTVMVASGVLLAAHYAFLIPGLQAQAVRAAQKRTAAGIMKNPVVDGVVVTDIQDLH-VDP
MGKGMPFMHGDLGVPLLMMGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIVVTDIDTMT-IDP
LDKGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIVAIDLDPVV-YDA
LGKGWPLSKMDIGVPLLAIGCYSQVNPITLTAALLLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVIDLDPIP-YDP
LDKGWPISKMDLGVPLLALGCYSQVNPLTLIAAVLLLVTHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTIDLDPVI-YDS
LGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPIS-YDP
MDSGIPFSSIELSVPLLALGCWTQITPCSLILACVLLSTHYAILLPGMQAQAARDAQRRTAAGIMKNAVVDGIVATDIPPLDGAGP
MDKGYPFMKLRLPLILMAVGALNNINAVALLLGLACAVFHWSLVLPGLRAKLAKMALRRTYHGVTKNAMVDGTLTNDLDEGEDMPE
MDKGYPFMRLRIPLVLLLAGAINNLTAITAGLGFLCAMIHWALVLPGLKAKLAKQALRRTYHGVTKNAVVDGMCTNDLDAGDDMPE
MDKGIPFIKMNMSVVILLLSAWNGITLLPLFAGMGAAALHWGFILPGLRAQAAKAAQKRVYHGVAKNPVVDGNPTADIDDAPGMPA
MDKGIPFMKMNISVIILLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSKLAQRRVFHGVAKNPVVDGNPTVDIEEAPEMPA
LGGGTPFFGVAGHVIALGVTSLVGATPLSLGLGVALAAFHLAIVASGLEAELTQRAHRVFFSAMVKNPMVDGDVINPFPDGEPKPV
LGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAILHLAVVTSGLEAELTQRAHRAFFSAMVKNPMVDGEVINPIPDGEPKPA
LGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAALHLAVVTSGLEAELTQRAHRAFFSAMVKNPMVDGEIINPIPDGDPKPA
LGGGTPFFGVAGHVLALGIASLVGATPTSLILGVGLAAFHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGDGEAKPA
LGGGAPFFGVAGHVMTLGVVSLVGATPTSLIVGIGLAAFHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGEGEAKPA
LGGGAPFFGVAGHVMTLGVVSLIGATPTSLMVGVGLAALHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGEGEAKPA
LGGGTPFFGVAGHVLTLGVVSLVGATPTSLVVGVGLAAFHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGDGEVKPA
LREGMGNVSIGLPELALGMSVVRGMTPVTLVLGGLAGVAHWCWFYPIHEAALTMKANKIVAQSMAKNTQVDGEVIYQLEEKIAKTE
LRNGFVSTSMSLGDLSLFISFCRNMSPLTTVTGLVLAAIHWLWFYPMHEASLTSKAHKMVAQSTAKNVAFDGEGIIDFHTEEVDTS
LPTGGAWMNLRMGDLTLLATTLKGMTCFNLLGGLTFAFIHWFWFFPLHEAAESAKAHKIVTQSLSKNNMVDGEVIYQLDEVRAETE
LPFGAAWLSLGLGDLTLAVGAFRNMSCLTLVGGVLLALAHWTWFYPLHAAAESSKAHKIVTQSLSKNTMVDGETIYQLDQTSAETE
VSSIGGFRFDEIPFRAVLPSGFAIVAIASLPSVVVGLLAAGVFMAIMYCQNKWNATPKILTALDAR----DQRHDRPTEITSRVPL
VSTVGGFRLDRIPFRAVLPSGFAIVTTSSLSSSLVGLAASSLFLTIAYYQNKWNATPKIISAMDAR----DQKHDRPTDITNRVPL
L
G PF
MDL VLLL LGCW QVTP TLV GVVLA
HYAYMLPGLQA A R AQKRTAAGIMKNPVVDGIV
DI ELE
78
Figure 3-1 (continued). Flaviviral NS4B complete amino acid alignment. Residues exhibiting complete
conservation (yellow), high-identity (blue), high-homology (green), or high-variability (white) are denoted.
79
(173)
ALFV(170)
MVEV(171)
JEV(168)
USUV(171)
W NV(168)
BAGV(167)
ILHV(168)
ROCV(169)
SLEV(171)
BSQV(167)
IGUV(167)
KEDV(167)
ZIKV(165)
DENV1(164)
DENV2(163)
DENV3(163)
DENV4(160)
KOKV(164)
ENT V(165)
Y OKV(170)
SEPV(164)
Y FV(166)
ALKV(167)
DTV(167)
POW V(167)
LGT V(167)
LIV(167)
TBEV(167)
OHFV(167)
APOIV(169)
MODV(169)
MMLV(169)
RBV(173)
CFAV(166)
KRV(169)
Consensus(173)
173
180
190
200
210
220
230
240
250
264
MMQKRLGQILLIGVSVTALLVNPRVTTVREAGVLCSAALLTLWDN--SASAVWNSTTATGLCHVMRGSWLAGASIAWTLIKNAEKPTFKR-QMQKRLGQILLVLASVAAVCVNPRITTIREAGILCTAAALTLWDN--NASAAWNSTTATGLCHVMRGSWIAGASIAWTLIKNAEKPAFKR-LMQKKVGQVLLIGVSVAAFLVNPNVTTVREAGVLVTAATLTLWDN--GASAVWNSTTATGLCHVMRGSYLAGGSIAWTLIKNADKPSLKR-LMQKKVGQILLIGVSAAALLVNPCVTTVREAGILISAALLTLWDN--GAIAVWNSTTATGLCHVIRGNWLAGASIAWTLIKNADKPACKR-IMQKKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWEN--GASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR-ITEKKFGQVILIVVALCSVFLKPGTTSLTEFGMLTSAASVNLDKG---QRTDMECTTAVAMCHLMRKNWLTGASLAWTISRNLQNGSMRR-ITEKKLGQILLILLCGASLLVKFDTMVLVEAGVLTTSAMATLIEG--NANTVWNSTVAVGVCHLMRGAWLAGPSIGWTIVRNLENPKLKR-IVEKKMGQVMLLIISALAILLNPDTMTVVEGGVLITAALATLLEG--NANTVWNSTVAVGVCHLMRGGWAAGPSIGWTIIRNLEAPKVKR-MTEKKMGQILLIAAAVLAVLVRPGICSIKEFGVLGSAALVTLIEG--TAGVVWNCTTAVGLCNLMRGGWLAGMSITWTVYKNVDKPKGKR-LVEKKLGQYILLAVAIAAAVLRQDLQSWSECATLSAAAAATLWEG--SPGKIWNASTACSLVNIFRGHTLAAVPFMFTILRNTGNTGKR--AIEKKLGQVLLISIALAAAFMRQDIIGWRECGILASAGIGTLWEG--TPSKFWNASIASSLCNIIRGSHLAALPFLFTLIRNMTKPNKR--QTEKTMGQVLLMAVALASACLTPSTWTCGEAVALMSAAAGTLWEG--NPGRMWNSSTACSLANVFRGSMLAGAGLMYTVTRNVSSTKR---QVEKKMGQVLLIAVAISSAVLLRTAWGWGEAGALITAATSTLWEG--SPNKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKRR--KFEKQLGQIMLLILCTSQILLMRTTWALCESITLATGPLTTLWEG--SPGKFWNTTIAVSMANIFRGSYLAGAGLAFSLMKSLGGGRR---KFEKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPVSTLWEG--NPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTSTRR---KFEKQLGQVMLLVLCAVQLLLMRTSWALCEVLTLATGPITTLWEG--SPGKFWNTTIAVSMANIFRGSYLAGAGLALSIMKSVGTGKR---KFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEG--NPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRR---LTEKKLGQLLLFAAAVTGVVITRSPRSWSELGVLGSAVGSTLIEG--SAGKFWNATTVTAMCNLFRGSYLAGVPLTYTIIRNS-NPSNKR-LFEKQLGTIVLMVLSLANVFTLRSTLATTEAVVLITSSLPQLVNG--VPSPVWNTQVAVGVAGLLRGNYLALISTGHALWSVRGNRR----KFEKQLGTFVLITLVFLNVILNHNVKAFLEGMVLVSASLQPLLTG--VPNPYWNQQIAVGVAGLMRGNYMAAVGMAHALWNAQANRR----MYEKKLALIILFILATVNLILTRTPFSIAELVVLGSAALGPLLEG--NTNAYWNGPIAVAFTGLMRGNYYATIGLMYNGWLAKQTRR----LYEKKLALYLLLALSLASVAMCRTPFSLAEGIVLASAALGPLIEG--NTSLLWNGPMAVSMTGVMRGNYYAFVGVMYNLWKMKTGRR----LYERRMSLILAIALCMVSVVLNRTAASMTEAGAVGLAALGQLVHP--ETETLWTMPMACGMAGLVRGSFWGLLPMGHRLWLKTTGTRR---LYERKMSLLLAVGLCIAAVALNRTAAAMTEAGAVAVAALGQLLRP--GEESWWTMPMACGMAGLVRGSLWGLLPVLHRIWLRTQGARR---LYERKMSLFLAIGLCIAAVALNRTAAAMTEAGAVAVAALGQLLRP--EEESWWTMPMACGMAGLVRGSLWGLLPVLHRIWLRTQGARR---LYERKLSLILALVLCLASVVMNRTFVAVTEAGAVGVAAAMQLLRP--EMDVLWTMPVACGMSGVVRGSLWGLLPLGHRLWLRTTGTRR---LYERKMSLVLAIVLCLVSVVMNRTVASMTEAAAVGLAATGQLLRP--EADTLWTMPVACGMSGVVRGSLWGFLPLGHRLWLRASGGRR---LYERKMSLVLATVLCLMSVVMNRTVASITEASAVGLAAAGQLLRP--EADTLWTMPVACGMSGVVRGSLWGFLPLGHRLWLRASGGRR---LYERKMSLILAMILCFMSVVLNRTVPAVTEASAVGLAAAGQLIRP--EADTLWTMPVACGLSGVVRGSLWGFLPLGHRLWLRTSGTRR---DHEKKFSYGVAVILNLINLALNQNLWSLLECAMAILAALCYIR----GQPTLCTLPVVAGLGKILRGDYLGILPVALHAWRSTSENRR---SAERKTSFGVAILLAVINVVVVREPWAMLEAGLVLLAAIKFAADG--AKETLITLPVLSGIGALIRGDYFGVAPILLNLYLQTTSNRR---TNERNFSLGVAGCLALLNIVMCRKPWTVLEALMIISVVAKNYLDP--KAETFWTLPVASGLSALLRNEFLGLVPIGYRVWKHLSPGRR---RSEKIFSMVTGFTLTAINVFTLRKAWAVLECVMVGMVLIKYLIEP--KGTTFWTLPVVSGLTSLVRGDFFGLIPISFRVWLYARSDRR---ENTRSIMYAFCLIFSLFWAFCTRSPGDFLRGSLVVGASMWQILHPRSKIHDVMDFGSMVSAIGLLEMNYLFYRFMHIAARALGAVAPFNQFR
DNTRSVMYAFGMFFSMAWVFCTRSRWDAGLCTLVVIACMWQILHPRSRVHDVMDFGSMLSAIGIFELDFLFYKFIHIAARGLGGMPPFNQFR
L EKKLGQILLIVLAL AVVL RT
AV EAGVL SAAL
LLEG
VWN TVA GL
LMRGSYLAGL LAWTL K
RR
79
Figure 3.2. Phylogenetic tree generated by neighbor-joining analysis (PAUP) of
aligned amino acid sequences of the NS4B proteins from different flaviviruses with
bootstrap resampling analysis (500 replicates). Cell fusing agent virus (CFAV) and
Kamiti river virus (KRV) together were used as the outgroup.
Bootstrap
96
MVEV
100
100
100
ALFV
JEV
USUV
WNV
80
BAGV
63
100
ILHV
ROCV
SLEV
70
IGUV
57
100
BSQV
76
KEDV
Mosquitoborne
ZIKV
97
DENV1
DENV3
100
100
77
DENV2
DENV4
KOKV
100
ENTV
YOKV
100
100
100
SEPV
YFV
ALKV
80
100
DTV
POWV
100
LGTV
99
98
64
100
LIV
Tickborne
TBEV
OHFV
APOIV
100
MODV
67
99
MMLV
Nonvector
RBV
KRV
CFAV
80
Insect
Figure 3-3. Phylogenetic tree generated by neighbor-joining analysis (PAUP) of
aligned NS4B amino acid sequences of 115 West Nile virus strains and 1 Japanese
encephalitis virus (used as the outgroup). Branches are drawn to scale.
NJ
AB185914
AB185915
AB185916
AB185917
AF260967
AY289214
DQ080061
DQ164198
DQ164205
DQ374651
DQ411035
DQ411034
DQ411033
DQ411032
DQ411031
DQ411030
DQ411029
DQ377180
DQ377179
DQ377178
DQ374653
DQ374652
DQ374650
DQ211652
DQ176637
DQ164206
DQ164204
DQ164201
DQ164200
DQ164199
DQ164197
DQ164196
DQ164195
DQ164194
DQ164193
DQ164191
DQ164190
DQ164189
DQ164188
DQ164186
DQ080070
DQ080069
DQ080068
DQ080066
DQ080065
DQ080064
DQ080063
DQ080060
DQ080059
DQ080058
DQ080057
DQ080056
DQ080055
DQ080054
DQ080053
DQ080052
DQ080051
DQ066423
DQ005530
AY848697
AY848696
AY848695
AY842931
AY795965
AY712947
AY278441
AY268133
AF533540
AF481864
AF404756
AF404755
AF404754
AF404753
AF206518
AF202541
AF196835
DQ118127
DQ080062
DQ080071
DQ080072
DQ164202
DQ164187
DQ164192
DQ080067
AY712948
AF260969
AF317203
AF404757
AY262283
AY268132
AY277252
AY278442
AY701412
AY701413
AY712945
AY712946
AY646354
AF260968
AY603654
AY490240
AY660002
DQ164203
AY274504
AY274505
D00246
AY532665
M12294
NC 001563
DQ318019
AY688948
DQ318020
DQ116961
DQ176636
AY277251
0.005 changes
81
Lineage Ia
Kunjin
Lineage Ib
Lineage 2
AY765264
JEV
Rus98
Lineage 3?
Rabensburg
Lineage 4?
Figure 3-4. Predicted secondary structure of the West Nile virus NS4B protein
generated using the SOSUI hydrophobicity plotting program (Hirokawa et al.,
2003). Four transmembrane domains (TMDs) are predicted with the third TMD
(amino acid residues 104-126) predicted to serve as the primary helix.
1
255
36
60
98
103
74
205
127
181
82
Figure 3-5. Predicted topology of the West Nile virus NS4B protein generated using
the ConPredII hydrophobicity plotting program (Arai et al., 2004). Five
transmembrane domains are predicted with amino acid residues 106-126 predicted
to serve as the primary helix (green). Predicted endoplasmic reticulum (ER) lumenal and cytoplasmic regions are also denoted.
1
G
M
E
N
W
NH2
L
D
K
T
K
S
L
S
S
I
D
F
ER Lumen
Membrane
G
Q
R
I
E
S
M
F
G
N
E
E
F
K
L
V
L
D
W G
L
C
R
G
Q
P
A
V
A
T
A
L
T A
L
T
W
L
V
S
L
T
L
A
V
Y
S
T
A
V
A
V
G
A
T
V
I
T
D
L
A
V
L
V
F
F
L
P
C
T
F
H
P
G
Y
R
L
A
L
A
Y
K
L
M
T
H
F
V
L
L
P
I
S
A
G
T
L
R T
T
S
W
N
T
R
A
S
A
Q
I
S
Q
A
D Y
Q
A
I
A
G
N V
E
S
I
A
M
M R
37
101
59
Cytoplasm
105
79
127
83
G I
L
E
A
W
N
I
T
E
L
G
A
T
N
T
R
T
A
A
W
A
V
V
S
A
V
I
T
A
S
K
G
V
L C
S
H
P
I
N
M
R
V
G
V
G
V
W
A
L
A
S
L
C
S
L
V
S
L
I
I
L
T
M
W
I
T
Q
L
G
I
V
K
K
K
N
M
Q
E
K
L
M
E
P
E
I
G
P
L
R
P
K
V
R
T T
D
193
V D
V
G
A
I
N
V
K
A
225
171
T
247
COOH
255
CHAPTER 4
DISRUPTION OF RESIDUES IN THE CENTRAL HYDROPHOBIC
REGION OF THE WNV NS4B PROTEIN
4.1 Abstract
West Nile Virus (WNV) is a member of the genus Flavivirus in the family
Flaviviridae. The WNV genome is a positive-sense RNA molecule approximately 11kb
in length encoding a single polyprotein that is cleaved by a combination of viral and host
proteases to produce three structural and seven nonstructural proteins. NS4B is a small
hydrophobic protein approximately 27kD in size that is hypothesized to participate both
in viral replication and evasion of host innate immune defenses. Utilizing site-directed
mutagenesis of a WNV NY99 infectious clone, amino acid substitutions were introduced
into the NS4B protein primarily targeting a central hydrophobic region where mutations
have been frequently observed in various attenuated or passage-adapted flavivirus strains.
Four recombinant mutant viruses were produced, and none showed any alteration in
cellular multiplication or mouse neuroinvasive phenotypes as compared to wild-type
recombinant NY99 WNV.
84
4.2 Introduction
West Nile virus (WNV) is a mosquito-transmitted member of the genus
Flavivirus, family Flaviviridae. This genus includes other mosquito-borne viruses of
public health importance: yellow fever (YF), Japanese encephalitis (JE), and the four
dengue viruses (DEN1-DEN4). Secondary structure predictions of the flavivirus NS4B
protein generated by the ConPredII program (Arai et al., 2004) suggest that it is a very
hydrophobic protein with five transmembrane domains (Fig. 4-1). Experimental
evidence suggests that there are three membrane-spanning domains in the DEN2 NS4B
protein that are capable of targeting a cytosolic marker protein to intracellular membranes
while the two N-terminal putative trans-membrane domains predicted by some computer
programs may not be biologically significant (Miller et al., 2006).
A number of publications have described mutations in the NS4B protein in
attenuated or passage-adapted mosquito-borne flaviviruses suggesting that this protein
plays an important role in replication and pathogenesis. A single coding mutation
(P101L) in DEN4 virus NS4B conferred a small-plaque phenotype in C6/36 cells, while
at the same time increasing plaque size in Vero cells two-fold and Huh-7 cells three-fold
(Hanley et al., 2003). Subsequent studies have suggested that DEN4 virus NS4B
interacts with the NS3 protease and that the P101L substitution ablates this interaction
(Umareddy et al., 2006). Pletnev et al. (2002) described DEN4 NS4B T105I and L112S
substitutions that occurred in a chimeric virus expressing WNV structural proteins in a
DEN4 virus backbone. Blaney Jr. et al. (2003a) noted NS4B V109A, L112S/F, and
G119S mutations in DEN4 virus passaged in Vero cells. The live attenuated JE virus
vaccine strain SA14-14-2 encodes an I106V substitution in NS4B (Ni et al., 1995) while
a live attenuated JEV veterinary vaccine, strain ML17, has three mutations in NS4B
(N191K, V192I, T208S) (Shah et al., 2006). A hamster viscerotropic Asibi strain of
YFV, generated by seven passages through hamsters, accumulated seven amino acid
substitutions in the polyprotein, including a V98I substitution in NS4B (McArthur et al.,
2003). Interestingly, YFV vaccine strains also display a mutation in NS4B at I95M
85
(Hahn et al., 1987; Wang et al., 1995). When the NS4B proteins from different
flaviviruses are aligned, it becomes clear that these mutations are all located in a similar
central hydrophobic region of the protein (Fig. 4-2).
For the purposes of this study, five amino acid substitutions were engineered into
the central hydrophobic region of the WNV NS4B protein. A WNV L97M substitution
corresponding to the I95M mutation observed in YFV vaccine strains was constructed
(Fig. 4-2). Also, a WNV A100V substitution was engineered corresponding to the YFV
V98I mutation observed in the hamster-passaged Asibi strain. WNV L108P and T116I
substitutions were constructed corresponding to mutations encoded by Vero cell-adapted
DEN4 viruses. Finally, a V106I substitution was engineered corresponding to an I106V
mutation observed in the live attenuated JEV vaccine strain SA14-14-2. Recombinant
mutant viruses were then assayed for differences in phenotypic characteristics as
compared to recombinant wild-type NY99 WNV.
4.3 Results
4.3.1 Rescue of recombinant viruses
Utilizing the two-plasmid infectious clone of WNV NY99 (Beasley et al., 2005),
mutant viruses containing a L97M, A100V, L108P, or T116I substitution in the NS4B
protein were rescued. Sequencing of the NS4B region of recombinant viruses was
conducted to confirm the presence of the mutation of interest. The original virus yield
from the transfection was used in all subsequent studies with no further passaging. All
recombinant viruses generated infectivity titers in excess of 6 log10 pfu/mL by 5 days
post-transfection. Repeated attempts to obtain a recombinant virus encoding a V106I
substitution were unsuccessful.
86
4.3.2 Temperature sensitivity assay
Each recombinant mutant virus was investigated for temperature sensitivity by
plaque assay in Vero cells at both 37°C and 41°C. Wild-type virus and the recombinant
mutant viruses all exhibited comparable levels of plaquing efficiency at both
temperatures (Table 4-1).
4.3.3 Multiplication kinetics in cell culture
Growth curves of wild-type WNV NY99 and the four recombinant mutants were
undertaken at a moi of 0.01 in Vero cells at both 37°C and 41°C (Fig. 4-3). None of the
recombinant mutant viruses exhibited any alteration in multiplication kinetics as
compared to wild-type NY99 virus at either temperature. Growth curves were also
conducted in mouse neuroblastoma Neuro2A cells and mosquito C6/36 cells (Fig. 4-4)
grown at 37°C or 28°C, respectively. Recombinant viruses containing L97M, A100V,
L108P, and T116I substitutions multiplied at levels comparable to wild-type in both cell
lines.
4.3.4 Mouse neuroinvasiveness phenotype
A mouse model has been developed to investigate neuroinvasiveness of WNV via
the intraperitoneal (ip) inoculation of three-week-old female NIH Swiss mice (Beasley et
al., 2002). The L97M, A100V, L108P, and T116I mutants were as virulent as wild-type
WNV following ip inoculation in terms of lethality and average survival time (Table 4-1).
4.4 Discussion
Amino acid substitutions were engineered into the central hydrophobic region of
the WNV NS4B protein. Recombinant mutant viruses encoding L97M, A100V, L108P,
or T116I substitutions exhibited cell multiplication and mouse virulence phenotypes
comparable to recombinant wild-type NY99 WNV. All viruses multiplied comparably in
87
monkey kidney Vero cells at both 37°C and 41°C as well as in mouse neuronal Neuro2A
cells and mosquito C6/36 cells. In addition, mouse intraperitoneal LD50 values and
average survival times were similar for wild-type and recombinant mutant viruses. Thus,
none of the engineered substitutions conferred any alteration in assayed phenotypic
properties as compared to wild-type WNV. However, repeated attempts to rescue
recombinant virus encoding the V106I substitution failed suggesting that this mutation
may be lethal to the virus.
Various amino acid substitutions in the central hydrophobic region of the NS4B
protein have been noted in the literature with a variety of either attenuated or passageadapted flavivirus strains (Fig. 4-2). While many substitutions in the central hydrophobic
region have been published, most occur in the context of other substitutions scattered
throughout the genome so the relative contribution to observed phenotypes conferred by
the NS4B mutations is poorly understood. Each of the substitutions in this study targeted
such a previously published flaviviral mutation. For example, the L97M substitution
corresponds to a yellow fever virus (YFV) mutation that occurs in vaccine strains. Both
17D and French neurotropic vaccine (FNV) strains encode an I95M substitution as
compared to the wild-type Asibi YFV strain (Wang et al., 1995). The WNV L97 residue
is conserved in each of the WN strains for which NS4B sequence data is available
(Appendix 1). The WNV NS4B A100V substitution corresponds to a YFV V98I
substitution that occurred in Asibi YFV passaged seven times through hamster liver
resulting in a virus exhibiting a novel viscerotropic phenotype in hamsters (McArthur et
al., 2003). The A100 residue is conserved in all published WN sequences except for two
lineage 2 viruses and the related Rabensburg virus that encode A100V substitutions
(GenBank M12294, NC001563, and AY765264). The engineered V106I substitution
corresponds to an I106V substitution that was observed in the Japanese encephalitis virus
(JEV) SA14-14-2 vaccine strain (Ni et al., 1995). The V106 residue is conserved in all
sequenced WNV strains, and this was the only tested substitution for which no
recombinant virus could be rescued (Appendix 1). The L108P substitution corresponds
to a P101L substitution occurring in a DEN4 strain that exhibited reduced infectivity in
88
mosquito cells and increased infectivity in Vero and Huh7 cells (Hanley et al., 2003).
This substitution represents a rare situation where the observed substitution occurred
without any additional confounding mutations allowing one to conclude that the P101L
substitution directly conferred the observed alterations in phenotype. While the WNV
L108 residue is conserved in each sequenced strain, disruption of this residue conferred
no noticeable alteration in phenotype as compared to wild-type WNV. The engineered
T116I substitution corresponds to a V109A mutation observed in a DEN4 strain that had
been passage-adapted in Vero cells. The WNV T116 residue is highly conserved in all
lineage I WN strains including the Kunjin subtype. The lineage II WN and Rabensburg
viruses encode a T116A substitution except for the Madagascar-AnMg798 strain which
encodes a T116I substitution (Appendix 1).
While the central region of the NS4B protein is often the site of amino acid
substitutions in various flaviviruses, it is only recently that a potential mechanism of
action for such mutations has recently been proposed. Umareddy et al. (2006) found that
the DEN4 NS4B protein was capable of interacting with the NS3 viral protease and was
involved in positively modulating NS3 helicase activity by dissociating the NS3 protein
from RNA. When the previously described DEN4 P101L substitution (Hanley et al.,
2003) was introduced, the mutant NS4B protein could no longer interact with the NS3
protein. It was hypothesized that the NS4B protein increased NS3 helicase activity by
leading to increased dissociation from RNA and allowing the formation of new
complexes. The implications of the DEN4 NS4B P101L substitution ablating the
interaction with the NS3 protein in vitro remain unclear as DEN4 virus expressing such a
substitution actually displayed increased infectivity in Huh7 and Vero cells. It is thought
that the NS4B protein may serve as a scaffold upon which other members of the
replication complex such as NS3 and NS5 assemble. The central hydrophobic region of
NS4B may play a critical role in such interactions. It is also likely that there are other
components involved including as yet unidentified cellular proteins. The failure of the
engineered substitutions to attenuate the resulting recombinant viruses could be explained
the possibility that these mutations did not sufficiently alter the hydrophobic character of
89
this region as introduced substitutions were relatively conservative. It is likely that this
region mediates critical protein interactions within the replication complex, however
amino acid identity is likely not as important as the overall helical hydrophobicity profile
allowing for the protein interactions to occur. Since the NS4B protein has recently been
found to regulate NS3 helicase activity, any introduced amino acid substitution that
confers an alteration in helicase regulation could also lead to attenuation of mutant
viruses. Subsequent experimentation will be necessary to better elucidate the role of the
NS4B protein in the viral replication complex.
90
Table 4-1. Temperature sensitive and mouse virulence phenotypes of central
hydrophobic mutants.
a
Virus seeds were plaque titrated in Vero cells under agar overlay at 37°C and 41°C.
The relative change [log10 (titer at 41°C/titer at 37°C)] in viral titer at the higher
temperature indicates the degree of temperature sensitivity.
b
Median lethal viral dose (LD50) following intraperitoneal (i.p.) administration of virus.
AST = average survival time
c
Infectious clone-derived wild-type WNV, strain NY99, and central hydrophobic
mutants in NS4B were tested. L97M = leucine-to-methionine substitution at amino acid
redidue NS4B-97.
a
b
Temperature sensitivity
c
Virus
Mouse virulence
Viral
Viral
Relative change
Titer/mL at 37°C
Titer/mL at 41°C
in viral titer
i.p. LD50
i.p. AST
(log10 pfu)
[log10(pfu41C/pfu37C]
(pfu)
(days ± SD)
(log10pfu)
NY99
6.5
6.7
0.2
0.5
7.4±0.9
L97M
6.5
6.8
0.3
0.4
7.6±1.0
A100V
7.0
6.7
-0.3
0.7
7.2±0.4
L108P
6.8
7.0
0.2
<0.1
7.6±1.5
T116I
6.9
7.0
0.1
0.7
8.0±1.8
91
Fig. 4-1. A model for the NS4B protein was produced based on hydrophobicity
plots. The L97 residue is predicted to reside within the second transmembrane
domain while L108 and T116 residues localize to the third primary transmembrane
domain. A100 and V106 residues are predicted to reside at the junction of an ERlumenal ectodomain and transmembrane-spanning regions. Recombinant viruses
encoding L97M, A100V, L108P, and T116I substitutions (orange positions) were
successfully produced. Repeated attempts to construct the V106I virus (yellow
position) were unsuccessful.
1
G
M
E
N
W
NH2
L
D
K
T
K
S
L
S
S
I
D
F
ER Lumen
Membrane
G
Q
R
I
E
S
M
F
G
N
E
E
F
K
L
V
L
D
W G
L
C
R
G
Q
P
A
V
A
T
A100 A
L L108
T A
L
T
W
L
V
S
T
L97 A L
L
V
Y
S
T
A
V
A
T116
V
G
A
T
V
T
T
D
L
A
V
L
V
F
F
L
P
C
T
F
H
P
G
Y
R
L
A
L
A
Y
K
L
M
T
H
F
V
L
L
P
I
S
A
G
T
L
R T
T
S
W
N
T
R
A
S
A
Q
I
S
Q
A
D Y
Q
A
I
A
G
N V
E
S
I
A
M
M R
37
101
59
Cytoplasm
105
79
127
G I
L
E
A
W
N
I
T
E
L
G
A
T
N
T
R
T
A
A
W
A
V
V
S
A
V
I
T
A
S
K
G
V
L C
S
H
P
I
N
M
R
V
G
V
G
V
W
A
L
A
S
L
C
S
L
V
S
L
I
I
L
T
M
W
I
T
Q
L
G
I
V
K
K
K
N
M
Q
E
K
L
M
E
P
E
I
G
P
L
R
P
K
V
R
T T
D
193
V D
V
G
A
I
N
V
K
A
225
171
T
247
COOH
255
Fig. 4-2. Various amino acid substitutions in the central hydrophobic domain have
been observed in attenuated or passage-adapted flaviviruses. Such substitutions
92
(orange) are plotted on a flaviviral NS4B amino acid alignment (panel A) and
compared to the corresponding residue (purple) in the WNV sequence (panel B).
A.
aa 68
ALFV
MVEV
USUV
WNV
KUNV
JEV
SLEV
BAGV
ILHV
Mosquito- ROCV
IGUV
borne
BSQV
KOKV
ZIKV
DENV1
DENV2
DENV3
DENV4
KEDV
YFV
SEPV
YOKV
ENTV
POWV
DTV
TickALKV
borne
LGTV
OHFV
TBEV
LIV
Non-Vector APOIV
MODV
RBV
Insect
CFAV
KRV
(68)
(69)
(69)
(66)
(66)
(66)
(69)
(65)
(66)
(67)
(65)
(65)
(62)
(64)
(63)
(62)
(62)
(59)
(66)
(64)
(62)
(68)
(63)
(65)
(65)
(65)
(65)
(65)
(65)
(65)
(67)
(67)
(71)
(68)
(71)
Central Hydrophobic Domain
aa 135
TTSLASISAQAGTLFTLPKGIPFSNIDMTVALVFLGCWGQITLTTLLTAVVLGVVHYGYLLPGWQAEALR
TTSLASINAQAGSLFTLPKGIPFTDFDLSVALVFLGCWGQVTLTTLIMATILVTLHYGYLLPGWQAEALR
TTSLASISAQAGSLFNLPRGLPFTELDFTVVLVFLGCWGQVSLTTLITAAALATLHYGYMLPGWQAEALR
NTSLTSINVQASALFTLARGFPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMR
NTSLTSINVQASALFTLARGFPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMR
TTSLASINSQAGSLFVLPRGVPFTDLDLTVGLVFLGCWGQITLTTFLTAMVLATLHYGYMLPGWQAEALR
NFSLTAIASQAGVLLGLTNGMPFTAMDLSVPLLVLGCWNQMTLPSLAVAVMLLAIHYAFMIPGWQAEAMR
NFSLMAVTAQAGALFGLGKGMPFVAIDLSTPLLLLGCWGQFTMTTTLTTIMLLIIHYAFLIPGWQAEAMR
SFSLTAITAQASALFGLSAGYPFVGIDLAVGFLLLGCYGQYNLPTAVATGLLLLAHYGYMIPGWQAEAMR
SFSLTAITSQASALFGLNSGYPFVGVDLSVVFLLVGCYGQYNLPTTMATIGLLVGHYAFMIPGWQAEAMR
NVSLSAIAAQAGNLFMMKNGHPFTQLDWAVPLLALGCWSTMTPLALVAATLLLLVHYAYMIPGWQAMGAR
NFSLMAMANQAGSLFGMQTGAPFSKMDWAVPAIVVGCWQQLTPATLMTALVLLAVHYIYMIPGWQAGAAR
NISLMAIASQAGTLFSMDSGIPFSSIELSVPLLALGCWTQITPCSLILACVLLSTHYAILLPGMQAQAAR
NYSLMAMATQAGVLFGMGKGMPFMHGDLGVPLLMMGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAAR
NISLTAIANQAAILMGLDKGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQAKATR
NVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNPITLTAALLLLVAHYAIIGPGLQAKATR
NVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLALGCYSQVNPLTLIAAVLLLVTHYAIIGPGLQAKATR
NLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATR
NFSLTAIGNQAGILFGMGTGVPFYKWDWGVPLLLLGCATQITPTVMVASGVLLAAHYAFLIPGLQAQAVR
NLSLSGIAQSASVLSFMDKGIPFMKMNISVIILLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSK
NISLSGITQNAQVLGLMDKGIPFIKMNMSVVILLLSAWNGITLLPLFAGMGAAALHWGFILPGLRAQAAK
AISLQGISQGAAALFQMDKGYPFMRLRIPLVLLLAGAINNLTAITAGLGFLCAMIHWALVLPGLKAKLAK
AISLQGITQSAAAFFQMDKGYPFMKLRLPLILMAVGALNNINAVALLLGLACAVFHWSLVLPGLRAKLAK
RLVNSSVAAGTQAMRDLGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAALHLAVVTSGLEAELTQ
RLVNSSVAAGTQAMKDLGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAILHLAVVTSGLEAELTQ
QLVNSSVASGAQAMRDLGGGTPFFGVAGHVIALGVTSLVGATPLSLGLGVALAAFHLAIVASGLEAELTQ
QLVNSAVASGAQAMRDLGGGTPFFGVAGHVLALGIASLVGATPTSLILGVGLAAFHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGTPFFGVAGHVLTLGVVSLVGATPTSLVVGVGLAAFHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGAPFFGVAGHVMTLGVVSLIGATPTSLMVGVGLAALHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGAPFFGVAGHVMTLGVVSLVGATPTSLIVGIGLAAFHLAIVVSGLEAELTQ
RVVTGAISGRSDLLGQLREGMGNVSIGLPELALGMSVVRGMTPVTLVLGGLAGVAHWCWFYPIHEAALTM
RIVAGSVSGKLDTLNGLRNGFVSTSMSLGDLSLFISFCRNMSPLTTVTGLVLAAIHWLWFYPMHEASLTS
RVITGTVAGKHDMVNLLPFGAAWLSLGLGDLTLAVGAFRNMSCLTLVGGVLLALAHWTWFYPLHAAAESS
QENWTARMYAMKHPEMVSSIGGFRFDEIPFRAVLPSGFAIVAIASLPSVVVGLLAAGVFMAIMYCQNKWN
QENWIAQMYVMKHPEMVSTVGGFRLDRIPFRAVLPSGFAIVTTSSLSSSLVGLAASSLFLTIAYYQNKWN
B.
Published central region NS4B
mutations (orange)
Corresponding WNV
position (purple)
Citation
YFV I95M
YFV V98I
DEN4 P101L
DEN4 chimera T105I, L112S
DEN4 V109A, L112S/F, G119S
JESA14-14-2 I106V
L97
A100
L108
V112, F119
T116, F119, V126
V106
Wang et al., 1995
McArthur et al., 2003
Hanley et al., 2003
Pletnev et al., 2002
Blaney Jr. et al., 2003a
Ni et al., 1995
Fig. 4-3. Multiplication kinetics of recombinant wild-type and mutant viruses in
monkey kidney Vero cells (MOI of 0.01) at 37°C (panel A) and 41°C (panel B).
93
Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of triplicate
samples.
A.
Vero37
9
8
7
6
5
4
Titer (log10PFU/mL)/mL)
3
2
Wt
1
A100V
0
L108P
L97M
0
24
48
96
T116I
B.
Vero41
9
8
7
6
5
4
3
2
Wt
1
A100V
0
L108P
L97M
0
24
48
96
T116I
Time (hours)
Fig. 4-4. Multiplication kinetics of recombinant wild-type and mutant viruses in
mouse Neuro 2A (panel A) and mosquito C6/36 (panel B) cells at an MOI of 0.01.
94
Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of triplicate
samples.
A.
Neuro2A
8
7
6
5
4
3
Wt
2
L97M
Titer (log10PFU/mL)/mL)
1
A100V
0
0
24
48
96
L108P
T116I
B.
C6/36
8
7
6
5
4
3
Wt
2
L97M
1
A100V
0
L108P
0
24
48
Time (hours)
CHAPTER 5
95
96
T116I
DISRUPTION OF THE CYSTEINE RESIDUES IN THE WNV NS4B
PROTEIN
5.1 Abstract
West Nile virus (WNV) NS4B is a small hydrophobic nonstructural protein that is
hypothesized to participate both in viral replication and evasion of host innate immune
defenses. The protein encodes four cysteine residues (residues 102, 120, 227 and 237).
Since cysteines are often critical for the function of proteins, each of the four cysteine
residues found in WNV NS4B were mutated to serines by site-directed mutagenesis.
While three of these substitutions had little effect on replication or mouse virulence
phenotypes, the C102S mutation was associated with a temperature-sensitive phenotype
at 41oC as well as attenuation of the neuroinvasive and neurovirulence phenotypes in
mice.
5.2 Introduction
96
As cysteine residues are often critical for the structure and function of a protein,
they serve as promising targets when attempting to disrupt protein function, i.e., in this
case to attenuate the virulence phenotype of WNV. Cysteine residues can mediate
catalytic reactions, be involved in disulfide bonds, or can under go lipid modification
such as palmitoylation. For example, disruption of a cysteine residue was found to highly
attenuate the alphavirus Sindbis, and this phenotype was found to be conferred at least in
part by ablation of a palmitoylation site (Ahola et al. 2000). Also, cysteine residues are
located within the catalytic motif of phosphatases encoded by certain poxviruses and
echoviruses that may be involved in counteracting the immune response by
dephosphorylating STAT1 (Najarro et al. 2001 and Peters et al., 2000). The WNV NS4B
protein contains four such residues at positions 102, 120, 227 and 237. Secondary
structure predictions of the flavivirus NS4B protein utilizing the ConPred II program
(Aria et al. 2004) suggest that NS4B C102 may reside near the junction of an ectodomain
and two transmembrane regions while C120, C227, and C237 residues localize within
transmembrane regions (Fig. 5-1). In addition, examination of amino acid alignments of
flaviviral NS4B proteins revealed that C102 and C120 residues localize to a central
region where mutations are frequently observed in attenuated or passage-adapted strains
of JE, YF and DEN4 viruses (Figs. 5-2 and 5-3). A single coding mutation (P101L) in
DEN4 virus NS4B conferred a small-plaque phenotype in C6/36 cells, while at the same
time increasing plaque size in Vero cells two-fold and Huh7 cells three-fold (Hanley et
al., 2003). Pletnev et al. (2002) described DEN4 NS4B T105I and L112S substitutions
that occurred in a chimeric virus expressing WNV structural proteins in a DEN4 virus
backbone. Blaney et al. (2003a) noted a NS4B L112F mutation in a DEN4 virus passaged
in Vero cells. The live attenuated Japanese encephalitis virus (JEV) vaccine strain SA1414-2 has an I106V substitution in NS4B (Ni et al., 1995). A viscerotropic Asibi strain of
YFV generated by passaging seven times through hamsters accumulated seven amino
acid substitutions in the translated polyprotein including a V98I substitution in NS4B
(McArthur et al., 2003). Interestingly, YFV vaccine strains also display a mutation in
97
NS4B at I95M (Hahn et al., 1987; Wang et al., 1995). When the NS4B proteins from
different flaviviruses are aligned, it becomes clear that these mutations are all located in a
similar region of the protein (Fig. 5-3).
While C102 is conserved throughout all members of the DEN and JE serogroups,
C120 is unique to WN and Kunjin viruses. Both C227 and C237 are located in the Cterminal region of the protein. The C227 residue is conserved within the JE serogroup
while C237 is again unique to WN and Kunjin viruses. Since cysteines are often critical
for proper protein function, the role of the four cysteine residues in the NS4B protein was
investigated by mutating each of them to a serine using reverse genetics.
5.3 Results
5.3.1 Rescue of recombinant viruses
Utilizing the two-plasmid infectious clone of WNV NY99 (Beasley et al. 2005),
mutant viruses containing a C102S, C120S, C227S, C237S, or C102A substitution in the
NS4B protein were rescued. Sequencing of the NS4B region of recombinant viruses was
conducted to confirm the presence of the mutation of interest. The complete genome of
the C102S virus was sequenced, and no additional mutations were detected. The original
virus yield from the transfection was used in all subsequent studies with no further
passaging. All recombinant viruses generated infectivity titers in excess of 5 log10
pfu/mL by 5-6 days post-transfection.
5.3.2 Temperature sensitivity assay
Each recombinant mutant virus was investigated for temperature sensitivity by
plaquing in Vero cells at both 37°C and 41°C. Wild-type virus and the C120S, C227S,
and C237S mutant viruses all showed comparable levels of plaquing efficiency at both
temperatures (Table 5-1). In contrast, the C102S mutant exhibited an infectivity titer of
98
5.7 log10 pfu/mL at 37°C, but no plaques (< 0.7 log10 pfu/mL) were detectable at 41°C,
equivalent to a reduction in efficiency of plaquing of >5.0 log10 pfu/mL. However, the
C102S mutant was not temperature sensitive at 39.5°C and plaqued as well at this
temperature as it did at 37°C (Table 5-1).
5.3.3 Multiplication kinetics in cell culture
Growth curves of wild-type WNV NY99 and the four cysteine mutants were
undertaken at a moi of 0.01 in Vero cells at both 37°C and 41°C (Fig. 5-4). Other than
the C102S mutant, the cysteine mutants grew as well as wild-type virus at both 37°C and
41˚C. Although the C102S mutant grew comparably to wild-type virus at 37°C, its peak
infectivity titer was approximately 5 log10 lower than wild-type WNV at 41°C. Growth
curves were also conducted in mouse neuroblastoma Neuro2A cells and mosquito C6/36
cells (Fig. 5-5) grown at 37°C or 28°C, respectively. Recombinant viruses containing
C102S, C120S, C227S, and C237S substitutions multiplied at levels comparable to wildtype in both cell lines at those temperatures.
5.3.4 C102S mutant is attenuated for neuroinvasiveness and neuroinvirulence in
mice
The Barrett lab have previously described a mouse model to investigate
neuroinvasiveness and neurovirulence of WNV via intraperitoneal (ip) or intracerebral
(ic) inoculation of 3-4-week-old NIH Swiss mice, respectively (Beasley et al., 2002). The
C120S, C227S, and C237S mutants were as virulent as wild-type WNV following ip
inoculation in terms of lethality and average survival time (Table 5-2). In contrast, the
C102S mutant was attenuated when inoculated by the ip route with no mice showing
clinical signs of infection following inoculation with virus doses as high as 10,000 pfu.
The C102S mutant was also found to be attenuated for neurovirulence. The C102S
mutant failed to kill any mice at an inoculum of 1000 pfu whereas wild-type recombinant
WNV had a LD50 value of 0.2 pfu, indicating at least 5,000-fold attenuation. Although
99
the C102S mutant was highly attenuated, it remained capable of inducing a protective
immune response with an ip median protective dose (PD50) value of 0.4pfu (Table 5-2).
5.3.5 Reversion of the C102S mutation
To generate temperature sensitive revertants, growth curve samples harvested at
the 48 hour timepoint from either 37°C or 41°C were tested for the presence of revertants
by picking plaques at 41°C, amplifying in Vero cells at 37°C, and determining the
efficiency of plaquing at 37°C versus 41°C. Plaque picks were identified that contained a
S102C reversion, lacked temperature sensitivity at 41°C, and displayed
neuroinvasiveness and neurovirulence characteristics similar to those of wild-type WNV
(Table 5-1).
5.3.6 C102A substitution is associated with a virulent phenotype in mice
A C102A substitution was engineered to determine if the attenuation of C102S
was due to disruption of a disulfide bond. The C102A substitution was associated with a
less drastic temperature sensitive phenotype, i.e., a reduction in efficiency of plaquing of
only 1.9 log10 pfu/mL at 41°C versus 37°C (Table 5-1). Sequencing of plaque picks
taken at 41°C show that the C102A substitution was retained in marked contrast to
C102S. In addition, this virus exhibited a neuroinvasive phenotype similar to wild-type
WNV with an ip LD50 value of 0.7 pfu. Brains were harvested from selected animals
succumbing to C102A virus infection, and viral RNA was isolated. Sequencing of RTPCR products of the NS4B gene revealed that the designed C102A substitution was
present indicating that this substitution retained the neuroinvasive phenotype of the wildtype WNV.
5.3.7 RNA and protein levels in wild-type and C102S virus-infected Vero cell
culture
To determine where the block in viral replication occurred with respect to the
C102S virus at 41°C, intracellular viral RNA and protein levels were assayed in Vero
100
cells at both 37°C and 41°C. Quantitative real-time RT-PCR results indicated
comparable levels of viral RNA synthesis for both wild-type and C102S viruses in Vero
cells at 37°C (Fig. 5-6). In contrast, there was a sharp reduction in synthesis of viral
RNA levels in C102S virus-infected cells compared to wild-type virus-infected cells at
41°C. Unexpectedly, initial intracellular RNA levels for the C102S virus-infected cells
appeared significantly higher than infectivity viral titers would indicate. This was
attributed to the presence of non-replicating viral particles in the inoculum. Viral protein
levels were measured by Western blot utilizing an anti-WNV E protein domain III
antiserum to probe cell lysates generated from virus-infected Vero cells (Fig. 5-7). Viral
E protein levels for both wild-type and C102S virus-infected cells were comparable at
37°C, while viral protein levels were sharply reduced in C102S virus-infected cells
compared to wild-type virus-infected cells at 41°C. β-actin was used as an internal
standard, and these protein levels were similar in all samples.
5.4 Discussion
In this study the role of cysteine residues in the function of the flavivirus NS4B
protein was investigated using WNV as a model. Although there are four cysteine
residues (102, 120, 227 and 237) only the cysteine-to-serine mutation at residue 102
altered the phenotypic properties of the virus. Specifically, mutation of residue 102
attenuated mouse virulence and induced a temperature sensitive phenotype. As described
in the introduction, there is evidence to suggest that the central region of NS4B plays a
role in the virulence phenotype of flaviviruses. However, this is the first time a single
engineered amino acid substitution in this region has been shown to directly confer an
attenuated phenotype in an animal model. Examination of a hydrophobicity plot (Fig. 51) of NS4B utilizing predictions generated by the ConPredII program (Arai et al. 2004)
suggests that NS4B C102 may reside near the junction of an ectodomain and two
101
transmembrane regions while C120, C227, and C237 residues localize within
transmembrane regions. Interestingly, this cysteine residue is conserved in all members
of the JE and DEN genetic groups (Fig. 5-1) suggesting that the C102S mutation at this
locus may attenuate all of these viruses.
The C102S mutant was found to be attenuated by at least 10,000-fold for mouse
neuroinvasiveness and at least 5,000-fold for mouse neurovirulence, compared to the
wild-type WNV. Such high levels of attenuation for mice have been reported associated
with chimeric constructs such as the WNV prM-E/DEN4 chimera (Pletnev et al., 2002)
and WNV prM-E/YFV 17-D chimera (Monath et al., 2001), which express the prM-E
structural genes of WNV in the attenuated genetic background of the heterologous DEN4
virus and YFV, respectively. The fact that a single nucleotide change in WNV can lead
to such a dramatic attenuated phenotype implies that the NS4B protein encodes a critical
function in virulence that may not always be readily identifiable in cell culture. The
multiplication of the C102S mutant was comparable to wild-type WNV in Vero,
Neuro2A, and C6/36 cells at permissive temperatures. However, the C102S mutant
displayed a reduced replication phenotype in Vero cells at 41°C, though not at 39.5°C,
relative to wild-type WNV and the other three mutants studied. Analysis of intracellular
viral RNA and protein levels correlated closely with viral infectivity titers for wild-type
WNV. In contrast, the C102S virus exhibited higher than expected initial RNA levels
relative to observed titers. Low levels of viral RNA were synthesized in C102S virusinfected cells at 41°C between 0 and 24 hours, however viral RNA levels at this
temperature decreased between 24 and 48 hours. This is presumably due to a degradation
of the input viral RNA. Western blots showed comparable levels of viral E protein for
both wild-type and C102S virus-infected cells at 37°C, while an E protein band was not
visualized at 41°C in the C102S virus-infected cells. In addition, a band with a ~33kD
mobility was visualized with the WNV anti-E domainIII antiserum at 41°C beginning at
12h for both wildtype and C102S infected cells, while this band was not visible in the
37°C blots. However this band was also observed in mock-infected cells grown at 41°C,
indicating that presence of virus was not required for induction of this product. In
102
combination these data suggest that reduced viral RNA synthesis is at least partially
responsible for the observed temperature sensitive phenotype. Alternatively, the disparity
between observed RNA levels and lack of detectable viral titers with the C102S mutant at
early timepoints could suggest blockage of viral assembly or budding.
The NS4B C102A substitution was associated with a less drastic temperature
sensitive phenotype at 41°C when compared to C102S and a neuroinvasive phenotype
comparable to wild-type WNV (Tables 5-1 and 5-2). This demonstrated that a cysteinespecific mechanism such as disulfide-bonding or palmitoylation was not responsible for
the mouse attenuation phenotype. This can be contrasted with results observed regarding
palmitoylation of cysteine residues in the distantly related HCV NS4B protein. Genotype
1a HCV encodes three cysteine residues in NS4B (C187, C257, and C261), and two of
these (C257 and C261) were found to be palmitoylated when the protein was
overexpressed using a mammalian expression vector and vaccinia virus-infected Huh7
cells (Yu et al., 2006). In addition, palmitoylation of the highly conserved C257 and
C261 residues was observed upon direct transfection of NS4B-encoding mRNA. An
introduced C261T substitution was found to completely inhibit HCV subgenomicreplicon colony formation while the C257A substitution was found to confer no
noticeable change in phenotype as compared to wild-type HCV replicons. It is important
to note in this study that palmitoylation of the HCV NS4B protein was never directly
observed in replicon-infected cells. In addition, it is possible that the mechanism of
colony inhibition associated with disruption of the C261T residue is more a response to
reductions in NS4B-5A cleavage efficiency than lack of palmitoylation as C261 resides
within the NS3 cleavage site directly adjacent to the first residue of the NS5A protein.
Addition of the specific palmitoylation inhibitor, 2-bromopalmitate, was found to inhibit
HCV RNA replication in a dose-dependent manner although levels of cellular GAPDH
RNA also were inhibited to some degree (Yu et al., 2006). Palmitoylation of C257 and
C261 residues was also found to increase polymerization of HCV NS4B following
overexpression of the protein. It is still unclear that palmitoylation of the HCV NS4B
protein is required for successful replication during the course of natural infection.
103
Rather it could be that particular residues are required to mediate a critical yet currently
unidentified homo- or heterotypic protein-protein interaction. Umareddy et al. (2006)
have suggested that DEN4 NS4B interacts with the NS3 protein in the replication
complex and presented evidence that the NS4B protein may exist as an oligomer. Such
findings are consistent with results observed in this study since the attenuating WNV
NS4B C102S substitution resides within the central hydrophobic region where various
other substitutions in related flaviviruses occur (Fig. 5-3) some of which also exhibit
documented phenotypic effects.
Munoz-Jordan et al. (2003) described inhibition of STAT-1 phosphorylation in
response to DEN2 infection or overexpression of DEN2 NS4B implying that altering the
IFN-signaling response is one of the functions of NS4B although similar results were not
observed with JE or TBE NS4B proteins (Best et al., 2005). Subsequent studies
(Munoz-Jordan et al., 2005) found that DEN2 NS4B amino acid residues 75-125 were
critical for mediating this function. The C102 residue is located within this region and
hence may be involved in these signaling pathways. The interactions between virus and
host are undoubtedly complex, but the identification of an attenuated phenotype
associated with the C102S mutation provide a platform to further study the role of the
NS4B protein. In addition, engineering attenuating mutations into the NS4B protein may
prove to be a useful strategy in designing new flaviviral vaccines given that reversion was
not seen in vivo.
104
Table 5-1. Temperature sensitive phenotypes of recombinant wild-type WNV and
the five cysteine mutants.
a
Virus seeds were plaque titrated in Vero cells under agar overlay at 37°C and 41°C.
The relative change [log10 (titer at 41°C/titer at 37°C)] in viral titer at the higher
temperature indicates the degree of temperature sensitivity.
b
Infectious clone-derived wild-type WNV, strain NY99, and five cysteine mutants in
NS4B were tested. C102S = cysteine-to-serine mutation at amino acid residue NS4B-102.
c
S102C revertant was isolated from a plaque pick at 41°C following extended incubation
of the C102S virus at 41°C
(log10 pfu)
(log10 pfu)
Viral
Titer/mL at 41°C
(log10 pfu)
6.5
5.9
5.4
5.9
5.2
6.2
6.5
6.4
5.7
n.d.
5.9
n.d.
6.0
n.d.
6.7
<0.7
5.2
5.5
5.3
4.3
6.3
Viral
Titer/ml at 37°C
Virusb
NY99
C102S
C120S
C227S
C237S
C102A
S102Crevc
Temperature sensitivitya
Viral
Titer/ml at 39.5°C
105
Relative change
in viral titer
[log10 (pfu41°C/pfu37°C)]
0.2
> -5.2
-0.2
-0.4
0.1
-1.9
-0.2
Table 5-2. Mouse virulence phenotypes of recombinant wild-type WNV and the five
cysteine mutants.
a
Median lethal viral dose (LD50) or median protective viral dose (PD50) following
intraperitoneal (i.p.) or intracerebral (i.c.) administration of virus. AST = average
survival time
b
Infectious clone-derived wild-type WNV, strain NY99, and five cysteine mutants in
NS4B were tested. C102S = cysteine-to-serine mutation at amino acid residue NS4B-102.
c
n.d. = not done
d
S102C revertant was isolated from a plaque pick at 41°C following extended incubation
of the C102S virus at 41°C
Mouse virulencea
Virusb
NY99
C102S
C120S
C227S
C237S
C102A
S102Crevd
i.p. LD50
i.p. AST
i.p. PD50
i.c. LD50
i.c. PD50
(pfu)
(days ± SD)
(pfu)
(pfu)
(pfu)
0.5
>10,000
0.7
2
5
0.7
2
7.4±0.9
>35
8.0±1.0
9.4±2.4
8.6±1.1
7.8±1.4
8.2±1.0
n.d.c
0.4
n.d.
n.d.
n.d.
n.d.
n.d.
0.2
>1,000
n.d.
n.d.
n.d.
n.d.
1.2
n.d.
1.2
n.d.
n.d.
n.d.
n.d.
n.d.
106
Fig. 5-1. A model for the NS4B protein was produced based on hydrophobicity
plots. C102 is predicted to reside at the junction of a ER-lumenal ectodomain and a
transmembrane domain. C120, C227, and C237 residues are predicted to localize
within membrane-spanning regions.
1
G
M
E
N
W
NH2
L
D
K
T
K
S
L
S
S
I
D
ER Lumen
Membrane
F
G
Q
R
I
E
S
M
F
G
N
E
E
F
K
L
V
L
D
W G
L
C
R
G
Q
P
A
V
A
T
A
L
T A
L
T
W
L
V
S
L
T
L
A
V
Y
S
T
A
V
A
V
G
A
T
V
I
T
D
L
A
V
L
V
F
F
L
P
C
T
F
H
P
G
Y
R
L
A
L
A
Y
K
L
M
T
H
F
V
L
L
P
I
S
A
G
T
L
R T
T
S
W
N
T
R
A
S
A
Q
I
S
Q
A
D Y
Q
A
I
A
G
N V
E
S
I
A
M
M R
C102
37
101
105
193
59
79
127
225
C227
C237
C120
Cytoplasm
G I
L
E
A
W
N
I
T
E
L
G
A
T
N
T
R
T
A
A
W
A
V
V
S
A
V
I
T
A
S
K
G
V
L C
S
H
P
I
N
M
R
V
G
V
G
V
W
A
L
A
S
L
C
S
L
V
S
L
I
I
L
T
M
W
I
T
Q
L
G
I
V
K
K
K
N
M
Q
E
K
L
M
E
P
E
I
G
P
L
R
P
K
V
R
T T
D
V D
V
G
A
I
N
V
K
A
171
T
247
COOH
255
Fig. 5-2. Complete NS4B amino acid alignments including both tick-borne and
mosquito-borne flaviviruses show conservation of the WNV C102 residue within the
107
DEN and JE genetic groups. This residue is not found in the tick-borne flaviviruses
or yellow fever virus. In contrast, the WNV C120 and C237 residues are only found
in WNV and Kunjin virus while C227 is found throughout the JE genetic group.
Langat
TBE
Powassan
OHF
YFVasibi
YFV17D
DEN1
DEN3
DEN2
DEN4
JEV
MVEV
Kunjin
WNV382-99
SLE
Usutu
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
Langat
TBE
Powassan
OHF
YFVasibi
YFV17D
DEN1
DEN3
DEN2
DEN4
JEV
MVEV
Kunjin
WNV382-99
SLE
Usutu
Consensus
(87)
(83)
(83)
(83)
(83)
(82)
(82)
(81)
(80)
(80)
(77)
(84)
(87)
(84)
(84)
(87)
(87)
(87)
(173)
Langat (169)
TBE (169)
Powassan (169)
OHF (169)
YFVasibi (168)
YFV17D (168)
DEN1 (166)
DEN3 (165)
DEN2 (165)
DEN4 (162)
JEV (170)
MVEV (173)
Kunjin (170)
WNV382-99 (170)
SLE (173)
Usutu (173)
Consensus (173)
1
10
20
30
40
50
60
70
86
NEMGLLEKTKADLAALFARDQGETVR----WGEWTNLDIQPARSWGTYVLVVSLFTPYMLHQLQTRIQQLVNSAVASGAQAMRDLG
NEMGFLEKTKADLSTALWSEREEPRP----WSEWTNVDIQPARSWGTYVLVVSLFTPYIIHQLQTKIQQLVNSAVASGAQAMRDLG
NELGYLEQTKTDISGLFRREDQGGMV----WDAWTNIDIQPARSWGTYVLIVSLFTPYMLHQLQTKIQRLVNSSVAAGTQAMRDLG
NEMGFLEKTKADLSAVLWSEREEPRV----WSEWTNIDIQPAKSWGTYVLVVSLFTPYIIHQLQTRIQQLVNSAVASGAQAMRDLG
NELGMLEKTKEDLFGKKNLIPSSAS-----PWSWPDLDLKPGAAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMD
NELGMLEKTKEDLFGKKNLIPSSAS-----PWSWPDLDLKPGAAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMD
NEMGLLETTKKDLGIGH--VAAENQH----HATMLDVDLRPASAWTLYAVATTVITPMMRHTIENTTANISLTAIANQAAILMGLD
NEMGLLETTKRDLGMS---KEPGVVS----PTSYLDVDLHPASAWTLYAVATTVITPMLRHTIENSTANVSLAAIANQAVVLMGLD
NEMGFLEKTKKDLGLG---NIATQQP----ESNILDIDLRPASAWTLYAVATTFITPMLRHSIENSSVNVSLTAIANQATVLMGLG
NEMGLIEKTKTDFGFY---QVKT-------ETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLG
NEYGMLEKTKADLKSMFVGKTQASG---LTGLPSMALDLRPATAWALYGGSTVVLTPLLKHLITSEYVTTSLASINSQAGSLFVLP
NEYGMLERTKTDIRNLFGKSLIEENEVHIPPFDFFTLDLKPATAWALYGGSTVVLTPLIKHLVTSQYVTTSLASINAQAGSLFTLP
NEMGWLDKTKSDISGLFGQRIETKEN---FSIGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLA
NEMGWLDKTKSDISSLFGQRIEVKEN---FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLA
NEMGLLEKTKSDIAKLFGSQPGSVGFAIRTTPWDISLDIKPATAWALYAAATMVMTPLIKHLITTQYVNFSLTAIASQAGVLLGLT
NEYGMLERTKSDLGKIFSSTRQPQSALPLPSMNALALDLRPATAWALYGGSTVVLTPLIKHLVTSEYITTSLASISAQAGSLFNLP
NEMGLLEKTKADL LF
W LDLRPATAWALYAVATTVLTPLLKH I S Y NLSLSAIASQA AL LG
87
100
110
120
130
140
150
160
172
GGTPFFGVAGHVLALGIASLVGATPTSLILGVGLAAFHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGDGEAKPALY
GGAPFFGVAGHVMTLGVVSLIGATPTSLMVGVGLAALHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGEGEAKPALY
GGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAALHLAVVTSGLEAELTQRAHRAFFSAMVKNPMVDGEIINPIPDGDPKPALY
GGTPFFGVAGHVLTLGVVSLVGATPTSLVVGVGLAAFHLAIVVSGLEAELTQRAHKVFFSAMVRNPMVDGDVINPFGDGEVKPALY
KGIPFMKMNISVIILLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSKLAQRRVFHGVAKNPVVDGNPTVDIEEAPEMPALY
KGIPFMKMNISVIMLLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSKLAQRRVFHGVAENPVVDGNPTVDIEEAPEMPALY
KGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIVAIDLDPVV-YDAKF
KGWPISKMDLGVPLLALGCYSQVNPLTLIAAVLLLVTHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTIDLDPVI-YDSKF
KGWPLSKMDIGVPLLAIGCYSQVNPITLTAALLLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVIDLDPIP-YDPKF
KGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPIS-YDPKF
RGVPFTDLDLTVGLVFLGCWGQITLTTFLTAMVLATLHYGYMLPGWQAEALRAAQRRTAAGIMKNAVVDGMVATDVPELERTTPLM
KGIPFTDFDLSVALVFLGCWGQVTLTTLIMATILVTLHYGYLLPGWQAEALRAAQKRTAAGIMKNAVVDGIVATDVPELERTTPQM
RGFPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIM
RGFPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIM
NGMPFTAMDLSVPLLVLGCWNQMTLPSLAVAVMLLAIHYAFMIPGWQAEAMRAAQRRTAAGIMKNAVVDGIVATDIPDLSPATPMT
RGLPFTELDFTVVLVFLGCWGQVSLTTLITAAALATLHYGYMLPGWQAEALRAAQRRTAAGIMKNAVVDGLVATDVPELERTTPLM
KG PF MDLSVLLLALGCW QVTPTTLI AV LA LHYAIILPGLQAEATR AQKRTAAGIMKNPVVDGIV DV ELE
PLY
C102
173
180
190
C120
200
210
220
230
240
258
ERKLSLILALVLCLASVVMNRTFVAVTEAGAVGVAAAMQLLRPEMDVLWTMPVACGMSGVVRGSLWGLLPLGHRLWLRTTGT--RR
ERKMSLVLATVLCLMSVVMNRTVASITEASAVGLAAAGQLLRPEADTLWTMPVACGMSGVVRGSLWGFLPLGHRLWLRASGG--RR
ERKMSLFLAIGLCIAAVALNRTAAAMTEAGAVAVAALGQLLRPEEESWWTMPMACGMAGLVRGSLWGLLPVLHRIWLRTQGARR-ERKMSLILAMILCFMSVVLNRTVPAVTEASAVGLAAAGQLIRPEADTLWTMPVACGLSGVVRGSLWGFLPLGHRLWLRTSGTRR-EKKLALYLLLALSLASVAMCRTPFSLAEGIVLASAALGPLIEGNTSLLWNGPMAVSMTGVMRGNYYAFVGVMYNLWKMKTGRR--EKKLALYLLLALSLASVAMCRTPFSLAEGIVLASAALGPLIEGNTSLLWNGPMAVSMTGVMRGNHYAFVGVMYNLWKMKTGRR--EKQLGQIMLLILCTSQILLMRTTWALCESITLATGPLTTLWEGSPGKFWNTTIAVSMANIFRGSYLAGAGLAFSLMKSLGGG--RR
EKQLGQVMLLVLCAVQLLLMRTSWALCEVLTLATGPITTLWEGSPGKFWNTTIAVSMANIFRGSYLAGAGLALSIMKSVGTG--KR
EKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPVSTLWEGNPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTST--RR
EKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTP--RR
QKKVGQVLLIGVSVAAFLVNPNVTTVREAGVLVTAATLTLWDNGASAVWNSTTATGLCHVMRGSYLAGGSIAWTLIKNADKPSLKR
QKRLGQILLVLASVAAVCVNPRITTIREAGILCTAAALTLWDNNASAAWNSTTATGLCHVMRGSWIAGASIAWTLIKNAEKPAFKR
QKKVGQVMLILVSLAALVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
QKKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
EKKMGQILLIAAAVLAVLVRPGICSIKEFGVLGSAALVTLIEGTAGVVWNCTTAVGLCNLMRGGWLAGMSITWTVYKNVDKPKGKR
QKKVGQILLIGVSAAALLVNPCVTTVREAGILISAALLTLWDNGAIAVWNSTTATGLCHVIRGNWLAGASIAWTLIKNADKPACKR
EKKLGQILLLVLCLAAVLVNRTV AV EAGILATAALLTLWE A LWN TIAVGMA VMRGSYLAGL LAWTLIKN
KR
C227
C237
Fig. 5-3 Various amino acid substitutions in the central hydrophobic domain have
been observed in attenuated or passage-adapted flaviviruses. Such substitutions
108
(yellow) are plotted on a flaviviral NS4B amino acid alignment (panel A) and
compared to the corresponding residue (purple) in the WNV sequence (panel B).
WNV C102 and and C120 residues are plotted in orange.
A.
aa 68
ALFV
MVEV
USUV
WNV
KUNV
JEV
SLEV
BAGV
ILHV
ROCV
Mosquito- IGUV
BSQV
borne
KOKV
ZIKV
DENV1
DENV2
DENV3
DENV4
KEDV
YFV
SEPV
YOKV
ENTV
POWV
DTV
TickALKV
borne
LGTV
OHFV
TBEV
LIV
APOIV
Non-Vector MODV
RBV
CFAV
Insect
KRV
(68)
(69)
(69)
(66)
(66)
(66)
(69)
(65)
(66)
(67)
(65)
(65)
(62)
(64)
(63)
(62)
(62)
(59)
(66)
(64)
(62)
(68)
(63)
(65)
(65)
(65)
(65)
(65)
(65)
(65)
(67)
(67)
(71)
(68)
(71)
Central Hydrophobic Domain
aa 135
TTSLASISAQAGTLFTLPKGIPFSNIDMTVALVFLGCWGQITLTTLLTAVVLGVVHYGYLLPGWQAEALR
TTSLASINAQAGSLFTLPKGIPFTDFDLSVALVFLGCWGQVTLTTLIMATILVTLHYGYLLPGWQAEALR
TTSLASISAQAGSLFNLPRGLPFTELDFTVVLVFLGCWGQVSLTTLITAAALATLHYGYMLPGWQAEALR
NTSLTSINVQASALFTLARGFPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMR
NTSLTSINVQASALFTLARGFPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMR
TTSLASINSQAGSLFVLPRGVPFTDLDLTVGLVFLGCWGQITLTTFLTAMVLATLHYGYMLPGWQAEALR
NFSLTAIASQAGVLLGLTNGMPFTAMDLSVPLLVLGCWNQMTLPSLAVAVMLLAIHYAFMIPGWQAEAMR
NFSLMAVTAQAGALFGLGKGMPFVAIDLSTPLLLLGCWGQFTMTTTLTTIMLLIIHYAFLIPGWQAEAMR
SFSLTAITAQASALFGLSAGYPFVGIDLAVGFLLLGCYGQYNLPTAVATGLLLLAHYGYMIPGWQAEAMR
SFSLTAITSQASALFGLNSGYPFVGVDLSVVFLLVGCYGQYNLPTTMATIGLLVGHYAFMIPGWQAEAMR
NVSLSAIAAQAGNLFMMKNGHPFTQLDWAVPLLALGCWSTMTPLALVAATLLLLVHYAYMIPGWQAMGAR
NFSLMAMANQAGSLFGMQTGAPFSKMDWAVPAIVVGCWQQLTPATLMTALVLLAVHYIYMIPGWQAGAAR
NISLMAIASQAGTLFSMDSGIPFSSIELSVPLLALGCWTQITPCSLILACVLLSTHYAILLPGMQAQAAR
NYSLMAMATQAGVLFGMGKGMPFMHGDLGVPLLMMGCYSQLTPLTLIVAIILLVAHYMYLIPGLQAAAAR
NISLTAIANQAAILMGLDKGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQAKATR
NVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNPITLTAALLLLVAHYAIIGPGLQAKATR
NVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLALGCYSQVNPLTLIAAVLLLVTHYAIIGPGLQAKATR
NLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATR
NFSLTAIGNQAGILFGMGTGVPFYKWDWGVPLLLLGCATQITPTVMVASGVLLAAHYAFLIPGLQAQAVR
NLSLSGIAQSASVLSFMDKGIPFMKMNISVIILLVSGWNSITVMPLLCGIGCAMLHWSLILPGIKAQQSK
NISLSGITQNAQVLGLMDKGIPFIKMNMSVVILLLSAWNGITLLPLFAGMGAAALHWGFILPGLRAQAAK
AISLQGISQGAAALFQMDKGYPFMRLRIPLVLLLAGAINNLTAITAGLGFLCAMIHWALVLPGLKAKLAK
AISLQGITQSAAAFFQMDKGYPFMKLRLPLILMAVGALNNINAVALLLGLACAVFHWSLVLPGLRAKLAK
RLVNSSVAAGTQAMRDLGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAALHLAVVTSGLEAELTQ
RLVNSSVAAGTQAMKDLGGGTPFFGVAGHVVALGVTSLVGATPTSLALGVALAILHLAVVTSGLEAELTQ
QLVNSSVASGAQAMRDLGGGTPFFGVAGHVIALGVTSLVGATPLSLGLGVALAAFHLAIVASGLEAELTQ
QLVNSAVASGAQAMRDLGGGTPFFGVAGHVLALGIASLVGATPTSLILGVGLAAFHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGTPFFGVAGHVLTLGVVSLVGATPTSLVVGVGLAAFHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGAPFFGVAGHVMTLGVVSLIGATPTSLMVGVGLAALHLAIVVSGLEAELTQ
QLVNSAVASGAQAMRDLGGGAPFFGVAGHVMTLGVVSLVGATPTSLIVGIGLAAFHLAIVVSGLEAELTQ
RVVTGAISGRSDLLGQLREGMGNVSIGLPELALGMSVVRGMTPVTLVLGGLAGVAHWCWFYPIHEAALTM
RIVAGSVSGKLDTLNGLRNGFVSTSMSLGDLSLFISFCRNMSPLTTVTGLVLAAIHWLWFYPMHEASLTS
RVITGTVAGKHDMVNLLPFGAAWLSLGLGDLTLAVGAFRNMSCLTLVGGVLLALAHWTWFYPLHAAAESS
QENWTARMYAMKHPEMVSSIGGFRFDEIPFRAVLPSGFAIVAIASLPSVVVGLLAAGVFMAIMYCQNKWN
QENWIAQMYVMKHPEMVSTVGGFRLDRIPFRAVLPSGFAIVTTSSLSSSLVGLAASSLFLTIAYYQNKWN
B.
Published central region NS4B
mutations
Corresponding WNV
position
Citation
YFV I95M
YFV V98I
DEN4 P101L
DEN4 chimera T105I, L112S
DEN4 V109A, L112S/F, G119S
JESA14 I106V
L97
A100
L108
V112, F119
T116, F119, V126
V106
Wang et al., 1995
McArthur et al., 2003
Hanley et al., 2003
Pletnev et al., 2002
Blaney Jr. et al., 2003
Ni et al., 1995
Fig. 5-4. Multiplication kinetics of recombinant wild-type and cysteine mutant
viruses in monkey kidney Vero cells (MOI of 0.01) at 37°C (panel A) and 41°C
109
(panel B). Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of
triplicate samples.
A.
Wt
Vero37
C102S
Titer (log10PFU/mL)/mL)
9
8
7
6
5
4
3
2
1
0
C120S
C227S
C237S
0
12
24
48
72
96
Wt
Vero41
B.
C102S
9
8
C120S
7
C227S
6
C237S
5
4
3
2
1
0
0
12
24
48
72
Time (hours)
110
96
Fig. 5-5. Multiplication kinetics of recombinant wild-type and cysteine mutant
viruses in mouse Neuro 2A (panel A) and mosquito C6/36 (panel B) cells at an MOI
of 0.01. Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of
triplicate samples.
A.
Wt
Neuro2A
C102S
8
C120S
7
C227S
6
C237S
5
4
3
2
1
Titer (log10PFU/mL)/mL)
0
0
12
24
B.
48
72
96
Wt
C6/36
8
C102S
7
C120S
6
C227S
5
C237S
4
3
2
1
0
0
12
24
48
Time (hours)
111
72
96
Fig. 5-6. C102S and Wild-type viral RNA levels were assayed in Vero cells at 37°C
(panel A) and 41°C (panel B). Taqman quantitative real-time RTPCR was
conducted on total cellular RNA preparations using primers localizing to the WNV
3’-UTR. Data was converted to RNA genome equivalents (GEQ) utilizing a
standardized curve and compared to observed plaque titers.
A.
9
9
Wtrtpcr
8
8
Wtplaque
7
7
C102Srtpcr
6
6
C102Splaque
5
5
4
4
3
3
2
2
1
1
0
0
12
24
48
B.
9
9
Wtrtpcr
8
Wtplaque
8
7
C102Srtpcr
7
6
C102Splaque
6
5
5
4
4
3
3
2
2
1
1
0
0
0
12
24
Time (hours)
112
48
RNA (log10GEQ/mL)
Titer (log10PFU/mL)/mL)
0
Figure 5-7. C102S and Wild-type viral protein levels were assayed in Vero cells at
37°C and 41°C. Protein levels were assayed from crude cellular lysates, and
Western blots were probed with rabbit anti-domainIII polyclonal antibody. β-Actin
levels were used as an internal standard.
Vero cells 37˚C
Wt
Vero cells 41˚C
C102S
Wt
C102S
E→
~50kD
~
~33kD
~
→
~45kD
~
0
12
24 48
0
12 24 48
0
12
Time (hours)
113
24
48
0
12
24 48
CHAPTER 6
DISRUPTION OF A CONSERVED N-TERMINAL MOTIF IN THE
WNV NS4B PROTEIN
6.1 Abstract
WNV NS4B is a small hydrophobic protein approximately 27kD in size that is
hypothesized to participate both in viral replication and evasion of host innate immune
defenses. Utilizing site-directed mutagenesis of a WNV NY99 infectious clone, amino
acid substitutions were introduced into the NS4B protein targeting residues located in the
N-terminal domain that exhibit a high degree of conservation among both mosquito- and
tick-borne flaviviruses.
Out of four engineered substitutions (D35E, P38G, W42F, and
Y45F), the NS4B P38G substitution was found to be associated with temperaturesensitive and small-plaque phenotypes. Furthermore, this mutation was found to
attenuate neuroinvasiveness greater than 10,000,000-fold compared to the wild-type
NY99 virus in a mouse model. Following extended incubation at 41°C, mutants
encoding putative compensatory substitutions in the NS4B protein were selected and
exhibited a reduction in the temperature-sensitive phenotype and return to a virulent
phenotype in the mouse model.
114
6.2 Introduction
NS4B is approximately 27kD in size and is cleaved from the translated
polyprotein by a combination of viral and host proteases (Chambers et al., 1989;
Preugschat et al., 1991). Within the family Flaviviridae, WNV NS4B exhibits ~35%
identity with other mosquito-borne flaviviruses such as YF virus and members of the
DEN serogroup. Accumulation of Kunjin virus NS4B in the perinuclear region along
with induction of membrane proliferation has been described, and there is evidence that
NS4B can translocate into the nucleus (Westaway et al., 1997). Recently DEN2 virus
NS4B was found to inhibit the interferon-signaling cascade at the level of nuclear signal
transducer and activator of transcription (STAT) phosphorylation (Munoz-Jordan et al.,
2004, 2005). NS4B has been found to colocalize with other components of the viral
replication complex such as NS3 and double-stranded RNA (Miller et al., 2006).
Secondary structure predictions of the flavivirus NS4B protein generated by the
CONPRED program (Hirokawa et al., 2003) suggest that it is a very hydrophobic protein
with five transmembrane domains (Fig. 6-1). Experimental evidence suggests that there
are three membrane-spanning domains in the DEN2 NS4B protein that are capable of
targeting a cytosolic marker protein to intracellular membranes while the two N-terminal
putative trans-membrane domains predicted by some computer programs may not be
biologically significant (Miller et al., 2006).
For the purposes of this study, various amino acid substitutions were engineered
into a highly conserved N-terminal motif of the NS4B protein that is found in both tickborne and mosquito-borne flaviviruses. Out of four engineered substitutions (D35E,
P38G, W42F, and Y45F), three of the mutations had no noticeable effect on the
phenotypic properties examined. However, the P38G substitution was found to confer
small-plaque and temperature-sensitive phenotypes in cell culture and attenuation of the
neuroinvasive phenotype in mice.
115
6.3 Results
6.3.1 Design of amino acid substitutions
The alignment of different flaviviral NS4B amino acid sequences highlights a
series of highly conserved amino acids in the N-terminal portion of NS4B proteins of
both mosquito- and tick-borne flaviviruses (Fig. 6-2). For WNV these residues
correspond to D35, P38, W42, and Y45. Residues W42 and Y45 are perfectly conserved
in all mosquito- and tick-borne flaviviruses based on available sequence data. The D35
residue is found in every mosquito- or tick-borne flavivirus examined except for the
Brazilian mosquito-borne flavivirus Ilheus, which encodes a glutamic acid residue at this
position, and Rocio virus, which encodes an alanine (Kuno and Chang, 2005). The P38
residue is conserved in every flavivirus examined except for Ilheus, which encodes an
alanine at this residue. Utilizing the ConPredII hydrophobicity plotting program (Arai et
al., 2004), these residues are predicted to reside either within the ER lumen or in a
secondary transmembrane domain. To determine the importance of these residues in
viral multiplication and virulence phenotypes, D35E, P38G, W42F, and Y45F mutations
were engineered into the WNV NS4B protein via site-directed mutagenesis of an
infectious clone.
6.3.2 Rescue of recombinant viruses
Utilizing the two-plasmid infectious clone of WNV NY99 (Beasley et al., 2005)
and site-directed mutagenesis, mutant viruses containing either D35E, P38G, W42F, or
Y45F substitution in the NS4B protein were rescued following transfection. Sequencing
of the NS4B region from recombinant viruses was conducted to confirm the presence of
the mutation of interest and absence of any additional mutations. Virus stocks produced
from the initial transfection were used for all subsequent studies with the exception of the
P38G virus. Following transfection, titers of P38G virus were not sufficiently high (~2
log10 pfu/mL) for subsequent studies so this virus was passaged once in Vero cells. Upon
sequencing of the NS4B region, the P38G virus was found to contain an additional T116I
116
substitution that was not present in the original DNA plasmid so this virus will henceforth
be denoted as P38G/T116I. Subsequent full-length genomic sequencing identified no
additional mutations in the virus. A T116I substitution alone was engineered into the
WNV infectious clone to test the relative contribution of T116I towards observed
phenotypes of this mutation outside the context of the P38G mutation. The NS4B region
was sequenced for all recombinant mutant viruses, and no other nucleotide changes were
identified in any virus. The P38G/T116I mutant virus was found to exhibit a smallplaque phenotype (<1 mm versus 4 mm for the parental virus) at both 37°C and 41°C
when compared to wild-type or other mutant viruses. Recombinant virus encoding only
the P38G substitution was not recovered. In addition, a recombinant virus encoding a
P38A substitution with no additional NS4B mutations was recovered, and this virus did
not exhibit the small-plaque phenotype observed with the P38G/T116I virus suggesting
alanine is better tolerated than glycine at this position.
6.3.3 Recombinant P38G/T116I virus is temperature sensitive and exhibits a smallplaque phenotype
Each recombinant mutant virus was investigated for temperature sensitivity by
plaquing in Vero cells at both 37oC and 41oC. D35E, P38A, W42F, and Y45F mutant
viruses showed comparable levels of plaquing efficiency to wild-type at both
temperatures (Table 6-1). In contrast, the P38G/T116I virus was found to exhibit a
reduction in efficiency of plaquing of 3.8 log10 pfu/mL at 41°C as compared to 37°C
while there was no reduction in plaquing efficiency in Vero cells at 39.5°C.
6.3.4 Multiplication kinetics of recombinant viruses in cell culture
Growth curves of wild-type WNV NY99 and the mutant viruses were compared
at a moi of 0.01 in Vero cells at both 37oC and 41oC (Fig. 6-3 A and B, respectively). In
addition, growth curves of recombinant viruses were undertaken in mouse neuroblastoma
Neuro2A cells at 37°C and mosquito C6/36 cells at 28°C (Fig. 6-4 A and B,
respectively). All mutant viruses were indistinguishable from wild-type virus in each cell
117
line tested with the exception of the P38G/T116I virus. The P38G/T116I virus exhibited
a delay in multiplication in Vero cells at 41oC, but not at 37°C, with infectivity titers for
the P38G/T116I virus at 41oC significantly lower than those for wild-type virus at every
timepoint except 96 hours (Fig. 6-3B). The P38G/T116I virus showed no differences in
multiplication kinetics from wild-type virus in Neuro2A and C6/36 cells.
6.3.5 P38G/T116I virus is attenuated for neuroinvasiveness but not neurovirulence
in mice
Our laboratory has previously described a mouse model to investigate
neuroinvasiveness and neurovirulence of WNV via intraperitoneal (ip) or intracerebral
(ic) inoculation of three-week-old female NIH Swiss mice, respectively (Beasley et al.,
2002). All recombinant viruses were as virulent as wild-type WNV following ip
inoculation in terms of lethality and average survival time with the exception of the
P38G/T116I virus (Table 6-2). The P38G/T116I mutant was found to be attenuated for
neuroinvasiveness with an ip LD50 value of greater than 10,000 pfu; however, when
inoculated via the ic route, the P38G/T116I mutant remained as lethal as wild-type WNV
with an ic LD50 value of less than 0.1 pfu. The recombinant virus containing only the
T116I substitution retained a comparable neuroinvasive phenotype to wild-type virus
indicating that the P38G mutation was primarily responsible for attenuation.
6.3.6 Isolation of P38G/T116I derivatives encoding compensatory mutations
As shown in Fig. 6-3B, the P38G/T116I virus exhibited delayed multiplication in
Vero cells at 41°C with infectivity titers finally approaching those of wild-type virus by
96 hours post-infection. Given that a multiplication pattern characteristic of delayed
multiplication was observed, a virus sample from the 96 hour timepoint was investigated
to test for the selection of virus that was either a revertant or contained compensatory
mutations. Serial dilutions of P38G/T116I-derived virus grown in Vero cells at 41°C
from the 96 hour timepoint were inoculated via the ip route into female three-week-old
NIH Swiss mice to test for virulence phenotypes of the virus. Sporadic mortality was
118
observed, but no specific dose was associated with either uniform mortality or survival.
Brains were taken from four representative mice that showed clinical signs of WNV
disease, and viral RNA was isolated and sequenced. Upon sequencing of the NS4B
region, viral RNA from two of the mice was found to encode an additional NS4B A95T
substitution in addition to the original P38G and T116I substitutions. Viral RNA from
the third mouse encoded an additional NS4B V110A mutation while viral RNA isolated
from the fourth mouse encoded an I224V mutation in addition to P38G and T116I
mutations. Upon analysis of the consensus sequencing chromatogram from the original
96 hour 41°C virus sample, heterogeneity was observed at nucleotides G/A7198
corresponding to amino acid A95 and T/C7244 corresponding to amino acid V110 but
not at positions G7162, C7214, and A7585 corresponding to amino acids A83, A100, and
I224, respectively. Taken together, these data suggest the presence of a mixed population
of viruses at the 96 hour timepoint following growth in Vero cells at 41°C (Fig. 6-5).
To identify any additional mutations that may have been selected for during
multiplication at 41°C, eight plaques were picked from virus at the 96 hour timepoint at
41°C. Nucleotide sequencing of the NS4B region of these eight plaques revealed four
isolates encoding the I224V substitution, and one isolate each encoding the following
substitutions: A83S, A95T, A100V, and V110A. Thus, A95T, V110A, and I224V
substitutions were identified both in plaque picks from the 96 hour timepoint sample in
cell culture and in mouse brain, while A83S and A100V substitutions were identified
only by plaque picking. Interestingly, virus encoding either a direct G38P or I116T
reversion was never detected.
6.3.7 Analysis of compensatory mutants
Viruses containing the P38G/T116I substitutions plus a putative compensatory
substitution (i.e., A83S, A95T, A100V, V110A, or I224V) were analyzed to determine if
the temperature-sensitive and mouse neuroinvasive-attenuated phenotypes of the
parental virus were conserved. Viruses encoding A95T, A100V, V110A, or I224V
substitutions in addition to P38G/T116I were found to be no longer temperature-sensitive
119
in Vero cells at 41°C with less than a 1 log10 decrease in titer at 41°C compared to 37°C
(Table 6-3). The P38G/T116I + I224V virus exhibited a noticeable increase in plaque
size (4mm) compared to the original P38G/T116I parental virus stock (<1mm). The
P38G/T116I/A83S virus was still found to be temperature-sensitive with a 1.3
log10pfu/mL decrease in titer 41°C versus 37°C, although this virus was less temperaturesensitive than the parental P38G/T116I virus. Viruses encoding the putative
compensatory substitutions in addition to the P38G/T116I substitutions were serially
inoculated into goups of three-week-old female NIH Swiss mice via the intraperitoneal
route (Table 6-3). Viruses encoding additional A95T, A100V, V110A, and I224V
substitutions were found to exhibit a mouse-neuroinvasive virulent phenotype with ip
LD50 values greater than 2 log10 pfu lower than observed for the attenuated P38G/T116I
parental virus. The P38G/T116I/A83S virus was found to retain the attenuated
phenotype with an ip LD50 value of greater than 1,000 pfu. Full-length genomic
sequencing was conducted on each virus encoding compensatory substitutions
(P38G/T116I + A83S, A95T, A100V, V110A, or I224V), and no additional mutations
were observed.
6.3.8
Mutation rate of the P38G/T116I virus when passaged in Vero cells at 37°C
or 41°C
To better understand the significance of the observed compensatory substitutions,
viral RNA was isolated from the parental P38G/T116I virus that had been passaged once
in Vero cells post-transfection plus virus samples taken from Vero cell supernatants at 96
hours post-infection at 37°C and 41°C. Viral RNA was subjected to RT-PCR utilizing
NS4B-specific primers, and PCR products were sequenced directly to obtain the
consensus sequence. In addition, PCR products were cloned and 21-23 clones were
sequenced for each product to assay the prevalence of nucleotide changes and deduced
amino acid substitutions. The basal variability of the parental P38G (C7027G/C7028G)/
T116I (C7262T) viral RNA was analyzed and compared to the selection of specific
mutants following 96 hours in Vero cells at either 37°C or 41°C. Cloned PCR fragments
120
from the parental P38G/T116I virus were found to exhibit few differences with 19/22
(86%) sequenced fragments exhibiting no nucleotide changes and 21/22 (95%) fragments
encoding no amino acid substitutions (Table 6-4). Two parental cDNA clones exhibited
silent nucleotide changes, and a single cDNA clone encoded a H58Y (C7087T) amino
acid substitution. In contrast, 22/23 (96%) cDNA clones from the 96-hour 41°C virus
sample exhibited nucleotide changes leading to amino acid substitutions. Fifteen of 23
(65%) sequenced clones encoded the A95T (G7198A) substitution, 4/23 (17%) encoded
the A100V (C7214T) substitution, 2/23 (9%) encoded the I224V (A7585G) substitution,
and 1/23 (4%) encoded a R84Q (G7166A) substitution. In addition, T48A (A7057G),
L56P (T7056C), V155I (G7378A), I168V (A7414G), and C227R (T7594C) substitutions
were each detected in one cDNA clone (4% prevalence) from the 96-hour 41°C virus and
occurred in conjunction with either an A95T or A100V substitution (Table 6-4). Seven
of 23 (30%) cDNA clones exhibited additional silent nucleotide changes in addition to
coding amino acid substitutions. Sequenced cDNA clones from the 96-hour 37°C virus
sample exhibited an intermediate frequency of substitutions with 14/21 (67%) exhibiting
no nucleotide changes and 16/21 (76%) encoding no amino acid substitutions. Two of 21
(10%) clones were found to encode T109A (A7240G) and I245V (A7648G)
substitutions. One of 21 (5%) clones encoded a V217I (G7564A) substitution, and 1/21
(5%) clones encoded a N189S (A7481G) substitution. Finally, one (5%) clone was found
to lack the parental T116I substitution, but this fragment encoded an additional M177L
(A7444T) substitution. A total of 50 nucleotide changes occurred in 66 sequenced cDNA
clones, and 49/50 (98%) were transition mutations. The single A7444T transversion led
to the deduced M177L amino acid substitution in the only fragment lacking the parental
T116I (C7262T) substitution. Three of 50 (6%) nucleotide changes occurred in cDNA
clones from the parental virus, 36/50 (72%) occurred in clones from the 96 hour 41°C
virus, and 11/50 (22%) occurred in clones from the 96 hour 37°C virus.
121
6.3.9 RNA and protein levels in wild-type and P38G/T116I virus-infected Vero cell
culture
To determine where the block in viral replication occurred with respect to the
multiplication of P38G/T116I virus at 41°C, intracellular viral RNA and protein levels
were assayed in Vero cells at both 37°C and 41°C. Quantitative real-time RT-PCR
results indicated comparable levels of viral RNA synthesis for both wild-type and
P38G/T116I viruses in Vero cells at 37°C (Fig. 6-6). In contrast, there was a sharp
reduction in synthesis of viral RNA levels in P38G/T116I virus-infected cells compared
to wild-type virus-infected cells at 41°C. Viral protein levels were measured by Western
blot utilizing a rabbit anti-WNV E protein domain III antiserum to probe cell lysates
generated from virus-infected Vero cells (Fig. 6-7). Viral E protein levels for both wildtype and P38G/T116I virus-infected cells were comparable at 37°C, while viral protein
levels were sharply reduced in P38G/T116I virus-infected cells compared to wild-type
virus-infected cells at 41°C. β-actin was used as an internal standard, and these protein
levels were similar in all samples.
6.4 Discussion
In this study we have investigated the role of amino acid substitutions in the
NS4B protein and their role in certain phentotypic properties using WNV as a model.
Targeted N-terminal residues (D35, P38, W42, and Y45) were highly conserved
throughout both tick-borne and mosquito-borne flaviviruses. Most engineered
substitutions were well-tolerated with only one of four recombinant mutant viruses tested
exhibiting an attenuated phenotype in mice. This mutant, containing an engineered P38G
substitution and an additional T116I substitution, was found to exhibit small-plaque
morphology and attenuation of neuroinvasiveness in mice. The P38G/T116I mutant
exhibited at least 10,000,000-fold attenuation for mouse neuroinvasiveness when
inoculated via the ip route, yet was as neurovirulent as wild-type WNV when injected via
122
the ic route. Mutant recombinant WNV containing the T116I substitution only exhibited
comparable growth and virulence phenotypes to wild-type WNV suggesting that the
P38G substitution was primarily responsible for observed differences in phenotypes
(Tables 4-1 and 4-2).
Given that virus encoding only the P38G substitution was never isolated, an
additional substitution (such as T116I) may be required for viability of the virus. To
better address this issue, a P38A substitution was also engineered to determine if viruses
encoding this mutation would be viable. As previously described, the Brazilian flavivirus
Ilheus encodes an alanine at the homologous residue suggesting that this substitution
could be better tolerated (Figueiredo et al., 2000). In fact, the P38A substitution was
well-tolerated, and this recombinant virus exhibited multiplication and virulence
phenotypes similar to wild-type virus. It was also found that the attenuated phenotype
conferred by the P38G substitution could be compensated for by a variety of additional
substitutions (Table 6-3). Multiplication of the P38G/T116I virus at 41°C resulted in
identification of variants containing additional putative compensatory mutations at the 96
hour timepoint sample. Viruses containing A95T, A100V, V110A, or I224V
substitutions in addition to the original P38G and T116I substitutions exhibited mouse
virulence characteristics similar to wild-type WNV and were no longer temperaturesensitive. However, virus containing an additional A83S substitution was still found to
be attenuated in mice and exhibited a temperature-sensitive phenotype in Vero cells at
41°C, although this virus was less temperature-sensitive than the parental P38G/T116I
virus (Table 6-3). These data suggest that the same underlying mechanism may be
responsible for both the temperature-sensitive phenotype in cell culture and the
neuroinvasive phenotype in mice.
Full-length genomic sequencing was conducted on each virus encoding
compensatory substitutions (P38G/T116I + A83S, A95T, A100V, V110A, or I224V), and
no additional mutations were observed. While the attenuated phenotype of the
P38G/T116I virus was reversed by a variety of compensatory mutations, a direct G38P
revertant was never detected, rather, the engineered substitution appeared to be quite
123
stable. This is presumably due to the engineered glycine differing from proline by two
transversion nucleotide changes (C7027G/C7028G), such that direct reversion would be a
rare event. This is in marked contrast to the previously described C102S (G7220C) virus,
where direct S102C (C7220G) revertants (a single nucleotide change) were associated
with reversal of temperature-sensitive and attenuation phenotypes. Each of the identified
compensatory mutations associated with the engineered P38G virus (A83S (G7162T),
A95T (G7198A), A100V (C7214T), V110A (T7244C), T116I (C7262T), I224V
(A7585G)) represents a single nucleotide change.
It is not yet entirely clear how the
compensatory substitutions exert their effects. None of the compensatory mutations were
highly conserved among the mosquito-borne flaviviruses. The NS4B A83S substitution
has been found to occur naturally in a virus strain isolated from human plasma in Ohio in
2002 (Davis et al., 2005) In addition, the related naturally mouse-attenuated Rabensburg
virus differs from the NY99 strain of WNV at positions A95S, A100V, and T116A
(Bakonyi et al., 2005).
A lineage 2 isolate from Nigeria also encodes A100V and
T116A substitutions while an attenuated lineage 2 strain from Madagascar (MAD78)
encodes the T116I substitution although it is unlikely that this mutation is directly
responsible for the attenuation of the virus (Yamshchikov et al., 2001; Keller et al.,
2006). In contrast, the V110 and I224 residues are highly conserved in all analyzed
WNV isolates but not in other members of the JE serogroup. Further research must be
conducted to better understand how perturbing the P38 residue can be compensated by
such a wide range of additional mutations. However, it is interesting that the A95, A100,
V110, and T116 residues all are located within the same hydrophobic region that was
originally targeted (Chapter 4). Upon alignment, the A100V substitution corresponds to
a V98I substitution previously identified in hamster-passaged YFV (McArthur et al.,
2003). This A100V substitution was found to have no effect on WNV multiplication or
virulence phenotypes when expressed alone (Table 4-2). The fact that this same
substitution was independently identified as a compensatory mutation in the attenuated
P38G/T116I virus provides strong evidence that the central hydrophobic region of the
NS4B protein plays a critical role in flavivirus multiplication and virulence
124
characteristics. It is noteworthy that A95T, A100V, and I224V substitutions were highly
enriched by incubating the parental P38G/T116I virus in Vero cells at 41°C for 96 hours.
These substitutions were not detected in the parental sample or the 96-hour 37°C sample
suggesting that these three substitutions confer a specific selective advantage in Vero
cells at increased temperature. Enrichment of specific substitutions was observed to a
much lesser degree in Vero cells at 37°C over 96 hours suggesting that the P38G/T116I
virus is stable under these conditions. The T109A/I245V double mutant was observed in
two cDNA clones (10% prevalence), suggesting that these substitutions may be selected
for at 37°C, however the parental sequence made up the majority of sequenced cDNA
clones. The T109 residue is highly conserved in WNV isolates but not in other members
of the JE serogroup. In contrast, the I245 residue is variable with related Rabensburg,
RUS98, and Kunjin viruses, as well as a lineage 1 Colorado 2003 strain isolated from a
magpie, exhibiting the I245V substitution. Intriguingly, a single cDNA clone lacking the
T116I substitution and encoding an additional M177L substitution was detected at 37°C.
The T116I substitution was not originally engineered but was detected in sequence from
the parental stock of the P38G virus. This clone suggests that a minor fraction of viral
RNA (<5%) quasispecies does not encode the T116I substitution but may encode other
substitutions allowing for growth in Vero cells, such as M177L. The lineage 2 Uganda
B956 and related Nigerian WNV strains display an M177I substitution at this residue
when compared to lineage 1 NY99-derived strains. The V110A and A83S substitutions
identified by plaque picking compensatory mutants were not detected by RT-PCR
although a single R84Q substitution was observed. In summary, addition of A95T,
A100V, V110A, and I224V substitutions to the P38G/T116I virus were found to
compensate for the observed temperature-sensitive and mouse-attenuated phenotypes. In
addition, relative amounts of RNAs encoding A95T, A100V, and I224V substitutions
increased during incubation in Vero cells at 41°C but not at 37°C suggesting that these
substitutions only confer increased viral fitness at increased temperature.
Umareddy et al. (2006) suggested that DEN4 NS4B interacts with the NS3
protein in the replication complex and presented evidence that the NS4B protein may
125
exist as an oligomer. It was also found that a single NS4B P101L substitution could
ablate the interaction between NS4B and NS3. If this is the case, the engineered P38G
substitution could also disrupt the ability of the NS4B protein to assemble in the
replication complex under some conditions. Putative compensatory substitutions such as
T116I, M177L, A95T, A100V, V100A, and I224V may restore the ability of the NS4B
protein to assemble and function properly. Lai et al. (2006) have shown that there are
molecular determinants in both N-terminal and C-terminal regions of the Hepatitis C
virus (HCV) NS4B protein that may influence the ability of NS4B to polymerize. In
addition, it has been shown that substitutions in predicted ER-lumenal regions of the
NS4B protein can nearly abolish HCV replicon colony formation although these regions
are likely separate from the cytoplasmic components of the replication complex
(Lindstrom et al., 2006). This again supports the idea that the NS4B protein may form
either oligimers or crucial complexes with certain cellular proteins. While HCV NS4B
displays little sequence homology with WNV NS4B, it is thought that these proteins may
exhibit similar topologies and functions within the cell (Lundin et al., 2003). The P38
residue is predicted to localize to the junction of a ER-lumenal region and a
transmembrane domain, and it will be critical to elucidate potential protein-protein
interactions to better explain the temperature sensitive and mouse attenuation phenotypes
observed in conjunction with disruption of this residue.
126
Table 6-1. Temperature sensitive phenotypes of recombinant wild-type WNV and
the N-terminal mutants.
a
Virus seeds were plaque titrated in Vero cells under agar overlay at 37°C and 41°C.
The relative change [log10 (titer at 41°C/titer at 37°C)] in viral titer at the higher
temperature indicates the degree of temperature sensitivity.
b
Infectious clone-derived wild-type WNV, strain NY99, and N-terminal mutants in
NS4B were tested. D35E = aspartic acid-to-glutamic acid mutation at amino acid residue
NS4B-38.
c
not done
Temperature sensitivitya
Viral
Viral
Titer/mL
at 41°C
Titer/ml at 39.5°C
(log10 pfu)
(log10 pfu)
Viral
Titer/ml at 37°C
Virusb
NY99
D35E
P38G/T116I
W42F
Y45F
P38A
(log10 pfu)
6.5
5.7
6.2
6.7
5.5
5.8
6.4
6.7
5.4
3.0
6.5
5.8
5.6
c
n.d.
5.8
n.d.
n.d.
n.d.
127
Relative change
in viral titer
[log10 (pfu41°C/pfu37°C)]
0.2
-0.3
-3.2
-0.2
0.3
-0.2
Table 6-2. Mouse virulence phenotypes of recombinant wild-type WNV and the Nterminal mutants.
a
Median lethal viral dose (LD50) or median protective viral dose (PD50) following
intraperitoneal (i.p.) or intracerebral (i.c.) administration of virus. AST = average
survival time
b
Infectious clone-derived wild-type WNV, strain NY99, and the N-terminal mutants in
NS4B were tested. D35E = aspartic acid-to-glutamic acid mutation at amino acid residue
NS4B-35.
c
n.d. = not done
Mouse virulencea
i.p. LD50
i.p. AST
i.p. PD50
i.c. LD50
Virusb
(pfu)
(days ± SD)
(pfu)
(pfu)
NY99
0.7
7.2±0.4
n.d.c
0.2
D35E
0.4
7.2±0.9
0.4
n.d.
P38G/T116I
>10,000,000
>35
n.d.
<0.1
W42F
<0.1
7.4±0.9
n.d.
n.d.
Y45F
<0.1
7.4±0.9
n.d.
<0.1
P38A
7
8.6±1.5
n.d.
n.d.
128
Table 6-3. Temperature sensitive and mouse virulence phenotypes of P38G/T116Iderived viruses encoding compensatory substitutions.
a
Virus seeds were plaque titrated in Vero cells under agar overlay at 37°C and 41°C.
The relative change [log10 (titer at 41°C/titer at 37°C)] in viral titer at the higher
temperature indicates the degree of temperature sensitivity.
b
Median lethal viral dose (LD50) or median protective viral dose (PD50) following
intraperitoneal (i.p.) or intracerebral (i.c.) administration of virus. AST = average
survival time
c
Infectious clone-derived wild-type WNV, strain NY99, parental P38G/T116I virus, and
P38G/T116I-derived viruses encoding compensatory substitutions in NS4B were tested.
+A83S = alanine-to-serine mutation at amino acid residue NS4B-83 in addition to P38G
and T116I substitutions.
a
Temperature sensitivity
c
Virus
Mouse virulence
b
Viral
Viral
Relative change
Titer/mL at 37°C
Titer/mL at 41°C
in viral titer
i.p. LD50
i.p. AST
(log10 pfu)
[log10(pfu41C/pfu37C]
(pfu)
(days ± SD)
(log10pfu)
NY99
7.2
7.0
-0.2
0.7
7.2±0.4
P38G/T116I
6.7
3.4
-3.3
>10,000,000
>35
+ A83S
4.7
3.4
-1.3
>1000
>21
+ A95T
5.0
4.6
-0.4
2
8.8±0.8
+A100V
5.5
5.1
-0.4.
8
11±1.2
+V110A
5.1
4.7
-0.4
4
9.6±1.3
+I224V
4.6
4.3
-0.3
5
11±2.0
129
Table 6-4. Analysis of the mutation rate of the P38G/T116I virus when passaged in
Vero cells at either 37°C or 41°C compared to variability in the parental stock.
Parental P38G/T116I
Virus
Nucleotide Amino acid
Change
change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
-a
T7015C
C7087T*
A7554G
N.A.
H58Y
N.A.
41°C 96-hour Virus
Nucleotide Change
A7158G, G7198A*, G7378A*
T7015C, C7214T*
G7198A*
G7198A*, T7488C, A7554G
G7198A*
G7198A*
G7198A*
G7198A*
G7198A*
G7198A*
C7214T*, A7227G
T7015C, C7214T*, T7594C*
G7166A*
G7198A*
G7198A*, C7410T, A7417G*
A7005G, G7198A*, T7488C
A7585G*
G7198A*
G7198A*
A7057G*, C7214T*
A7585G*
T7056C*, G7198A*
37°C 96-hour Virus
Amino acid
change
Nucleotide change
Amino acid
change
A95T, V155I
A100V
A95T
G7564A*
V217I
A95T
G7080A
A95T
A95T
A95T
G7080A, A7240G*, A7648G* T109A, I245V
A95T
A95T
A95T
A7218G
A100V
A100V, C227R
R84Q
A95T
A7481G*
N189S
A95T, I168V
A95T
I224V
A95T
A7240G*, A7648G*
T109A, I245V
A95T
T48A, A100V
T7262C*, A7444T*
I116T, M177L
I224V
N.A.
N.A.
L56P, A95T
N.A.
N.A.
-a : no amino acid substitution
130
Fig. 6-1. A model for the NS4B protein was produced based on hydrophobicity
plots. D35, P38, W42, Y45, and P54 residues (orange) are predicted to localize to
either the ER-lumen or the first putative transmembrane domain (pTMD1).
Compensatory substitutions (in yellow) tend to localize within pTMD2 or pTMD3
although the I224 residue is located at the junction of the ER-lumen and pTMD5.
1
G
M
E
N
W
NH2
L
D
K
T
K
S
L
S
S
I
D
F
ER Lumen
Membrane
S
M
F
G
N
E
E
F
K
L
V
L D35
D
W G
L
C
R
G
Q
P
A
V
A
T
P38 A100 A
L
T A
L
T
W42
W
L
V V110
S
T
Y45
A95 A L
L
V
Y
S
T
A
V
A
V
G
A
T
V
T T116
T
D
L
A
V
L
V
F
F
L
P
C
T
F
H
P
G
Y
R
L
A
L
A
Y
K
L
M
T
H
F
V
L
L
P
I
S
A
G
T
L
R T
T
S
W
N
T
R
A
S
A
Q
I
S
Q
A
D Y
Q
A
I
A
G
N V
E
S
I
A
M
M R
G
Q
R
I
E
37
101
105
193
A83
Cytoplasm
59
79
127
131
G I
L
E
A
W
N
I
T
E
L
G
A
T
N
T
R
T
A
A
W
A
V
V
S
A
V I224 I
T
A
S
K
G
V
L C
S
H
P
I
N
M
R
V
G
V
G
V
W
A
L
A
S
L
C
S
L
V
S
L
I
I
L
T
M
W
I
T
Q
L
G
I
V
K
K
K
N
M
Q
E
K
L
M
E
P
E
I
G
P
L
R
P
K
V
R
T T
D
171
V D
V
A
N
K
225
G
I
V
A
T
247
COOH
255
Fig. 6-2. NS4B amino acid alignments show conservation of the targeted N-terminal
residues within both mosquito- and tick-borne flaviviruses. W42, Y45, and P54
residues are completely conserved while the D35 residue is variable in Rocio and
Ilheus viruses. The P38 residue is conserved in all flaviviruses except for Ilheus
which encodes an alanine.
N-Terminal Motif
Insect Non-V
Tick
Mosquito
aa 1
ALFV
MVEV
USUV
WNV
KUNV
JEV
SLEV
BAGV
ILHV
ROCV
IGUV
BSQV
KOKV
ZIKV
DENV1
DENV2
DENV3
DENV4
KEDV
YFV
SEPV
YOKV
ENTV
POWV
DTV
ALKV
LGTV
OHFV
TBEV
LIV
APOIV
MODV
RBV
CFAV
KRV
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
D35 P38
W42 Y45
aa 87
NEFGMLEKTKEDLRHLFVGSKP-ANEA--ISMTTSMFTLDLRPATAWALYGGSTVVFTPMIKHIVTSQYVTTSLASISAQAGTLFTLPKGIP
NEYGMLERTKTDIRNLFGKSLIEENEV--HIPPFDFFTLDLKPATAWALYGGSTVVLTPLIKHLVTSQYVTTSLASINAQAGSLFTLPKGIP
NEYGMLERTKSDLGKIFSSTRQPQSAL--PLPSMNALALDLRPATAWALYGGSTVVLTPLIKHLVTSEYITTSLASISAQAGSLFNLPRGLP
NEMGWLDKTKSDISSLFGQRIEVKENF--SMGEF---LLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARGFP
NEMGWLDKTKSDISGLFGQRIETKENF--SIGEF---LLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARGFP
NEYGMLEKTKADLKSMFVGKTQASGLT--GLPSM---ALDLRPATAWALYGGSTVVLTPLLKHLITSEYVTTSLASINSQAGSLFVLPRGVP
NEMGLLEKTKSDIAKLFGSQPGSVGFA--IRTTPWDISLDIKPATAWALYAAATMVMTPLIKHLITTQYVNFSLTAIASQAGVLLGLTNGMP
NEMGWLEQTKKDVASLFGR----AHHQ--EPSRWEMPWPDLRPATAWAAYAGATTFLTPLLKHLIVTEYVNFSLMAVTAQAGALFGLGKGMP
NEMGWLETTKKDIGKLFRS---SGDTQ--EQSTWQSWAPEVRAATAWAGYAGLTVFLTPLFRHLITTQYVSFSLTAITAQASALFGLSAGYP
NEMGWLDTTKRDLGKLFSGP--SAVTT--SRWEPLKLALALKPATAWAGYAGMTMLLTPLFRHLITTQYISFSLTAITSQASALFGLNSGYP
NEMGYLEKTKNDIISLWGRSREQN------STLQEWFIMDIKPATAWTLYAVTTTILTPFIQHHITTHYANVSLSAIAAQAGNLFMMKNGHP
NEMGMLERTKQDLAGVFHKTERKS------TEFTLLTPPDLRPATAWSIYAIGTTLITPLIHHMITTHYANFSLMAMANQAGSLFGMQTGAP
NEMGWLEKTKADLSWVVRG------R---SSTTTPVVELDMKPATAWTLYALATTLLTPLFQHLIVTKYANISLMAIASQAGTLFSMDSGIP
NELGWLERTKNDIAHLMGRREEG-------ATMGFSMDIDLRPASAWAIYAALTTLITPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMP
NEMGLLETTKKDLGIGHVAAEN--------QHHATMLDVDLRPASAWTLYAVATTVITPMMRHTIENTTANISLTAIANQAAILMGLDKGWP
NEMGFLEKTKKDLGLG-NIATQ--------QPESNILDIDLRPASAWTLYAVATTFITPMLRHSIENSSVNVSLTAIANQATVLMGLGKGWP
NEMGLLETTKRDLGMS-KEPGV--------VSPTSYLDVDLHPASAWTLYAVATTVITPMLRHTIENSTANVSLAAIANQAVVLMGLDKGWP
NEMGLIEKTKTDFGFY-QVKT-----------ETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLGKGWP
NEAGLLERTKADIRGLLKKEEVNE-----PGWSLPRLELDLKPATTWTLYAVITIILSPFVQHSIITTYNNFSLTAIGNQAGILFGMGTGVP
NELGMLEKTKEDLFGKKNLIPSSASP-------WSWPDLDLKPGAAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMDKGIP
NEMGMLEKTKRDIFG--TTVVEEGKK-------WTFPELDLHPGAAWTVYVGLVTLVTPMLHHWIKVDYGNISLSGITQNAQVLGLMDKGIP
NENGYLEKTKEDLFGRRALNSSNVYAN---LPVEKWLSLDLQPATSWTLYAVIVGVLSPLYHHIEHVNYGAISLQGISQGAAALFQMDKGYP
NENGYLEKTKADIFGHKQMRTMPVNG--------SWMSFDLRPGSAWAVYAFVVGIFSPLYHHAESINYGAISLQGITQSAAAFFQMDKGYP
NELGYLEQTKTDISGLFRREDQGGMV------WDAWTNIDIQPARSWGTYVLIVSLFTPYMLHQLQTKIQRLVNSSVAAGTQAMRDLGGGTP
NELGYLERTKADIAGLFRYDTQGDRV------WDTWTNIDIQPARSWGTYVFIVSLFTPYMLHQLQTKIQRLVNSSVAAGTQAMKDLGGGTP
NEMGMLDKTKADLAGLMWHGEQRHPA------WEEWTNVDIQPARSWGTYVLIVSLFTPYMLHQLQTKIQQLVNSSVASGAQAMRDLGGGTP
NEMGLLEKTKADLAALFARDQGETVR------WGEWTNLDIQPARSWGTYVLVVSLFTPYMLHQLQTRIQQLVNSAVASGAQAMRDLGGGTP
NEMGFLEKTKADLSAVLWSEREEPRV------WSEWTNIDIQPAKSWGTYVLVVSLFTPYIIHQLQTRIQQLVNSAVASGAQAMRDLGGGTP
NEMGFLEKTKADLSTALWSEREEPRP------WSEWTNVDIQPARSWGTYVLVVSLFTPYIIHQLQTKIQQLVNSAVASGAQAMRDLGGGAP
NEMGFLEKTKADLSAMLWSGHEEHRQ------WSEWTNVDIQPARSWGTYVLVVSLFTPYIIHQLQTKIQQLVNSAVASGAQAMRDLGGGAP
NEMGFLERTKKDFREFFRKEVNMDGEP----TQWRIFDLDICPMVSWSLYVLLVTGLRPVCLHGLQMMTQRVVTGAISGRSDLLGQLREGMG
NELRWLENTKEDIKQLFGEKIHMGIS----SGGDFWKYIDLKPLSIWGTYATLVTFMRPQMLHNLRMFTQRIVAGSVSGKLDTLNGLRNGFV
NEMRLLENTKRDIMDLFKRDTTVNESPVFHYTWESLMEWDIRPLTIWATYVVFVTLARPQALHNLKMFTQRVITGTVAGKHDMVNLLPFGAA
WEMRMFPNIRSDLMELVKAVKEPEEVVNSGPSFPSWEIAQ---GKGATMLDSLQVFFFITVLSTKFLYWFQENWTARMYAMKHPEMVSSIGG
WEMRLFPNIRGDIMEMASAMKEPQETQSQASTISGSFFTSRVRGERATMLDSLQVFFFVTVLMNEFIIWVQENWIAQMYVMKHPEMVSTVGG
Fig. 6-3. Multiplication kinetics of recombinant wild-type and N-terminal
132
mutant viruses in monkey kidney Vero cells (MOI of 0.01) at 37°C (panel A) and
41°C (panel B). Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ±
standard deviation of triplicate samples. The P38G virus encodes both P38G and
T116I substitutions.
A.
Vero37
10
9
8
7
6
5
4
3
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1
0
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P38
D35
Titer (log10PFU/mL)/mL)
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96
Y45
W42
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Vero41
9
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P38
D35
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48
Time (hours)
133
72
96
Y45
W42
Fig. 6-4. Multiplication kinetics of recombinant wild-type and N-terminal mutant
viruses in mouse Neuro 2A (panel A) and mosquito C6/36 (panel B) cells at an MOI
of 0.01. Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± standard
deviation of triplicate samples. The P38G virus encodes both P38G and T116I
substitutions.
A.
Neuro2A
Titer (log10PFU/mL)/mL)
9
8
7
6
5
4
3
2
1
0
Time (hours)
Wt
P38
D35
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96
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Y45
W42
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C6/36
9
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P38
D35
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Y45
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Time (hours)
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T
44
C
A
G
C
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1567
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1567
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1461
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C7027G/
T
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Fragm ent
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GFragm ent
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Fragm ent
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0 Hours
G
Fragm ent
JW 3-6640
JW 3-6640
C
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C
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G
Fragm ent
JW 4-8155
TGCT CCAC CCC C TCG GG
CGG TCG AGGG TGG GT TT CCG AAC CAAA AATGT CC AAATA GCGC TTGCG GCCCCTTGTCGGCCGCT GATGGTGCGATA TCCGCCG ATCTCTG CCATT TG
GG CTTA T TT A GA G C AC GTT G C GT AG AG C
A
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592
593
594
595
596
597
598
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599
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601 602
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611
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612
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615
616
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618
619
620
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621
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622
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587 588
590 361
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600 373601374602375 603
604378605379606
358 589
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565497529
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583 584
481 482 483 484 485 486 487 488 489 490 491523492524493 525494526495527496528
498530
499568
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501 533
502 534
96 Hours
T
345
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355 356
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357
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0
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GTT CCCT CCCC CGTC GGCG NCNC TTT ATA CA
C GGG CCT GG
GN ACC GCTT GAC C G
T C G A AA TT AG
GG T
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359
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360590361591
564
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567
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570
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494 495 496 497
135
575
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578
Fragm ent
JW 1-6640
Fragm
ent
JW 1-6640
Fragm ent
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1567
135
T
Figure 6-5. Chromatogram data of sequenced PCR products of NS4B regions amplified from either
parental
at 41°C. The original P38G and
C A P38G/T116I
C T A T Tvirus
C AorCP38G/T116I
A C T C virus
G C incubated
G C G AforG 96G hours
Fragment
1567
T116I
amino acid and nucleotide positions are indicated in addition toJW those
associated with the
1-6640
T C A C C C T C A C C G T T A C G G T A A C
appearance of additional putative Gcompensatory
A G T 1567
G TGsubstitutions.
CC GT GA CG TC CA TG CC CC TG GG C A T T A G G C C T GFragm ent
G G G
579
580
581
582
583
584
585
586
587
530 542
531 543
532 544
533 545
534 535 547
536 537 538
539 540
572
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498 499
500 501
502 503546
504 573
505 574
506 575
507 508
588
541
510
589
542
511
590
543
512
G
591
G
G
592
544
Fig. 6-6. P38G/T116I mutant and wild-type viral RNA levels were assayed in Vero
cells at 37°C and 41°C. Taqman quantitative real-time RTPCR was conducted on
total cellular RNA preparations using primers localizing to the WNV 3’-UTR. Data
was converted to RNA genome equivalents (GEQ) utilizing a standardized curve
and compared to observed plaque titers. Each point is mean ± standard deviation of
triplicate samples. The P38G virus encodes both P38G and T116I substitutions.
A.
9
9
Wt RTPCR
8
P38G RTPCR
8
7
Wt Plaque
7
6
P38G Plaque
6
5
5
4
4
3
3
2
2
1
1
0
0
12
24
48
B.
9
8
9
Wt RTPCR
8
P38G RTPCR
7
P38G Plaque
7
6
Wt Plaque
6
5
5
4
4
3
3
2
2
1
1
0
0
0
12
24
Time (hours)
136
48
RNA (log10GEQ/mL)
Titer (log10PFU/mL)/mL)
0
Figure 6-7. P38G/T116I and wild-type viral protein levels were assayed in Vero
cells at 37°C and 41°C. Viral envelope (E) protein (~50 kD) levels were assayed
from crude cellular lysates, and Western blots were probed with rabbit antidomainIII polyclonal antibody. β-Actin (~45 kD) levels were used as an internal
standard.
.
Vero cells 37˚C
Vero cells 41˚C
Wt
P38G
Wt
P38G
~
~50kD
~
~
~45kD
0
12
24 48
0
12 24 48
0
12
24
48
0
12 24 48
Time (hours)
~
~
~
137
CHAPTER 7
ANALYSIS OF IMMUNE MECHANISMS POTENTIALLY
CONTRIBUTING TO ATTENUATION OF THE NS4B MUTANTS
7.1 Abstract
Site-directed mutagenesis of the West Nile virus NS4B protein resulted in the
identification of two highly attenuated viruses (C102S and P38G/T116I) that also
exhibited a temperature-sensitive phenotype. To better elucidate the mechanism of
attenuation, certain facets of the innate immune response were tested to determine if the
wild-type virus elicited a differential response from that induced by the attenuated
mutants. Mouse neuronal cell lines as well as macrophage and dendritic cell-derived cell
lines were utilized as each of these cell types are hypothesized to serve as critical
components of the natural WNV infection process. Multiplication kinetics and release of
cytokines were assayed in each of these cell lines upon infection with both wild-type and
attenuated viruses. In vivo experiments were also conducted in mice to better understand
the attenuated neuroinvasive phenotype and assay any differences in serum infectivity
titers between wild-type WNV and the NS4B mutants. The attenuated C102S and
P38G/T116I mutants showed decreased serum infectivity titers in mice relative to wildtype virus and failed to reach detectable titers in the brain. In addition, both attenuated
NS4B mutant viruses exhibited altered multiplication kinetics in murine macrophage and
dendritic cell-derived cell lines. Finally, DNA microarray studies led to the identification
of differentially expressed host genes in macrophage cell lines infected with either wildtype WNV or the attenuated C102S mutant virus that could lead to the elucidation of
critical cellular signaling pathways involved in mediating the antiviral response to WNV
infection.
138
7.2 Introduction
Two highly attenuated mutant viruses encoding amino acid substitutions in the
NS4B protein were generated using site-directed mutagenesis. The NS4B C102S and
P38G/T116I viruses exhibit attenuation of the neuroinvasive phenotype in mice and a
temperature-sensitive phenotype in Vero cells at 41°C. The mechanism of attenuation
responsible for the observed phenotypes has not yet been elucidated. It is possible that an
immune-related mechanism is involved, and experiments were undertaken to investigate
this hypothesis.
NS4B proteins from many members of the family Flaviviridae have been found to
be involved in disruption of IFN-signaling pathways. Namba et al. (2004) identified a
common V185F NS4B substitution while generating IFN-resistant HCV replicons by
long-term low dose exposure to IFN. DEN2 NS4B expression in human A549 cells was
found to enhance replication of the IFN-sensitive Newcastle disease virus, and
downregulation of IFN-β stimulated gene expression was observed (Munoz-Jordan et al.,
2003). Further analysis found that the STAT1 activation was partially blocked and that
the N-terminal 125 amino acids of the NS4B protein were sufficient to inhibit IFN
signaling (Munoz-Jordan et al., 2005). In these studies, YFV and WNV NS4B proteins
were also found to inhibit IFN signaling. In contrast, other studies have shown that the
NS5 protein rather than NS4B is responsible for blocking of STAT phosphorylation and
IFN signaling using LGT and JE encephalitis viruses (Best et al., 2005; Lin et al., 2006).
Liu et al. (2005) found that KUNV nonstructural proteins NS2A, NS2B, NS3, NS4A, and
NS4B but not NS1 or NS5 could each inhibit STAT1 and STAT2 activation in HEp2
cells. Thus, the exact role of the NS4B protein in altering the host immune response
remains unclear. STAT homologues are also present in mosquito cells, and infection of
C6/36 cells with JEV resulted in a decrease in STAT phosphorylation (Lin et al., 2004).
Thus, flaviviral manipulation of this signaling pathway is likely conserved in both the
mosquito vector and vertebrate host.
139
The vertebrate host immune response has evolved a wide array of components to
counteract viral infection. Following inoculation via mosquito bite, Langerhans dendritic
cells are thought to mediate the first round of WNV replication (Byrne et al., 2001).
Dendritic cells then migrate to lymph nodes resulting in viremia and infection of
peripheral tissues. A wide array of immune cells including CD8+ T-cell lymphocytes, Bcell lymphocytes, macrophages, and natural killer (NK) are critical in resolving WNV
infection (Samuel et al., 2006). West Nile virus may also reach the central nervous
system (CNS) via one of four routes: either through hematogenous spread, infection of
endothelial cells, direct axonal transport, or by infected immune cells (Solomon et al.,
2002). Once in the CNS, WNV may cause serious clinical symptoms such as neuronal
damage, flaccid paralysis, encephalitis, and ultimately death. Macrophages are known to
serve as important antigen-presenting cells, and depletion of macrophages has been found
to increase the severity of WNV infection in mice (Pisarev et al., 2003 and Ben-Nathan et
al., 1996). Diniz et al. (2006) found that NY99 WNV infection led to the rapid
destruction of neurons through apoptosis while astrocytes were permissive to persistent
infection suggesting that these cells could facilitate maintenance of WNV in the CNS.
Cellular signaling pathways are also known to be critical in mediating the antiviral
response. Toll-like receptor 3 (TLR3) is known to be involved in the recognition of viral
double-stranded RNA intermediates and mediates an antiviral response. Evidence also
suggests that TLR3 may be involved in disrupting the blood-brain barrier by inducing an
inflammatory response. In fact, TLR3-deficient mice exhibited increased WNV viral
load in peripheral tissues but showed a decreased viral load in the brain as well as
decreased neuropathology (Wang et al., 2004). Highly structured flaviviral RNA is also
involved in activating TLR7 signaling pathways in myeloid dendritic cells (Wang et al.,
2006). Retinoic acid-inducible gene 1 (RIG-1) is another early sensor of viral infection,
and WNV has evolved methods of delaying the host immune response mediated by this
pathway (Fredericksen et al., 2006). It is conceivable that a reduced capability of
modulating the host immune response could lead to the attenuation of mutant WNV
strains.
140
For the purposes of this study, the attenuated NS4B C102S and P38G/T116I
mutant viruses were compared to wild-type WNV by investigating multiplication in mice
as well as in neuronal, macrophage, and dendritic cell lines. In addition, the importance
of the TLR3 and RIG-1 signaling pathways in WNV infection were investigated using a
panel of human hepatocyte cell lines with variable expression phenotypes. Finally,
cytokine release and gene expression were assayed in response to WNV infection
utilizing specific cell lines. The attenuated NS4B mutants exhibited a set of unique
characteristics including reduced multiplication in mice, macrophages, and dendritic
cells. Also, alterations in gene expression were noted in response to infection by either
wild-type or C102S mutant WNV that could lead to elucidation of the molecular
determinants of attenuation.
7.3 Results
7.3.1 Multiplication of attenuated NS4B mutants in mice
Multiplication kinetics were compared in mice following intraperitoneal
inoculation with 100 pfu of either wild-type WNV, C102S mutant, or P38G/T116I
mutant in the NIH Swiss mouse model to determine serum and brain infectivity titers for
these viruses. The peak serum infectivity titer for wild-type WNV occurred on day 2
post-inoculation and reached 5.2 log10 pfu/mL (Table 7-1). Peak titers for the attenuated
C102S and P38G/T116I mutant viruses were 100-fold lower but also occurred on day 2
post-inoculation. Peak brain titers reached 6.8 log10 pfu/brain for the wild-type virus on
day 6 post-infection while virus infection was never detected in the brains of C102S or
P38G/T116I virus-infected mice. The limit of detection was 1.7 log10 pfu/mL for serum
and 2.7 log10 pfu/brain for the brain. To detect low levels of virus, RT-PCR amplification
was conducted on RNA isolated from mouse brain on day 6, when high titers of wildtype WNV were detected in the brain. No PCR product was detected for WNV RNA
from brains of mice infected with C102S or P38G/T116I virus while the brains of mice
141
infected with wild-type virus were positive for viral RNA. Thus while both attenuated
C102S and P38G/T116I viruses exhibited significant serum titers, there is no evidence
the virus ever reached the brain.
7.3.2 Multiplication kinetics in relevant murine cell types
Viral multiplication was assayed for wild-type WNV and the attenuated C102S
and P38G/T116I viruses using mouse neuronal Neuro2A, macrophage P388.D1, and
dendritic DC2.4 cell lines as these cell types are thought to be important in the course of
natural infection. Neuro2A cells were derived from strain A albino mice while P388.D1
and DC2.4 cells were derived from inbred BALB/c and C57BL/6 mice respectively.
Each cell type was infected with wild-type virus as well as the attenuated mutant viruses
at a moi of 0.1. C102S and P38G/T116I viruses multiplication kinetics were
indistinguishable from wild-type WNV in Neuro2A cells (Fig. 7-1A). In contrast, both
attenuated NS4B mutants exhibited a decrease in titer in macrophage P388.D1 cells
following the 48-hour timepoint (Fig. 7-1B). The C102S mutant infectivity titers
dropped to near the limit of detection (0.7 log10 pfu/mL) by 96 hours while the
P38G/T116I virus exhibited a less drastic but still noticeable decline (approximately 50fold) in titer compared to wild-type WNV. After infection of dendritic cell-derived
DC2.4 cells at an moi of 0.1, wild-type WNV as well as the attenuated mutants exhibited
marked decreases in titers following the 24-hour timepoint (Fig. 7-2A). At an moi of 5,
wild-type WNV maintained high titers in DC2.4 cells throughout the course of the
experiment while the attenuated C102S and P38G/T116I viruses again exhibited a
decline in titer following 12 hours (Fig. 7-2B). Overall, in both P388.D1 and DC2.4
cells, the C102S mutant exhibited the most attenuated phenotype in terms of decline in
infectivity titers while the P38G/T116I viruse expressed an intermediate phenotype.
7.3.3 Comparison of the neuroinvasive phenotype in both inbred and outbred mice
Both C102S and P38G/T116I viruses were previously found to exhibit an
attenuated neuroinvasive phenotype in female 3-4 week-old NIH Swiss mice. In
142
addition, both attenuated mutants exhibited altered multiplication kinetics in BALB/cderived P388.D1 cells and C57BL/6-derived DC2.4 cells. To test for conservation of the
attenuated neuroinvasive phenotype in inbred strains compared to outbred NIH Swiss
mice, the attenuated NS4B C102S and P38G/T116I mutants were inoculated into female
3-4 week old NIH Swiss, BALB/c, and C57BL/6 mice via the intraperitoneal route.
Wild-type WNV was found to exhibit comparable LD50 values and average survival
times in each mouse strain tested (Table 7-2). The C102S and P38G/T116I viruses were
found to be highly attenuated in each tested mouse strain with ip LD50 values greater than
10,000,000 pfu. However, both viruses were capable of inducing a protective immune
response against a subsequent challenge of wild-type WNV in each tested mouse strain
with PD50 values of less than 1 pfu. Thus, no difference was detected with respect to the
neuroinvasive phenotype of WNV in inbred BALB/c or C57BL/6 mice as compared to
the outbred NIH Swiss strain, and multiplication differences of the mutant viruses in the
BALB/c-derived P388.D1 cells and C57BL/6-derived DC2.4 cells were not due to the
genetic background of the host.
7.3.4 Multiplication kinetics in human hepatocyte-derived cell lines expressing
varying RIG-1 and TLR3 phenotypes
RIG-I and TLR3 signaling pathways are known to be critical mediators of the
antiviral immune response. Human hepatocyte-derived cell lines with differential RIG-I
and TLR3 phenotypes were obtained to determine if wild-type WNV as well as the
attenuated NS4B C102S and P38G/T116I mutant viruses exhibited altered multiplication
phenotypes in the different cell lines. Huh7 cells express RIG-I but not TLR3 while the
Huh7 Ft3.7 cells express both RIG-I and TLR3. Huh 7.5 cells do not express either RIGI or TLR3 while Huh7.5 iTLR3.16 cells express TLR3 but not RIG-I. In addition, Tantigen immortalized non-neoplastic PH5CH8 cells express high levels of both RIG-I and
TLR3. Each of the five cell lines was infected at a moi of 5 with wild-type WNV as well
as the attenuated C102S and P38G/T116I mutants, and differences in viral multiplication
143
kinetics were assayed. Peak wild-type WNV titers were approximately 3-fold higher
than either of the attenuated NS4B mutants in Huh 7 (Fig. 7-3A), Huh7 Ft3.7 (Fig. 7-3B),
and Huh7.5 iTLR3.16 (Fig. 7-4B) cells. No differences in titers were observed in Huh
7.5 cells that do not express either TLR3 or RIG-I (Fig. 7-4A). In contrast, a marked
difference in peak viral titer was observed in PH5CH8 cells (Fig. 7-5). The C102S
mutant exhibited greater than a 10-fold decrease in peak titer while the P38G/T116I
mutant exhibited greater than a 100-fold decrease in peak titer compared to wild-type
WNV.
7.3.5 Cytokine responses in West Nile virus-infected cells
Differences in cytokine expression in West Nile virus-infected versus uninfected
cells were assayed utilizing a Raybiotech antibody array membrane capable of detecting
levels of 62 different murine cytokines. Mouse neuronal Neuro2A, macrophage
P388.D1, and dendritic cell-derived DC2.4 cells were infected at an moi of 5 with wildtype WNV, and cytokine expression was determined for infected and uninfected cell
supernatants after 24 hours. Membranes were subjected to densitometry analysis to
account for variations in background intensity. Both macrophage P388.D1 and dendritic
DC2.4 cells expressed high levels of macrophage inflammatory protein (MIP), while
neuronal Neuro2A cells did not in both virus-infected and uninfected cells. Dendritic
DC2.4 cells were found to express high levels of IL-6 while P388 D1 cells did not (Fig.
7-6). Thus, each of the different cell types exhibited a unique and reproducible cytokine
expression pattern. However, no reproducible significant differences in cytokine
expression were observed in WNV-infected versus uninfected cells.
7.3.6 Gene expression in P388.D1 cells infected with either wild-type or NS4B
C102S mutant West Nile virus
The C102S virus was found to exhibit altered multiplication kinetics in P388.D1
cells as compared to wild-type WNV (Fig. 7-1B). DNA microarrays were utilized to
compare gene expression in mouse macrophage P388.D1 cells infected with either wild144
type or the attenuated NS4B C102S mutant WNV. Differences in the antiviral response
related to IFN-induced signaling pathways were hypothesized to serve as the primary
mechanism for the observed alterations in viral titers. P388.D1 cells were infected at an
moi of 5 with either wild-type WNV or the C102S mutant. RNA was isolated from cell
lysates 12 hours post-infection and gene expression was analyzed. Viral titers at the 12hour timepoint were 12,000,000 pfu/mL for wild-type WNV and 1,600,000 pfu/mL for
the C102S mutant. Genes that exhibited ≥ 2-fold change in expression between the wildtype and C102S virus-infected cells and a p-value of less than 0.001 were selected for
additional analysis utilizing the Ingenuity algorithm. A total of 73 genes were found to
be differentially expressed, and each of these genes were found to be upregulated in wildtype WNV-infected cells compared to C102S mutant-infected cells (Table 7-3).
Upregulated classes of genes primarily included those related to immunity, apoptosis, or
cell signaling. Seven genes were identified that were upregulated more than 3-fold in
response to wild-type infection as compared to C102S mutant infection. These include
interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), regulator of G-protein
signaling 3 (RGS3), viperin (RSAD2). The poorly characterized bolA-like 1 protein
exhibited 5.2-fold higher levels in wild-type virus-infected as compared to C102S
mutant-infected cells.
7.4 Discussion
Both attenuated NS4B mutants exhibited a series of phenotypic characteristics
distinct from those observed with the virulent wild-type NY99 strain of WNV. The
C102S and P38G/T116I viruses exhibited temperature-sensitive phenotypes in Vero cells
as well as attenuation of neuroinvasiveness in mice. Further studies showed that serum
viremias from C102S and P38G/T116I virus-infected mice demonstrated that both
viruses multiplied, but at lower levels than observed for the wild-type virus. In contrast,
neither detectable infectious virus nor viral RNA were detected in the brains of the NS4B
145
mutant-infected mice. Wild-type WNV was detected in the brain by day 4 postinoculation. Viremias associated with the attenuated C102S and P38G/T116I NS4B
mutants were different than those observed for the previously described attenuated
NY99/E154 mutant (Beasley et al., 2005). The NY99/E154 virus contains a mutation
that disrupted the glycosylation site in the envelope protein and exhibited significant
attenuation of the mouse neuroinvasive phenotype. This virus never reached detectable
levels in the serum (greater than 1.4 log10 pfu/mL) but exhibited an LD50 value of 126
pfu. In contrast, the NS4B C102S virus reached a peak titer of 3.4 log10 pfu/mL but still
had an LD50 value of greater than 10,000 pfu. These data indicate that the mechanism of
attenuation associated with the NS4B amino acid substitutions is distinct from that
associated with disruption of envelope protein glycosylation. It is hypothesized that
neither NS4B mutant virus was capable of reaching sufficiently high titers to cross the
blood-brain barrier.
To better understand the attenuated phenotypes of the NS4B mutants, the
multiplication kinetics of the C102S and P38G/T116I viruses were compared to those of
wild-type WNV in a set of relevant cell types thought to be important in the course of
natural infection. Following inoculation of a host by an infected mosquito, the first round
of replication is thought to occur in Langerhans dendritic cells (Byrne et al., 2001). In
addition, macrophages are known to serve as an important component of the antiviral
immune response involved in viral clearance (Ben-Nathan et al., 1996). Finally, WNV
effects many of its serious clinical correlates such as paralysis, meningitis, and
encephalitis by invading neuronal CNS cells either through hematogenous spread,
peripheral neurons, or infected immune cells (Diamond et al., 2003, Garcia-Tapia et al.,
2006, and Solomon et al., 2002). Accordingly, mouse neuronal Neuro2A cells,
macrophage P388.D1 cells, and dendritic cell-derived DC2.4 cells were used to compare
the multiplication kinetics of virulent wild-type WNV with those of the attenuated NS4B
mutants. It was found that both C102S and P38G/T116I viruses exhibited altered
multiplication kinetics in both P388.D1 and DC2.4 cells (Figs. 7-1 and 7-2). Wild-type
WNV was capable of maintaining relatively steady infectivity titers through 96 hours in
146
both P388.D1 and DC2.4 cells. Peak titers were much lower in these cell lines (5-6 log10
pfu/mL) as compared to other cell lines such as Neuro2A cells (7-8 log10 pfu/mL). Both
attenuated mutants exhibited a significant decrease in titer following 48 hours in P388 D1
cells and 24 hours in DC2.4 cells. At 96 hours C102S infectivity titers had decreased to
the limit of detection (0.7 log 10 pfu/mL) in both cell lines while the P38G/T116I virus
exhibited a less drastic decrease in titer. In contrast, the NS4B mutants multiplied
comparably to wild-type WNV in neuronal Neuro2A cells. The inability of the NS4B
mutant viruses to infect macrophage and dendritic cell lines effectively could explain the
attenuation of neuroinvasiveness observed in the mouse model as infected immune cells
are proposed to be one mechanism whereby transport of WNV into the CNS occurs
(Garcia-Tapia et al., 2006). Shirato et al. (2006) found that both E-protein glycosylated
and nonglycosylated variants of NY99 WNV reached peak titers at 18 hours postinfection in peritoneal macrophages isolated from BALB/c mice before dropping down to
undetectable titers by 72 hours. DC2.4 cells were established using dendritic cells from
the bone marrow of C57BL/6 mice (Shen et al., 1997). Wild-type WNV was capable of
maintaining steady infectivity titers in DC2.4 cells at an moi of 5 but not at an moi of 0.1
(Fig. 7-2 A and B). Infectivity titers of both C102S and P38G/T116I mutant viruses
declined to nearly undetectable levels by 48 hours and 96 hours respectively. In
summary, it appears that a decreased ability of the attenuated NS4B mutant viruses to
productively infect macrophages and dendritic cells could partially mediate the
attenuation of neuroinvasiveness observed in mice.
Following observation of the alteration of multiplication kinetics with the NS4B
mutants in P388.D1 and DC2.4 cells, the neuroinvasive phenotype was investigated using
WNV in the parental mouse strains of these cell lines. P388D.1 cells were derived using
BALB/c mice while the DC2.4 cells were obtained from C57BL/6 mice. Both BALB/c
and C57BL/6 mice are inbred strains while the NIH Swiss mouse model is an outbred
strain. The attenuated NS4B mutant viruses and wild-type WNV were tested for
neuroinvasiveness in 3-4 week-old females from each strain of mice. Both LD50 and
average survival times were comparable for wild-type WNV in each mouse strain (Table
147
7-2). In addition, the attenuated NS4B mutants exhibited LD50 values of greater than
10,000,000 in each strain. Both C102S and P38G/T116I were capable of inducing a
protective immune response against a lethal challenge of wild-type WNV in all mice
tested with PD50 values of less than 1 pfu. Thus, the outbred NIH Swiss strain is
comparable to the inbred BALB/c and C57BL/6 strains with respect to WNV
neuroinvasiveness.
Both TLR3 and RIG-1 mediated signaling pathways are known to be important
modulators of the antiviral immune response. RIG-1 null mouse embryo fibroblast
(MEF) cells exhibited a delayed response to WNV infection compared to Wt MEF cells
(Fredericksen et al., 2006). TLR3 is known to mediate WNV recognition in macrophages
and dendritic cells but may also lead to disruption of the blood-brain barrier resulting in
lethal encephalitis (Wang et al., 2004). A set of human hepatocyte cell lines with varying
TLR3 and RIG-1 phenotypes was used to elucidate the potential importance of these
signaling pathways with respect to the attenuated NS4B mutants. Huh7 cells express
RIG-I but not TLR3 while the Huh7 Ft3.7 cells express both RIG-I and TLR3. Huh 7.5
cells do not express either RIG-I or TLR3 while Huh7.5 iTLR3.16 cells express TLR3
but not RIG-I. In addition, T-antigen immortalized non-neoplastic PH5CH8 cells express
high levels of both RIG-I and TLR3 (Li et al., 2005). Although the attenuated C102S and
P38G/T116I viruses exhibited an approximate 3-fold decrease of infectivity titer in Huh7,
Huh7 Ft3.7, and Huh7.5 iTLR3.16 cells compared to wild-type WNV, the biological
significance of such slight decreases is doubtful (Figs. 7-3 and 7-4). Both C102S and
P38G/T116I mutant viruses exhibited alterations in multiplication kinetics using the
PH5CH8 cells with 10-fold and 100-fold decreases in peak titer respectively compared to
wild-type WNV (Fig. 7-5). Wild-type WNV grew equally well in Huh7, Huh7 Ft3.7,
Huh7.5, and Huh7.5 iTLR3.16 cell lines indicating the expression profile of RIG-1 and
TLR3 in human hepatocytes by itself does not significantly affect WNV multiplication
kinetics. Wild-type WNV did exhibit a 100-fold decrease in peak titer in the PH5CH8
cells compared to the other hepatocyte cell lines. The PH5CH8 cell line is a nonneoplastic hepatocyte cell line transformed with large T antigen and is quite distinct from
148
the Huh7- and Huh7.5-derived cell lines. PH5CH8 cells are known to respond
vigorously to poly(I-C) and can mediate an effective antiviral response (Li et al., 2005).
Thus, the mutant C102S and P38G/T116I mutants exhibited slight but noticeable
decreases in multiplication kinetics using Huh7, Huh7 Ft3.7, and Huh7.5 TLR3.16 cells
compared to wild-type WNV suggesting that these viruses are somewhat less capable of
modulating TLR3 and RIG-1 signaling pathways. Wild-type and NS4B mutant viruses
all exhibited a decrease in multiplication kinetics in PH5CH8 cells compared to the other
hepatocyte cell lines although the decrease was much more pronounced in the C102S
mutant and especially the P38G/T116I mutant. Robust TLR3 and RIG-1 signaling is
likely at least partially responsible for induction of an antiviral state in response to WNV
infection in these cells.
Expression of cytokines and chemokines is also known to be a critical component
of the antiviral immune response. TNFα and IL-1β were found to be up-regulated in
murine peritoneal macrophages (Shirato et al., 2006). RANTES, MIP-1α, MIP-1β, and
IP-10 were also found to be highly up-regulated in the brain of NY99 WNV-infected
mice at 7 days post-inoculation (Shirato et al., 2004). Utilizing cytokine array detection
membranes, levels of 62 cytokines were assayed in either WNV-infected or uninfected
mouse neuronal Neuro2A, macrophage P388.D1, and dendritic cell-derived DC2.4 cells.
Each cell line exhibited a unique and reproducible pattern of cytokine expression,
however infection with WNV did not lead to differences in cytokine expression with
respect to uninfected cells at either 24 or 72 hours post-infection (Fig. 7-6). Both
P388.D1 cells and DC2.4 cells expressed high levels of (macrophage inflammatory
protein) MIP cytokines as well as monocyte chemotactic protein-1 (MCP1). DC2.4 cells
expressed high levels of IL-6 while P388.D1 cells did not. As expected, Neuro2A cells
did not express MIP but did express relatively high levels of IL-6 and eotaxin-2. It is
possible that WNV infection does not induce sufficient cytokine release to be detected
using the bioarray membranes. In addition, the very process of maintaining these cell
lines could result in their activation leading to a masking effect of WNV-induced
cytokine expression. More sensitive assays such as ELISA or quantitative RT-PCR may
149
be necessary to elucidate the importance of individual cytokines in response to WNV
infection. It may also be advantageous to conduct these experiments in primary cells
instead of established cell lines.
Upon infection with WNV, distinct alterations in gene expression are known to
occur within cells (Koh et al., 2005). Utilizing DNA microarrays, differences in gene
expression were assayed utilizing the macrophage P388.D1 cell line infected with either
virulent wild-type WNV or the attenuated C102S mutant at an moi of 5. P388.D1 cells
were selected for this experiment since the C102S mutant was found to be incapable of
producing detectable infectivity titers in these cells for more than 72 hours while wildtype WNV maintained steady titers throughout the course of the experiment (Figure 71A). Genes that exhibited ≥ 2-fold change in expression between the wild-type and
C102S virus-infected cells were selected for further analysis (Table 7-3). Interestingly,
all selected genes exhibited up-regulation in wild-type virus cells in comparison to C102S
virus-infected cells. Many identified genes were involved in immunity, apoptosis, and
cell-signaling pathways. In addition, several identified genes in this study correspond to
genes identified when comparing mice infected with different WNV strains of varying
neurovirulence (Venter et al., 2005). For example, ubiquitin specific peptidase 18
(Usp18) was found to be expressed at higher levels (2.3-fold) in wild-type virus-infected
cells than in C102S mutant-infected cells. Similar results were observed in mouse liver
where Usp18 was up-regulated 4-fold in mice infected with highly neuroinvasive strains
of WNV compared to mice infected with WNV strains of lower neuroinvasiveness
(Venter et al., 2005). In fact, Usp18 is a negative regulator of IFN-signaling through the
Jak-Stat pathway (Malakhova et al., 2003). Thus, up-regulation of Usp18 could serve as
one way that wild-type WNV successfully modulates the immune response. Interferon
regulatory factor 7 (Irf7), tripartite motif protein 30 (Trim30), interferon-induced protein
with tetraticopeptide repeats 1 (Ifit1), Sec61 alpha 1 subunit 2 (Sec61a2), GTP binding
protein 1 (Gtbbp1), and histocompatibility 2, T region locus 24 (H2-T24) also represent
genes that were found to be differentially expressed in both this experiment and
previously in mice (Venter et al., 2005). The bolA-like protein 1 (Bola1) was found to be
150
most differentially expressed with 5.2-fold higher expression in wild-type virus-infected
cells compared to C102S-infected cells. BolA-like proteins are highly conserved in all
eukaryotes ranging from bacteria to vertebrates and are thought to bind nucleic acids and
serve as transcriptional regulators (Kasai et al., 2003). Eukaryotic translation initiation
factor 4, gamma 1 (Eif4g1) was also found to be differentially up-regulated in wild-type
virus-infected cells by 2-fold. Recently, it was shown that when cap-dependent
translation was inhibited, DENV could carry out translation by directly interacting with
eukaryotic initiation factors eIF4G and eIF4A through the 3’-UTR (Edgil et al., 2006).
Another identified gene coded for the eukaryotic translation initiation factor 2-alpha
kinase 2 (Eif2ak2). Eif2ak2 is involved in modulating activity of Eif2a that is a
eukaryotic initiation factor that mediates binding of initiator methionyl-tRNA to the 40S
ribosomal subunit (Zoll et al., 2002). Eif2a is also involved in regulating genes
responsible for actin organization in the cellular cytoskeleton and other morphogenic
processes (Komar et al., 2005). The identified differentially expressed capping protein
muscle Z-line beta (CapZb) gene is also involved in mediating actin organization
(Littlefield et al., 1998). It thus appears that wild-type WNV may be more capable of
modulating cellular translation machinery for optimal viral output than the attenuated
C102S mutant (Chapter 5).
A variety of genes involved in the IFN-signaling pathways were identified as
being differentially upregulated in wild-type virus-infected cells compared to C102Sinfected cells. Irf7, Ifit1, Ifi202b, Isg20, and Ifi44 genes were each identified as being
up-regulated in wild-type WNV-infected cells. Rgs3 and Gtbbp1 proteins were also
preferentially up-regulated, and these proteins are involved in regulating the cellular
GTP-signaling pathways. Pro-apoptotic caspase-3 (Casp3) was found to be expressed at
higher levels in wild-type WNV-infected cells suggesting the importance of apoptotic
processes during infection. The Rsad2 protein, also known as viperin/cig5, was found to
be up-regulated in wild-type-infected cells as well. Viperin is known to be a critical
antiviral protein and acts within the TLR3-signaling pathway. DNA microarray
experiments using polyriboinosinic polyribocytidylic acid (pIC)- stimulated human
151
astrocytes found that viperin was one of the most highly induced proteins in the TLR3
signaling network (Rivieccio et al., 2006). In this experiment, RNAi molecules directed
against viperin were also found to significantly reverse pIC-induced inhibition of HIV-1
replication suggesting viperin is a major modulator of the cellular antiviral response.
DNA microarray analysis led to the identification of a variety of genes that may be
critical mediators of WNV infection. Additional methods such as quantitative RT-PCR
will be utilized to confirm the significance of genes identified in this assay. It was
somewhat unexpected that all identified genes were up-regulated in wild-type virusinfected cells in comparison to C102S-infected cells. However, previous experiments in
mouse brain, spleen, and liver also showed that the vast majority of identified genes
exhibited increased expression in mice infected with highly neuroinvasive WNV strains
compared to mice infected with less neuroinvasive strains (Venter et al., 2005). It is
possible that attenuated WNV strains are less able to regulate cellular components
leading to overall disorganization and decreased gene expression overall. Further
experiments will be necessary to elucidate the exact function of identified genes in
mediating viral infection in host cells.
The attenuated C102S and P38G/T116I NS4B mutant viruses were both found to
exhibit a set of phenotypes distinct from that observed with wild-type WNV. The NS4B
mutants both exhibited reduced mouse serum titers compared to wild-type WNV and
were never observed in the brain. In addition, the NS4B mutant viruses were unable to
maintain infection in mouse macrophage P388 cells or dendritic DC2.4 cells in contrast
to wild-type WNV that maintained steady titers throughout the course of the experiment.
Expression of TLR3 and RIG-1 in human hepatocyte cell lines showed that these
signaling pathways led to a slight decrease in NS4B mutant multiplication kinetics as
compared to wild-type WNV virus. This difference was more pronounced in the nonneoplastic PH5CH8 cell line. Finally, DNA microarray experiments resulted in the
identification of differentially regulated genes that may be involved mediating infection
by virulent or attenuated WNV strains. Several potential mechanisms of attenuation have
thus been identified that may be responsible for the observed alterations in phenotype of
152
the NS4B mutants. The observed reduction in serum titers with respect to the attenuated
NS4B mutants would be expected to reduce direct hematogenous seeding of the CNS
resulting in attenuated neuroivasiveness. Also, the inability of the NS4B mutants to
persistently infect macrophages could lead to a decrease in crossing the blood-brain
barrier as infected immune cells are thought to be involved in carrying WNV into the
CNS. In addition, the attenuated mutants appear to be less capable of modulating cellular
antiviral responses in general shown both by the relative decrease in titer in response to
TLR3 and RIG-1 expression and the alterations in gene expression in macrophage P388
cells. Thus, attenuation of mouse neuroinvasiveness with the NS4B mutant viruses is
likely multifactorial with reductions in titer as well as modulation of host signaling
pathways each contributing to observed alterations in viral phenotype.
153
Table 7-1. Viremia and brain titers from mice inoculated with 100 pfu of either
wild-type WNV, the C102S mutant, or the P38G/T116I mutant virus via the
intraperitoneal route
a
No virus detected. The limits of detection were 1.7 log10 pfu/mL in the serum and 2.7
log10 pfu/brain.
NY99 ip Titer
Day
1
2
3
4
5
Mouse
Serum
Number (log10PFU/mL)
P38G/T116I ip Titer
Brain
Serum
Brain
Serum
Brain
(log10PFU/ Brain)
(log10PFU/mL)
(log10PFU/ Brain)
(log10PFU/mL)
(log10PFU/ Brain)
a
1
3.0
-
2.5
-
2.5
-
2
4.4
-
2.0
-
2.7
-
3
4.8
-
1.9
-
2.2
-
1
4.4
-
2.5
-
2.9
-
2
5.2
-
3.0
-
3.3
-
3
5.4
-
3.4
-
3.2
-
1
2.7
-
1.7
-
1.7
-
2
2.2
-
2.8
-
2.4
-
3
2.6
-
3.3
-
2.8
-
1
-
4.6
-
-
-
-
2
2.2
4.0
1.7
-
2.0
-
3
2.1
4.3
-
-
-
-
1
-
4.9
-
-
-
-
2
-
6.0
-
-
-
-
4.5
-
-
-
-
3
6
C102S ip Titer
1
-
6.3
-
-
-
-
2
-
6.8
-
-
-
-
3
-
7.4
-
-
-
-
154
Table 7-2. Virulence phenotypes of wild-type WNV and the attenuated NS4B
mutants in different mouse strains
a
Infectious clone-derived wild-type WNV, strain NY99 and NS4B mutants encoding
either C102S or P38G/T116I substitutions
b
Each virus was tested in 3-4 week-old female outbred NIH Swiss as well as inbred
BALB/c and C57BL/6 mouse strains
c
Median lethal viral dose (LD50) following intraperitoneal (ip) inoculation of virus
d
AST = average survival time
e
Median protective viral dose (PD50) against a 100 pfu intraperitoneal challenge of wild-
type NY99 virus
Virusa
Wt
C102S
P38G/T116I
Mouse Strainb
i.p. LD50c
i.p. ASTd
i.p. PD50e
(pfu)
(days ± SD)
(pfu)
NIH Swiss
0.7
7.4±0.4
n.d.
BALB/c
0.5
8.6±1.8
n.d.
C57BL/6
0.5
8.8±1.8
n.d.
NIH Swiss
>10,000,000
>35
0.7
BALB/c
>10,000,000
>35
0.5
C57BL/6
>10,000,000
>35
0.4
NIH Swiss
>10,000,000
>35
0.7
BALB/c
>10,000,000
>35
0.7
C57BL/6
>10,000,000
>35
0.4
155
Table 7-3. Differentially expressed genes in mouse macrophage P388.D1 cells
infected with either wild-type NY99 West Nile virus or the attenuated NS4B C102S
mutant virus at 12 hours post-infection (moi = 5)
a
Genes selected for additional analysis exhibited ≥ 2-fold change in expression in wild-
type WNV-infected cells compared to C102S virus-infected cells as determined by
Ingenuity analysis (p < 0.001)
Gene
Gene name
Usp18
Cdc23
Sirpa
Rgs3
Map2k3
Irf7
Trim30
H2-T24
Ifit1
Ifi202b
Isg20
Ifi44
Pik3cd
Bola1
CapZb
Ptdss2
Sec61a1
Eif2ak2
Casp3
Ubtd1
Eif4g1
Rsad2
Phip
Slc31a2
Gtbbp1
Vps37b
Psap
Ldlr
Bcat1
Pmm2
Baz1b
Polr3c
Rabac1
Kpnb1
Mbd3
Got2
LOC675851
Ubiquitin specific peptidase 18
CDC23 (cell division cycle 23, yeast, homolog)
Signal-regulatory protein alpha
Regulator of G-protein signaling 3
Mitogen activated protein kinase kinase 3
Interferon regulatory factor 7
Tripartite motif protein 30
Histocompatibility 2, T region locus 24
Interferon-induced protein with tetraticopeptide repeats 1
Interferon activated gene 202B
Interferon stimulated protein 20
Interferon-induced protein 44
Phosphatidylinositol 3-kinase catalytic delta polypeptide
bolA-like 1 (E. coli)
Capping protein (actin filament) muscle Z-line, beta
Phosphatidylserine synthase 2
Sec61 alpha 1 subunit
Eukaryotic translation initiation factor 2-alpha kinase 2
Caspase 3
Ubiquitin domain containing 1
Eukaryotic translation initiation factor 4, gamma 1
Radical S-adenosyl methionine domain containing 2, Viperin/cig5
Pleckstrin homology domain interacting protein
Solute carrier family 35 (UDP-galactose transporter), member 2
GTP binding protein 1
Vacuolar protein sorting 37B (yeast)
prosaposin
Low density lipoprotein receptor
Branched chain aminotransferase 1
Phosphomannomutase 2
Bromodomain adjacent to zinc finger domain, 1B
Polymerase (RNA) III (DNA directed) polypeptide C
Rab acceptor 1 (prenylated)
Karyopherin (importin) beta 1
Methyl-CpG binding domain protein 3
Glutamate oxaloacetate transaminase 2, mitochondrial
Similar to NADH dehydrogenase (ubiquinone) 1
156
Fold change induced by
wild-type WNV vs. the
attenuated C102S mutanta
2.3
2.1
2.8
3.1
2.4
2.3
2.0
2.5
3.4
2.2
2.2
2.0
2.5
5.2
3.7
3.3
3.6
2.5
2.4
2.4
2.0
3.7
2.0
2.1
2.2
2.1
2.2
2.0
2.0
2.7
3.3
2.9
2.4
2.7
2.1
2.8
2.0
Table 7-3 (continued). Differentially expressed genes in mouse macrophage
P388.D1 cells infected with either wild-type NY99 West Nile virus or the attenuated
NS4B C102S mutant virus at 12 hours post-infection (moi = 5)
a
Genes selected for additional analysis exhibited ≥ 2-fold change in expression in wild-
type WNV-infected cells compared to C102S virus-infected cells as determined by
Ingenuity analysis (p < 0.001)
Gene
Gene name
Add1
Arhgef2
Sfrs2
Scd2
Pip5k2c
Fosl1
Pcnxl3
Srf
Rtp4
Stom
Qscn6
Tmed2
Arf1
Hcfc1
Zfp313
Dhx9
Pitpnm1
P4ha1
Zfand3
Prkar2a
Cops7a
Nubp1
Smap1
Prmt3
Sbf1
Gpaa1
Parp14
Plag12a
Htf9c
Atp13a2LOC67
BC022687
BC057552
130018I05Rik
9430098E02Rik
1110007C09Rik
9530020G05Rik
Adducin 1 (alpha)
Rho/rac guanine nucleotide exchange factor (GEF) 2
Splicing factor, arginine-serine-rich 2
Stearoyl-coenzyme A desaturase
Phosphatidylinositol-4-phosphate 5-kinase, type III, gamma
Fos-like antigen 1
Pecanex-like 3
Serum response factor
Receptor transporter protein 4
Stomatin
Quiescin Q6
Transmembrane emp24 domain trafficking protein 2
ADP ribosylation factor 1
Host cell factor 1
Zinc finger protein 313
DEAH (Asp-Glu-Ala-His) box polypeptide 9
Phosphatidylinositol membrane-associated 1
Procollagen proline-4-hydroxlase, alpha 1 polypeptide
Zinc finger AN1-type domain 3
Protein kinase, cAMP dependent regulatory, type II alpha
COP9 (constitutive photomorphogenic) homolog, subunit 7a
Nucleotide binding protein 1
Stromal membrane-associated protein 1
Protein arginine-N-methyltransferase 3
SET binding factor 1
GPI anchor attachment protein 1
Poly (ADP-ribose) polymerase family, member 14
Phospholipase A2, group XIIA
Hpall tiny fragments locus 9c
Similar to ATPase type 13 2A
cDNA sequence BC022687
cDNA sequence BC057552
RIKEN cDNA 1300018I05
RIKEN cDNA 9430098E02
RIKEN cDNA 1110007C09
RIKEN cDNA 9530020G05
157
Fold change induced by
wild-type WNV vs. the
attenuated C102S mutanta
2.4
2.4
2.2
2.5
2.1
2.0
2.5
2.0
2.1
2.4
2.2
2.0
2.4
2.1
2.3
2.3
2.1
2.0
2.1
2.0
2.0
2.4
2.0
2.0
2.4
2.1
2.0
2.1
2.2
2.1
2.3
2.2
2.0
2.3
2.0
2.3
Fig. 7-1. Multiplication kinetics of recombinant wild-type and mutant viruses in
mouse neuronal Neuro2A cells (panel A) or macrophage P388 cells (panel B) at an
moi of 0.1. Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of
triplicate samples.
A.
Neuro2A
9
8
7
6
5
4
3
Wt
2
C102S
1
P38G/T116I
Titer (log10PFU/mL)/mL)
0
0
12
24
48
72
96
B.
P388 cells
6
5
4
3
2
Wt
1
P38G/T116I
C102S
0
0
12
24
Time (hours)
158
48
72
96
Fig. 7-2. Multiplication kinetics of recombinant wild-type and mutant viruses in
mouse dendritic cell-derived DC2.4 cells at an moi of 0.1 (panel A) or 5 (panel B).
Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of triplicate
samples.
A.
DC2.4 Cells (moi=0.1)
6
Wt
C102S
5
P38G/T116I
4
3
2
1
Titer (log10PFU/mL)/mL)
0
0
12
24
48
72
96
B.
DC2.4 Cells (moi=5)
6
5
Wt
4
C102S
P38G/T116I
3
Time
(hours)
159
2
1
0
Fig. 7-3. Multiplication kinetics of recombinant wild-type and mutant viruses in
human hepatocyte Huh7 (panel A) and Huh7 FT3.7 (panel B) infected at an moi of
5. Huh7 cells are RIG-I +/TLR3- while Huh7 FT3.7 cells are RIG-I+/TLR3+. Limit
of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of triplicate samples.
A.
Huh7
10
9
8
7
6
5
Wt
4
C102S
3
P38G
2
Titer (log10PFU/mL)/mL)
0
12
24
48
72
96
B.
Huh7 FT3.7
10
9
8
7
6
5
4
3
2
Wt
C102S
P38G
0
12
24
Time (hours)
160
48
72
96
Fig. 7-4. Multiplication kinetics of recombinant wild-type and mutant viruses in
human hepatocyte Huh7.5 (panel A) and Huh7.5iTLR3.16 (panel B) infected at an
moi of 5. Huh7.5 cells are RIG-I -/TLR3- while Huh7.5iTLR3.16 cells are RIG-I/TLR3+. Limit of detection was ≤0.7 log10 PFU/mL. Each point is mean ± SD of
triplicate samples.
A.
Huh 7.5
10
9
8
7
6
5
Wt
4
C102S
3
P38G
Titer (log10PFU/mL)/mL)
2
0
12
24
48
72
96
B.
Huh7.5 iTLR3.16
10
9
8
7
6
5
Wt
4
C102S
3
P38G
2
0
12
24
Time (hours)
161
48
72
96
Fig. 7-5. Multiplication kinetics of recombinant wild-type and mutant viruses in Tantigen immortalized non-neoplastic PH5CH8 hepatocytes infected at an moi of 5.
PH5CH8 cells are RIG-I+/TLR3+. Limit of detection was ≤ 0.7 log10 PFU/mL.
Each point is mean ± SD of triplicate samples.
PH5CH8 cells
8
Titer (log10PFU/mL)/mL)
7
6
5
4
Wt
3
C102S
P38G
2
0
12
24
48
Time (hours)
162
72
96
Figure 7-6. Raybiotech cytokine arrays were used to assay cytokine expression in
wild-type West Nile virus-infected (moi = 5) or uninfected mouse neuronal
Neuro2A, macrophage P388, or dendritic cell DC2.4 cell lines. Supernatants were
removed for analysis at 24 hours post-infection.
Media only
Neuro2A
P388
Mockinfected
Wt WNVinfected
163
DC2.4
CHAPTER 8
DISCUSSION
The flaviviral nonstructural NS4B protein is known to be critical for viral
replication, however its precise function remains unclear. The extremely hydrophobic
nature of this protein has led to unique difficulties related to expression and purification
that make direct structural and functional analyses challenging. Utilizing a WNV reverse
genetics system, engineering of amino acid substitutions into distinct regions of the
NS4B protein was undertaken to investigate the role of specific residues in the mouse
virulence and viral replication phenotypes. This dissertation has investigated phenotypic
alterations in recombinant West Nile virus strains encoding individual amino acid
substitutions in the NS4B protein as well as exploring the underlying molecular
mechanisms potentially responsible for such alterations in phenotype.
In the first aim of this dissertation, amino acid sequences of various flaviviral
NS4B proteins were subjected to alignment allowing for the identification of highly
conserved as well as variable regions in NS4B proteins throughout the flavivirus genus.
The WNV NS4B protein was also analyzed using hydrophobicity plotting programs to
produce a working model that would predict transmembrane regions in addition to
lumenal or cytoplasmic domains. Such sequence alignments and hydrophobicity plots
allowed for the identification of residues with a theoretical probability of contributing to
the function of NS4B, as well as facilitating interpretation of phenotypic alterations
observed in recombinant viruses encoding such amino acid substitutions. The second
aim of this research examined the phenotypic effects of engineered amino acid
substitutions targeting a highly conserved N-terminal domain, a variable central
hydrophobic region, and the four cysteine residues in the WNV NS4B protein. This
resulted in the identification of two highly attenuated recombinant viruses denoted as the
NS4B C102S and P38G/T116I mutants. Both mutant viruses exhibited temperature164
sensitive phenotypes in cell culture as well as attenuation of the neuroinvasive phenotype
in mice. While highly attenuated, the C102S and P38G/T116I mutant viruses were still
capable of inducing a protective immune response in mice against a lethal challenge of
wild-type WNV. In addition, upon extended incubation at a non-permissive temperature
(41°C), both viruses were capable of reverting to a virulent phenotype either through
direct reversion of the engineered amino acid substitution or by the appearance of
additional compensatory substitutions within the NS4B protein. The third aim
investigated putative underlying molecular mechanisms responsible for the attenuation of
the C102S and P38G/T116I viruses. Both NS4B mutants displayed reduced
multiplication kinetics both in mice and in cell types critical for mediating the antiviral
immune response. In addition, preliminary data was obtained that suggested the NS4B
mutants were less capable of successfully modulating certain host cell-signaling
pathways. A series of genes were identified that exhibited differential levels of
expression in wild-type WNV-infected cells compared to C102S mutant-infected cells
that may be involved in the viral manipulation of cellular processes. This chapter
discusses the novel findings of this research and speculates on the role of the NS4B
protein in the flaviviral replication complex within the context of the literature pertaining
to molecular virology.
Amino acid alignments and phylogenetic analyses of flaviviral and WN NS4B
proteins allowed for the identification of potentially critical residues and correlations
between closely and distantly related strains. The degree of NS4B amino acid identity
between both temporally and spatially diverse lineage 1a WNV strains (99% or greater)
indicate that the NS4B protein was already optimized for the hosts and vectors it would
encounter following its introduction into North America in 1999. Lineage 1a strains
isolated from Egypt in 1951 (Eg101) and Ethiopia in 1976 (Eth76) displayed only two
amino acid substitutions in the NS4B protein compared to the WNV 382-99 strain
isolated in New York in 1999. These NS4B S11N and V23A amino acid substitutions
observed in the Eg101 and Eth76 strains are also observed in a high proportion of lineage
2 WN strains including the original 1937 Uganda (B956) isolate (Appendix 1). Lineage
165
1b Kunjin virus encodes a V23T substitution while maintaining the S11 residue. The
Israel 1998 (Isr98) strain thought to be the immediate progenitor to early North American
isolates exhibited 100% NS4B amino acid identity with the WNV 382-99 strain, and this
still represents the consensus NS4B amino acid sequence of WN isolates as of 2006.
Thus at the time of its introduction into North America, the WNV NS4B protein was
already optimized suggesting that substitutions in other proteins were largely responsible
for adaptations to new hosts and vectors. Supporting this notion, certain naturally
occurring North American isolates encoding NS4B substitutions such as E249G have
actually been found to be attenuated by looking at small-plaque, temperature-sensitive,
and mouse attenuation phenotypes (Davis et al, 2004). In addition, a lineage 1a WNV
isolate from Kenya (Ken3829) exhibiting an E249D substitution (in conjunction with
other genomic mutations) exhibited decreased virulence in American crows (Brault et al.,
2004). While North American isolates are occasionally obtained that encode
substitutions in the NS4B protein, these substitutions do not appear to confer a beneficial
phenotype upon the virus and thus are not positively selected. It will be intriguing to
observe any potential adaptive substitutions within the NS4B protein as WNV continues
its migration throughout the Caribbean, Central, and South America.
The Eth76 WNV strain is known to be attenuated for neuroinvasiveness in mice,
and while this attenuation has been attributed in part to the lack of envelope (E) protein
glycosylation, it is possible that the NS4B amino acid substitutions could play a role as
well (Beasley et al., 2005). Introduction of NS4B S11A and V23A substitutions into the
WNV 382-99 infectious clone would allow for direct analysis regarding contributions of
these mutations to the mouse attenuation phenotype. A highly variable region in the Nterminal region of the WN NS4B protein was identified encompassing amino acid
residues 11-32 that was heavily mutated in lineage 2, 3, and 4 strains compared to lineage
1 strains. It would be interesting to conduct NS4B N-terminal swaps between members
of the largely mouse attenuated lineages 2, 3, and 4 with the highly virulent lineage 1a
viruses. Lineage 2 WNV strains also encode an insertion at NS4B position 27 which
encodes a threonine in each isolate except for Mad78 which encodes a glycine. The
166
potential significance of this insertion could also be analyzed utilizing the WNV 382-99
infectious clone.
Topological models generated using hydrophobicity plotting programs predicted
that the WNV NS4B protein is extremely hydrophobic with either four or five putative
transmembrane domains (TMDs). The consensus prediction ConPredII model prediction
is preferred as this model incorporates a variety of algorithms including the SOSUI
program (Arai et al. 2004). In addition, this model predicted five TMDs which correlates
with the current thinking that the N-terminus resides in the ER-lumen while the Cterminus localizes to the cytoplasm. Such topology has been predicted to occur in
relation to what is presently understood about the processing of various proteins within
the flaviviral genome (Lundin et al., 2003; Miller et al., 2006). The SOSUI prediction
model did predict that TMD3 encompassing amino acid residues 104-126 represented the
primary membrane-spanning helix due to its hydrophobic characteristics. The ConPredII
program also predicted the existence TMD3 (amino acids 106-126 in this algorithm) but
did not differentiate between primary and secondary helices. Thus, the preferred working
model incorporated the five TMDs predicted by the ConPredII program and designated
TMD3 as the primary helix (Fig. 3-5). Miller et al. (2006) were able to confirm the
ability of TMDs 3, 4, and 5 to localize eGFP to ER-membranes but could not confirm the
existence of TMDs 1 or 2 using DEN2 NS4B. It is likely that TMDs 1 and 2 occur in the
course of natural infection as they would exist in the context of the rest of the NS4B
protein. Intriguingly, DEN4 NS4B amino acids 91-136 (corresponding to WNV residues
98-143) were found to be critical in mediating the formation of high-order oligomers
(Umareddy et al., 2006). When these residues are plotted on the hydrophobicity model,
they overlap strongly with the primary TMD3. This suggests that a critical function of
this region is to support oligomerization of the NS4B protein. This central hydrophobic
region also corresponds to the location of various amino acid substitutions observed in a
variety of attenuated or passage-adapted flaviviral strains (Fig. 4-2). Thus, the various
amino acid substitutions targeting this region could exert noticeable phenotypic effects
167
upon different flaviviruses and may act by altering the conformation of high-order NS4B
multimers and disrupting the viral replication complex under certain conditions.
To experimentally investigate the importance of the central hydrophobic region,
amino acid substitutions corresponding to those identified in various attenuated or
passage-adapted flavivirus strains were engineered into the WNV NS4B protein using
site-directed mutagenesis of the two-plasmid WNV 382-99 infectious clone (Fig. 4-2).
Recombinant mutant viruses encoding NS4B L97M, A100V, L108P, and T116I
substitutions exhibited cell multiplication and mouse neurovirulence phenotypes
indistinguishable from those observed with wild-type recombinant WNV (Table 4-1).
This suggested that homologous substitutions based on those observed in various
passage-adapted flavivirus strains did not lead to attenuation of WNV. Amino acids
substitutions targeting the cysteine residues found that mutagenesis of C120, C227, and
C237 had no noticeable effect on tested phenotypes, suggesting that there are no disulfide
bridges in NS4B. In contrast, the C102S substitution localizing to the central
hydrophobic region was found to confer a highly temperature-sensitive phenotype in
addition to attenuation of mouse neuroinvasive and neurovirulence phenotypes (Table 51). The N-terminal D35E, W42F, and Y45F substitutions were found to exert no
noticeable effect on viral phenotype (Table 6-1). However, the P38G/T116I double
mutant exhibited a temperature-sensitive phenotype, a small-plaque phenotype, and
attenuation of the mouse neuroinvasive phenotype. Moreover, a series of putative
compensatory substitutions localizing to the central hydrophobic region including A95T,
A100V, V110A, and T116I mutations were each identified in conjunction with a
recombinant WNV strain encoding an engineered P38G substitution.
In summary, out of 14 engineered recombinant mutant viruses, two highly
attenuated NS4B mutants were identified. These are the first data to directly establish the
role of the NS4B protein in the mouse neuroinvasive phenotype. The C102S and
P38G/T116I viruses both exhibited mouse ip LD50 values of greater than 10,000,000 pfu
and PD50 values of less than 1 pfu (Tables 5-1 and 6-1). Thus, these viruses were greatly
attenuated and highly capable of inducing a protective immune response against wild168
type WNV 382-99, even at low doses. The C102 residue was found to be highly
conserved in all members of the JE and DEN serogroups while P38 was conserved in all
mosquito-borne, tick-borne, and non-vector flaviviruses except for the Brazilian Ilheus
virus. It is therefore likely that disruption of these residues would lead to attenuation of
numerous members of the flaviviral genus. While C102S and P38G substitutions highly
attenuated WNV, C102A and P38A substitutions did not alter the wild-type mouse
neuroinvasive phenotype. Introduction of attenuating NS4B substitutions in conjunction
with other attenuating mutations could serve as a promising strategy for developing new
flaviviral vaccine strains.
Recombinant mutant viruses encoding NS4B C102S and P38G/T116I
substitutions both exhibited temperature-sensitive phenotypes in Vero cells at 41°C.
However, extended incubation of these viruses at a non-permissive temperature (41°C)
led to the identification of revertant viruses for both mutants. The C102S virus exhibited
a direct S102C reversion, while the P38G/T116I virus expressed a series of additional
compensatory amino acid substitutions primarily within the central hydrophobic domain
(A83S, A95T, A100V, V110A, and I224V) (Table 6-2). Although different flavivirus
strains exhibiting temperature-sensitivity have been previously identified, this is the first
time amino acid substitutions within the NS4B protein have been found to be directly
responsible for this phenotype (Hollingshead et al., 1983; Eastman & Blair, 1985; Ledger
et al., 1992; Blaney Jr. et al., 2001; Blaney Jr. et al., 2003b; Rumyantsev et al., 2006;
Kinney et al., 2006). The Ken3829 WNV strain is known to be attenuated in crows
compared to the WNV 382-99 strain (Brault et al., 2004). Recombinant Ken3829 was
found to exhibit a temperature-sensitive phenotype in duck embryonic fibroblast (DEF)
cells at 44°C as compared to recombinant WNV 382-99 (Kinney et al., 2006). The
Ken3829 virus encodes 11 amino acid substitutions as compared to WNV 382-99 (Brault
et al., 2004), thus the mutation responsible for the temperature-sensitive phenotype has
not been confirmed. One candidate substitution is a NS3 P249T substitution that
localizes near a NS3 250 substitution that is known to determine temperature-sensitivity
in a DEN2 vaccine strain (Brault et al., 2004 and Butrapet et al., 2000). Alternatively, the
169
Ken3829 virus encodes a NS4B E249D substitution that could be responsible for the
observed temperature-sensitive phenotype. An NS4B E249G substitution has been
detected in a naturally occurring isolate exhibiting temperature-sensitive and mouse
attenuation phenotypes (Davis et al., 2004). Thus, additional studies will be required to
determine the genetic basis of the Ken3829 temperature-sensitive phenotype. Previous
studies have identified mutations in DEN4 virus NS1, NS2A, NS3, and NS5 proteins that
confer a temperature-sensitive phenotype (Blaney, Jr. et al., 2003 and Hanley et al.,
2002). Chemical mutagenesis of DEN4 virus resulted in the identification of NS4B
L112F, A116V, and A240V substitutions (corresponding to WNV residues F119, A123,
and M248) encoded by produced temperature-sensitive strains, however these
substitutions occurred within the context of other mutations scattered throughout the
genome (Blaney, Jr. et al., 2001). Thus the precise contributions of these amino acid
substitutions towards the observed temperature-sensitive phenotype remain unknown.
While various flavivirus strains exhibiting temperature sensitivity have been identified,
the mechanisms responsible for this phenotype have not been elucidated.
It is intriguing that each of the proteins found to encode substitutions conferring
the temperature-sensitive phenotype (NS1, NS2A, NS3, NS4B, and NS5) are
hypothesized to serve as components of the membrane-bound flaviviral replication
complex. NS1 is thought to be involved in negative-strand RNA synthesis and is capable
of specifically binding the NS4A integral membrane protein (Lindenbach et al., 1997).
The hydrophobic NS2A protein binds the 3’-UTR, and together with the NS5 polymerase
and NS3 protease is hypothesized to form the basis of the replication complex
(Mackenzie et al., 1998). The NS3 protein is known to encode helicase and NTPase
activities thought to be critical during viral RNA processing (Li et al., 1999). Recently,
DEN4 NS4B was found to enhance helicase activity by dissociating NS3 from singlestranded RNA utilizing an in vitro unwinding assay (Umareddy et al., 2006). It is likely
that the molecular mechanisms of the temperature-sensitive phenotype involve amino
acid substitutions which disrupt critical protein-protein interactions thereby resulting in
an inefficient viral replication complex at non-permissive temperatures.
170
A potential molecular mechanism explaining the temperature-sensitive phenotype
exhibited by the NS4B mutants will therefore be proposed. The rotavirus 35 kDa
nonstructural NSP2 protein is known to encode RNA-binding, helicase, and NTPase
activities (Taraporewala et al., 2002). This protein is known to form high-order
oligomers, and the octamer has been identified as the functional form of the protein
(Schuck et al., 2001). Upon binding of nucleotides and in the presence of Mg2+, the
NSP2 protein undergoes a conformational change into compact structures. NSP2
octamers bind ssRNA cooperatively with high affinity and are thought to facilitate viral
RNA packaging by removing secondary structure (Taraporewala et al., 1999). A
temperature-sensitive rotavirus mutant encoding a single NSP2 A152V substitution was
noted to exhibit decreased ssRNA and dsRNA replication and the production of mostly
empty virus particles at the nonpermissive temperature of 39°C. Upon further
examination, the A152V NSP2 protein was not capable of forming the functional octamer
unit effectively but instead formed large poorly organized aggregates. This resulted in a
drastic decrease in ssRNA-binding, helicase, and NTPase activities upon biochemical
analysis (Taraporewala et al., 2002). The flaviviral NS4B protein is also thought to form
high-order oligomers and modulates the helicase activity of the NS3 protein (Umareddy
et al., 2006). It is possible that NS4B is involved in viral RNA packaging, and that
enhancement of helicase activity serves to eliminate RNA secondary structure before
incorporation into viral nucleocapsids as occurs with rotaviral NSP2. It is also possible
that high-order NS4B oligomers serve as the functional form of the protein. Umareddy et
al. (2006) identified DEN4 NS4B amino acid residues 91-136 (homologous to WNV
residues 98-143) as being critical for multimer formation. The C102S substitution occurs
in this region which also overlaps with the predicted primary membrane-spanning TMD3
helix. It can be speculated that this substitution disrupts the formation of the potentially
mutimeric functional form of the protein at nonpermissive temperatures thereby
disrupting the protein’s ability to modulate helicase activity. While the temperaturesensitive P38G viral substitution does not occur in the proposed oligomeric-critical
domain, several putative compensatory substitutions (A95T, A100V, V110A, and T116I)
171
leading to increased multiplication or reversion of the temperature-sensitive phenotype
did localize to this region. It is also possible that residues in either the N-terminal or Cterminal region also mediate functional interactions that would manifest as temperaturesensitivity if disrupted. Future experiments should focus on identifying the functional
form of the NS4B protein utilizing biochemical purification techniques of WNV-infected
cells. Also, obtaining a structure for the NS4B protein would greatly aid in elucidating
its function.
An alternative molecular basis to the observed temperature-sensitive phenotype
conferred by the NS4B mutants could be an altered ability to modulate the host stress
response. Hepatitis C virus (HCV) NS4B has been found to specifically interact with the
CREB-RP/ATF6β heat shock protein (Tong et al., 2002). This protein is known to serve
as an ER-stress-induced transcription factor and may be involved in mediating certain
aspects of the antiviral response. Measles virus is known to exhibit a large-plaque
phenotype in response to expression of the 70kD heat shock protein (HSP72)
(Vasconcelos et al., 1998). Recently, HSP70 and HSP90 have been found to participate
in dengue virus entry into certain human cell lines by associating with lipid rafts in the
cell membrane (Reyes-del Valle et al., 2005). The roles of heat shock proteins and
cellular stress responses on flaviviral infection remain undefined but could serve as an
important mediator of viral replication and would be an interesting route to continue
these studies in the future.
Experiments analyzing viral multiplication mice found that the C102S and
P38G/T116I NS4B mutant viruses showed decreased serum titers in mice compared to
wild-type WNV (Table 7-1). In addition, NS4B mutant viral titers or RNA were never
detected in the brain. Both C102S and P38G/T116I viruses exhibited altered
multiplication kinetics compared to wild-type WNV in mouse P388 macrophage and
DC2.4 cell lines (Figs. 7-1 and 7-2). Evidence was also obtained that an inability to
counteract TLR3 and RIG-1 signaling pathways could be responsible for attenuation of
the NS4B mutants (Figs. 7-3, 7-4, 7-5). Analysis of cytokine expression neuronal
Neuro2A, macrophage P388, and dendritic cell DC2.4 cells did not identify any
172
differential expression in WNV-infected versus uninfected cells. More sensitive assays
such as ELISA may be necessary to detect alterations in cytokine expression, or perhaps
primary cell lines should be utilized for these experiments. Preliminary DNA microarray
gene expression experiments of wild-type versus C102S mutant WNV-infected
macrophage P388 cells led to the identification of several genes that may be critical in
mediating cellular antiviral responses. A variety of interferon-stimulated genes including
Ifit1, Irf7, Ifi202b, Isg20, and Ifi44 were found to be significantly up-regulated in wildtype-infected cells as compared to C102S-infected cells. The flaviviral NS4B protein has
been implicated in disrupting cellular IFN-signaling pathways at the level of STAT
phosphorylation, however the biological importance during the course of natural
infection remains undetermined (Munoz-Jordan et al., 2003 and 2005). Genes relating to
cellular translation factors, cytoskeleton organization, and ubiquitinization pathways were
also found to be preferentially up-regulated in wild-type WNV-infected cells (Table 7-2).
In summary, a combination of decreased multiplication in mice, reduced ability to
effectively multiply in certain immune cells, and ineffective modulation of cellular
antiviral responses likely serve mechanisms underlying the observed mouse attenuation
phenotype. Future experiments should determine if there are any differences in the
formation of viral replication complexes within macrophages or dendritic cells infected
with either wild-type WNV or the attenuated NS4B mutants. In addition, differentially
regulated genes identified in this study represent preliminary data and should be
confirmed by additional quantitation methods and further analyzed to elucidate critical
signaling pathways involved in viral replication.
Critical areas to explore with respect to the WNV NS4B protein include analyses
testing the importance of specific amino acid residues with respect to viral phenotype in
addition to elucidating precisely how the NS4B protein modulates viral replication.
NS4B amino acid substitutions identified in the original lineage 1a and attenuated lineage
2 strains should be introduced into the WNV 382-99 infectious clone to determine if
these mutations are capable of attenuating North American WNV on their own. New
isolates from the Caribbean, Central, and South America should be subjected to
173
sequencing of the NS4B gene to identify any new substitutions that may occur as WNV
encounters new hosts and vectors on its journey south. Concerted efforts should be
made to biochemically characterize the WNV NS4B protein in order to determine the
functional form of NS4B and elucidate the molecular basis of observed temperaturesensitive phenotypes. Obtaining a structure for NS4B will be absolutely critical to
improving our understanding of this protein’s role in the viral replication complex. The
difficulties in obtaining purified NS4B and antisera against the protein are well
recognized by the author and other flavivirologists and will require a substantial effort to
be successful.
Future experiments deriving from this body of work should seek to more
accurately elucidate the molecular mechanisms responsible for the observed temperaturesensitive phenotype in Vero cells and altered multiplication kinetics in P388.D1 and
DC2.4 cells with respect to the NS4B mutant viruses. Given that alterations in viral
multiplication were only observed in certain cell-types under specific conditions, it stands
to reason that there are cell-specific factors involved in the inhibition of NS4B mutant
viral replication. Initially, a combination of electron microscopy (EM) and
immunofluorescence studies could be utilized to identify any differences in intracellular
morphologies, especially relating to induced ER-membrane proliferation within C102S
mutant and wild-type virus-infected cells. In addition, such studies could be used to
analyze the number and morphology of viral RCs for mutant and wild-type viruses when
infecting cell types where alterations in multiplication kinetics have been observed. It is
conceivable that disruption of viral RC formation is responsible for the altered
multiplication kinetics observed with the NS4B mutants, and direct visual data supporting
this could lead to elucidation of the molecular mechanism(s) of attenuation.
Biochemically, the ability of the C102S and P38G/T116I mutant NS4B proteins to
interact with and modulate the activity of the NS3 helicase should be investigated.
Potential interactions between the NS4B protein and cellular heat-shock proteins should
also be explored, as modulation of host stress responses could be involved in allowing for
efficient viral replication. In addition, it will be necessary to confirm and expand on
174
observations pertaining to the NS4B protein’s role in modulating cellular antiviral
responses, especially in immunologically important cell types such macrophages and
dendritic cells. A better understanding of NS4B structure and function could result in the
development of new antiviral agents in addition to flaviviral vaccine strains.
175
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217
Appendix 1. NS4B amino acid sequence alignments from various West Nile virus isolates
218
AB185914
AB185915
AB185916
AB185917
AF260967
AY 289214
DQ080061
DQ164198
DQ164205
DQ374651
DQ411035
DQ411034
DQ411033
DQ411032
DQ411031
DQ411030
DQ411029
DQ377180
DQ377179
DQ377178
DQ374653
DQ374652
DQ374650
DQ211652
DQ176637
DQ164206
DQ164204
DQ164201
DQ164200
DQ164199
DQ164197
DQ164196
DQ164195
DQ164194
DQ164193
DQ164191
DQ164190
DQ164189
DQ164188
Consensus
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NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
218
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
219
DQ164186
DQ080070
DQ080069
DQ080068
DQ080066
DQ080065
DQ080064
DQ080063
DQ080060
DQ080059
DQ080058
DQ080057
DQ080056
DQ080055
DQ080054
DQ080053
DQ080052
DQ080051
DQ066423
DQ005530
AY 848697
AY 848696
AY 848695
AY 842931
AY 795965
AY 712947
AY 278441
AY 268133
AF533540
AF481864
AF404756
AF404755
AF404754
AF404753
AF206518
AF202541
AF196835
DQ118127
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
1
10
20
30
40
50
60
70
85
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
219
NEMGWLDKTKSDISSLFGQRIEVKENFSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
220
DQ080062
DQ080071
DQ080072
DQ164202
DQ164187
DQ164192
DQ080067
AY 712948
AF260969
AF317203
AF404757
AY 262283
AY 268132
AY 277252
AY 278442
AY 701412
AY 701413
AY 712945
AY 712946
AY 646354
AF260968
AY 603654
AY 490240
AY 660002
DQ164203
AY 274504
AY 274505
D00246
AY 532665
M12294
NC001563
DQ318019
AY 688948
DQ318020
DQ116961
DQ176636
AY 765264
AY 277251
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
1
10
20
30
40
50
60
70
86
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLSRG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLSRG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLSRG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLSRG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDVSSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FGMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLFGQRIEAKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLFGQRIEAKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLFGQRIDVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISSLFGQRIEVKEN-FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISGLFGQRIETKEN-FSIGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISGLFGQRIETKEN-FSIGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDISGLFGQRIETKEN-FSIGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYITTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLLGHKPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDIGSLLGHRPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDIGSLLGHRPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDIGSLLGHKPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKSDIGSLLGHKPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLLGHKPEARETTLGVESFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDISSLLGHKPETRETTLGVENFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDIGRLLGYKLEVKETGLGIESFLLDLRPATAWSLYAVATAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDKTKNDMSNLLGQKPGTTES-LGMADLLLDLKPATAWSLYAISTAFMTPLLKHLITSDYINTSLTSINVQASALFTLARG
NEMGWLDRTKSDIGTLWGQRTESRES-FGVESFLLDLKPATAWSLYAVSTAVMTPLLKHVITSDYINTSLTSINVQASALYSLARG
NEMGWLDKTKSDISSLFGQRIEVKEN FSMGEFLLDLRPATAWSLYAVTTAVLTPLLKHLITSDYINTSLTSINVQASALFTLARG
220
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
221
AB185914
AB185915
AB185916
AB185917
AF260967
AY 289214
DQ080061
DQ164198
DQ164205
DQ374651
DQ411035
DQ411034
DQ411033
DQ411032
DQ411031
DQ411030
DQ411029
DQ377180
DQ377179
DQ377178
DQ374653
DQ374652
DQ374650
DQ211652
DQ176637
DQ164206
DQ164204
DQ164201
DQ164200
DQ164199
DQ164197
DQ164196
DQ164195
DQ164194
DQ164193
DQ164191
DQ164190
DQ164189
DQ164188
Consensus
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
86
100
110
120
130
140
150
160
170
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERATPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERATPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERATPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
221
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
222
DQ164186
DQ080070
DQ080069
DQ080068
DQ080066
DQ080065
DQ080064
DQ080063
DQ080060
DQ080059
DQ080058
DQ080057
DQ080056
DQ080055
DQ080054
DQ080053
DQ080052
DQ080051
DQ066423
DQ005530
AY848697
AY848696
AY848695
AY842931
AY795965
AY712947
AY278441
AY268133
AF533540
AF481864
AF404756
AF404755
AF404754
AF404753
AF206518
AF202541
AF196835
DQ118127
Consensus
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
86
100
110
120
130
140
150
160
170
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNVVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
222
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
223
DQ080062
DQ080071
DQ080072
DQ164202
DQ164187
DQ164192
DQ080067
AY712948
AF260969
AF317203
AF404757
AY262283
AY268132
AY277252
AY278442
AY701412
AY701413
AY712945
AY712946
AY646354
AF260968
AY603654
AY490240
AY660002
DQ164203
AY274504
AY274505
D00246
AY532665
M12294
NC001563
DQ318019
AY688948
DQ318020
DQ116961
DQ176636
AY765264
AY277251
Consensus
(87)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(86)
(87)
(87)
(87)
(87)
(87)
(87)
(87)
(87)
(86)
(86)
(87)
87
100
110
120
130
140
150
160
171
FPFVDVGVSALLLAAGCWEQVTLTVTVTAATLLFCHYAYMVPGGQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLLCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLLCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTSATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNVVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAVGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNVVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAVGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNVVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAILLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPVMQ
FPFVDVGISSLLLAVGCWGQVTLTVAVTTAALLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAAALLFCHYAYMVPGWQAEAMRAAQRRTAAGIMKNAVIDGMVATDVPELERTTPVMQ
FPFVDVGVSALLLAAGCWGQVTLTVTVTAATLLFCHYAYMVPGWQAEAMRSAQRRTAAGIMKNAVVDGIVATDVPELERTTPIMQ
223
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
224
(171)
AB185914(171)
AB185915(171)
AB185916(171)
AB185917(171)
AF260967(171)
AY 289214(171)
DQ080061(171)
DQ164198(171)
DQ164205(171)
DQ374651(171)
DQ411035(171)
DQ411034(171)
DQ411033(171)
DQ411032(171)
DQ411031(171)
DQ411030(171)
DQ411029(171)
DQ377180(171)
DQ377179(171)
DQ377178(171)
DQ374653(171)
DQ374652(171)
DQ374650(171)
DQ211652(171)
DQ176637(171)
DQ164206(171)
DQ164204(171)
DQ164201(171)
DQ164200(171)
DQ164199(171)
DQ164197(171)
DQ164196(171)
DQ164195(171)
DQ164194(171)
DQ164193(171)
DQ164191(171)
DQ164190(171)
DQ164189(171)
DQ164188(171)
Consensus(171)
171
180
190
200
210
220
230
240
255
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKIGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSMTWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSMTWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSMTWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGIKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
224
Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
225
(171)
DQ164186(171)
DQ080070(171)
DQ080069(171)
DQ080068(171)
DQ080066(171)
DQ080065(171)
DQ080064(171)
DQ080063(171)
DQ080060(171)
DQ080059(171)
DQ080058(171)
DQ080057(171)
DQ080056(171)
DQ080055(171)
DQ080054(171)
DQ080053(171)
DQ080052(171)
DQ080051(171)
DQ066423(171)
DQ005530(171)
AY 848697(171)
AY 848696(171)
AY 848695(171)
AY 842931(171)
AY 795965(171)
AY 712947(171)
AY 278441(171)
AY 268133(171)
AF533540(171)
AF481864(171)
AF404756(171)
AF404755(171)
AF404754(171)
AF404753(171)
AF206518(171)
AF202541(171)
AF196835(171)
DQ118127(171)
Consensus(171)
171
180
190
200
210
220
230
240
255
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
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Appendix 1 (continued). NS4B amino acid sequence alignments from various West Nile virus isolates
226
(172)
DQ080062(171)
DQ080071(171)
DQ080072(171)
DQ164202(171)
DQ164187(171)
DQ164192(171)
DQ080067(171)
AY712948(171)
AF260969(171)
AF317203(171)
AF404757(171)
AY262283(171)
AY268132(171)
AY277252(171)
AY278442(171)
AY701412(171)
AY701413(171)
AY712945(171)
AY712946(171)
AY646354(171)
AF260968(171)
AY603654(171)
AY490240(171)
AY660002(171)
DQ164203(171)
AY274504(171)
AY274505(171)
D00246(171)
AY532665(172)
M12294(172)
NC001563(172)
DQ318019(172)
AY688948(172)
DQ318020(172)
DQ116961(172)
DQ176636(172)
AY765264(171)
AY277251(171)
Consensus(172)
172
180
190
200
210
220
230
240
256
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLIWVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIAWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLMKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMDKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMGKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMGKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQVMLILVSLAALVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQVMLILVSLAALVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQVMLILVSLAALVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQIILILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIMWTLIKNMEKPGLKR
KKVGQIILILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIMWTLIKNMEKPGLKR
KKVGQIILILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIMWTLIKNMEKPGLKR
KKVGQIMLILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIMWTLIKNMEKPGLKR
KKVGQIMLILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSIAWTLIKSMEKPVLKR
KKVGQIMLILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSMAAVVVNPSVRTVREAGILTTAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
KKVGQIMLILVSVAAVVVNPSVRTVREAGILTSAAAVTLWENGASSVWNATTAIGLCHVMRGGWLSCFSITWTLIKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTAREAGILITSASVTLWENGASSVWNATTAIGLCHVMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQIMLILVSTAAVVVNPSVKTVREAGILVTAAAVTLWENGASSVWNATTAIGLCHVMRGGWLSCLSITWTLVKNMEKPGLKR
KKVGQIMLILVSLAAVVVNPSVKTVREAGILITAAAVTLWENGASSVWNATTAIGLCHIMRGGWLSCLSITWTLIKNMEKPGLKR
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