AN ABSTRACT OF THE DISSERTATION OF
Su Jung Yang for the degree of
Doctor of Philosophy in
Microbiology presented
on December 22, 2006.
Title: Characterization of Vaccinia Virus A12L Protein: Its Proteolysis and
Functional Analyses in Virus Replication.
Abstract approved:
Dr. Dennis E. Hruby
In order to produce infectious virus progeny, vaccinia virus (VV)
undergoes morphogenic proteolysis to regulate the structural rearrangements of
virus particles. Several of the major structural precursor proteins of VV are
cleaved at a conserved Ala-Gly-X (where X is any amino acid) motif by the VV
I7L core protein proteinase at a step, which is necessary for formation of mature
virus particles. VV A12L encodes a 25kDa core protein, which is cleaved at an
AG/A site, yielding a 17kDa cleavage product. Both A12L precursor and the
cleavage product are localized to mature virions. The open reading frame (ORF)
of A12L contains two more AG/X (AG/K) sites, however, cleavage at these sites
has not been analyzed. Therefore, the aim of this study is to characterize the in
vivo processing of A12L proteolysis and elucidate the biological function of A12L.
The result of these studies would provide more details on the regulation and
participation of VV proteolysis during the morphogenic transitions.
Proteolytic processing of A12L produces multiple peptides, which do not
appear to utilize AG/K sites, but rather cleavages occur at both the N- and Cterminus. Of the three AG/X motifs in A12L, cleavage has only been
demonstrated at the AG/A site. The enzyme responsible for this cleavage has
been shown to be I7L. Immunoprecipitation studies have shown that A12L
associates with VV core and membrane proteins. A conditional mutant virus of
A12L was constructed and determined the essentiality of A12L in virus replication
and helped to elucidate its functions in the assembly of virus particles. An AG/A
site mutation abrogated the ability of the transfected A12L gene to rescue the
conditional mutant under non-permissive conditions, indicating that its proteolysis
at the AG/A site is required during viral replication. Next, we compared the protein
expression of A12L with D13L, an internal scaffolding protein, to investigate the
role of A12L in virus assembly. We showed that A12L is stably synthesized only in
the presence of D13L. Consequently, we established that A12L protein and its
proteolysis participate in viral assembly subsequent to D13L involvement.
©Copyright by Su Jung Yang
December 22, 2006
All Rights Reserved
CHARACTERIZATION OF VACCINIA VIRUS A12L PROTEIN:
ITS PROTEOLYSIS AND FUNCTIONAL ANALYSES IN VIRUS REPLICATION.
by
Su Jung Yang
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented December 22, 2006
Commencement June 2007
Doctor of Philosophy dissertation of Su Jung Yang
presented on December 22, 2006.
APPROVED:
Major Professor, representing Microbiology
Chair of the Department of Microbiology
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Su Jung Yang, Author
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to all the committee
members, Dr. Daniel Rockey, Dr. Theo Dreher, Dr. Thomas Wolpert, Dr. Machteld,
Mok for their help, guidance and encouragement.
I would like to appreciate for all of the support the laboratory members in
Dr. Hruby’s lab have shown me. Special thanks to Jennifer Yoder, the Lab
manager, who helped me to complete the writing of this thesis and for her helpful
discussions, which were valuable for conducting experiments. Dr. Chelsea Byrd,
a former graduate student in the same lab provided help with the writing and
editing of this thesis as well as with protocols and experimental procedures. I
would also like to say thanks for all the fun at American Society for Virology
conferences. Dr. Dina Alzhanova, a postdoctoral scientist, encouraged me a lot
when I struggled with experiments and taught me experimental skills for the
fluorescent microscopy. Thank you. Finally, I would like to thank Dr. Dennis Hruby,
a mentor who offered me a great opportunity to work in his lab and always
encouraged to have fun with A12L protein project. To be honest, I had a hard
time with this protein, but I was able to discover what the real meaning of
“research” and “dance of joy” are and how much I need to believe in myself. I
would like to express my sincere appreciation to Dr. Hruby for being my mentor
and for setting the example of being a good scientist.
I also would like to mention that I had unforgettable time being a teaching
assistant with all the graduate students during 2001 and 2002. I would like to say
thank you to everyone for being such good friends and sharing great times with
me.
Despite the fact that I came to the US by myself, I happened to have
family here. With help from all the church members especially in the college
group, I learned about God and how to love others. That became the source of
my strength not only working in the lab but also with the rest of life. Finally, I
would like to express my sincere love and appreciation to my family, Han Seob
Yang, Soon Ok Whang and Su Chul Yang, Su Hee Yang, Su Seon Yang, and
friends in Korea, who have shown even stronger faith in me than myself and
given me a consistent support and love. I cannot imagine standing here and
receiving a Ph.D degree without their love. Thank you all, who always stand
behind me and pray for me. I would like to say that I love you all.
TABLE OF CONTENTS
PAGE
1. INTRODUCTION
1.1 Vaccinia virus life cycle · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
1.2 Virus assembly and dissemination · · · · · · · · · · · · · · · · · · · · · · · ·
1.3 Proteolysis in virus replication · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
1.4 Vaccinia virus proteolysis · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
1.5 VV contextual proteolytic processing · · · · · · · · · · · · · · · · · · · · · ·
1.6 Vaccinia virus proteases · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
1.7 Vaccinia virus A12L core protein · · · · · · · · · · · · · · · · · · · · · · · · · ·
1
3
5
9
11
14
15
2. CHARACTERIZATION OF A12L PROTEOLYSIS AND ITS PARTICIPATION
IN VIRUS ASSEMBLY.
2.1 Abstract · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 21
2.2 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
2.3 Material and methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
2.4 Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
2.5 Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
22
26
32
43
3. ESSENTIALITY OF A12L IN VIRAL REPLICATION AND
MORPHOLOGICAL TRANSITION.
3.1 Abstract · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
3.2 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
3.3 Material and methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
3.4 Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
3.5 Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
61
62
67
71
76
4. PARTICIPATION A12L IN VACCINIA VIRUS ASSEMBLY SUBSEQUENT
TO D13L INVOLVEMENT
4.1 Abstract · ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
4.2 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
4.3 Material and methods · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
4.4 Results · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
89
90
94
96
TABLE OF CONTENTS (Continued)
PAGE
4.5 Discussion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
99
5. CONCLUSION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
106
6. BIBLOGRAPHY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
110
LIST OF FIGURES
PAGE
FIGURE
Chapter 1.
1.1 Vaccinia virus life cycle. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
17
1.2 Diagram of VV virion-associated proteins and
their cleavage products. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 19
Chapter 2.
2.1
Multiple cleavage products of A12L protein. 49
2.2
Kinetic analysis and pulse chase of A12L protein. · · · · · · · · · · · · · 51
2.3
Proteolysis of A12L. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 53
2.4
The AG/A site cleavage by the VV proteinase I7L. · · · · · · · · · · · · · 55
2.5
N-terminal proteolysis of A12L. · · · · · · · · · · · · · · · · · · · · · · · · · · · · 56
2.6
Identification of A12L-derived peptides. · · · · · · · · · · · · · · · · · · · · · 57
2.7
Association of A12L with other VV proteins. · · · · · · · · · · · · · · · · · · 58
2.8
Subcellular localization of A12L protein. · · · · · · · · · · · · · · · · · · · · · 60
Chapter 3.
3.1
Tet-dependent replication of vvtetOA12L and
one-step growth curve. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 81
3.2
Essentiality of A12L protein in VV replication. · · · · · · · · · · · · · · · · 83
LIST OF FIGURES (Continued)
PAGE
FIGURE
3.3
Morphology defects in the absence of A12L expression. · · · · · · · 85
3.4
Morphology defects by abrogated AG/A cleavage of A12L. · · · · · 87
Chapter 4.
4.1
Rifampicin-resistant loci in D13L ORF. · · · · · · · · · · · · · · · · · · · · · 101
4.2 Sensitivity of RifR viruses to different concentrations of
rifampicin. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 103
4.3
Protein expression of D13L and A12L in the presence of Rif. · · · 105
LIST OF TABLES
PAGE
TABLE
Chapter 2
2.1 Diagram of VV virion-associated proteins and
their cleavage products. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
46
2.2 The predicted molecular weights and pI’s of potential
A12L cleavage products. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
48
Chapter 3
3.1 Schematic diagram of a conditional mutant virus of A12L. · · · · · · 79
CHAPTER 1. INTRODUCTION
1
VACCINIA VIRUS LIFE CYCLE
Vaccinia virus (VV), one of the most well-studied animal viruses, belongs
to the Orthopoxvirus genus of the Poxviridae family, distinguished by their
cytoplasmic site of replication, temporal regulation of gene expression, and
unique morphology. VV possesses a large double-stranded DNA genome of
approximately 190 kilobase pairs (kbp) and produces at least 250 gene products
during the viral replication cycle. However, the virus resides in the cell cytoplasm
from the time the virus enters the host cell until the progeny viruses are produced.
Thus, in order to promote viral replication in the cytoplasm, VV needs to encode
its own enzymes for transcription as well as DNA replication, which suggests a
high level of independence from host cells. In addition, VV acquires its infectivity
by assembling multiple virion forms, which require complex morphogenic
pathways. The large number of gene products and the complex virion assembly
suggest that VV possesses tightly regulated gene expression systems to control
individual genes.
Upon binding of virions to cell surfaces, the viral entry into the host cells
is mediated differently depending on the form of the virus, intracellular mature
virus (IMV) or extracellular enveloped virus (EEV). IMV are hypothesized to enter
the cells by plasma membrane fusion in the presence of a single membrane
(Chang, 1976) or by uncoating outside of the cell in the presence of multiple
membranes (Krijnse-Locker et al. 2000). EEV, with an extra membrane, is
proposed to enter the cells by acidification of the virions in intracellular vesicles
(Ichihashi, 1996) or by shedding the membranes at the cell surface (Krijnse-
2
Locker et al. 2000).
Regardless of the mechanism of viral entry, once a virus is inside the cell,
VV accomplishes coordinating the processes of genome replication and virus
assembly through three consecutive stages categorized as the early,
intermediate, and late gene expression as described at Fig 1.1 (Broyles, 2003).
Once the core is uncoated, the mRNAs and VV-encoded enzymes packaged
within the core particles are transported near to cellular ribosomes by
microtubules (Carter et al. 2003, Mallardo et al. 2001). The enzymes in VV core
include: DNA-dependant RNA polymerase; early transcription factors; capping
and methylating enzymes; polyA polymerase; and DNA topoisomerase type I.
With these enzymes, the viral mRNAs are modified with 3’-capping and 5’polyadenylation and promote the first stage of gene expression. Proteins
participating in DNA replication, nucleotide biosynthesis and intermediate
transcription
are
synthesized
as
early
class
genes,
which
represent
approximately half of the viral genome. DNA replication begins in concert with
intermediate gene expression. Newly replicated viral DNA provides templates for
intermediate gene expression, of which the products, A1L, A2L, and G8R act as a
set of late gene transcriptional factors. At the late stage, virion structural proteins,
enzymes for DNA packaging and early transcription factors are expressed by
virtue of the 5’-polyA leader sequences. Then, the late gene products undergo
different
post-translational
modifications
morphogenesis at the post-replicative stage.
initiating
virion
assembly
and
3
VIRUS ASSEMBLY AND DISSEMINATION.
The assembly of virus progeny commences with the coalescence of viral
DNA, enzymes, lipids and structural proteins to form pre-virion particles, which
have crescent-shaped lipid membranes surrounding electron-dense areas called
viral factories (viroplasm) (Fig. 1.1). Unlike other viruses such as vesicular
stomatitis virus or influenza virus, VV membranes appear to be assembled in
virus cytoplasmic foci (viroplasm) into uniform, spherical, immature virus particles
(IV). The gene product of D13L, a 65kDa late protein, is reported to act as a
transitory scaffolding protein and is responsible for the formation of spicules,
giving the IV particle its characteristic morphology (Mark et al. 1991, Sodeik et al.
1994).
This non-infectious IV develops an internal core surrounding the viral
DNA and becomes associated with host-originated membranes acquired from the
intermediate compartment (IC), which is located between the endoplasmic
reticulum (ER) and the Golgi complex. The association of the IC membrane with
the IV particles gives rise to the development of the first infectious form of virus
with a brick-shaped core, referred to as intracellular mature virus (IMV) (Sodeik
et al. 1994). However, the origin and numbers of the membranes around the IMV
is controversial. In contrast to IC-originated double membranes, a single
membrane is postulated to be synthesized de novo. (Dales, 1968) The evidence
that IC resident proteins were not detected in purified virions, and that inhibitors
causing a blockage of the transport from ER to IC and Golgi did not disrupt IMV
membranes, suggests that IV may not use host-derived IC, but rather
4
synthesizes membranes from its own expression. (Krauss et al. 2002, Husain
and Moss, 2003) Regardless of the numbers and origins of the membranes, the
structural transformation of IV to produce IMV appears to involve the proteolytic
processing of certain polypeptide precursors (Lee and Hruby, 1994, Moss and
Rosenblum, 1973) and the participation of some proteins required for DNA
packing and core condensation (Morgan, 1976, Wilcock and Smith, 1996,
Cassetti et al. 1998).
Although IMV particles are infectious, they are not sufficient for longrange infection or efficient viral spread. An additional envelope is required for viral
spread. A portion of IMV is transported to the microtubule-organizing centre
(MTOC) and enwrapped in double layers of membrane cisternae, derived from
the trans-Golgi network or endosomes (Schemilz et al. 1994, Hollinshead et al.
2001, Rietdorf et al 2001, Ward and Moss, 2001), forming intracellular enveloped
virus (IEV). Consequently, IEV particles obtain two more membranes surrounding
the IMV and are migrated to the cell surface by microtubules, where the
outermost IEV membrane is fused with the plasma membrane. As a result, the
outer layer of the membrane is lost and the virus becomes exposed on the cell
surface in a manner similar to the exocytosis of intracellular vesicles. Depending
on the location and role in virus dissemination, this virion can be referred to as an
extracellular-enveloped virus (EEV) or cell-associated enveloped virus (CEV). If
the virion is released into the external medium mediating the long-range spread
of viruses, it is called EEV whereas if it is retained on the cell surface, it is named
CEV (Law et al. 2002). CEV stimulates actin polymerization beneath the plasma
5
membrane where CEV is attached (Van Eijl et al. 2000) and the growing actin
tails become the source of propelling the virus particles away, infecting
surrounding cells. Some poxviruses such as Cowpox virus (CPV) are capable of
producing another infectious form of IMV particles called A-type inclusion bodies
(ATI) (Patel et al. 1986). ATI is a late gene product, which forms the cytoplasmic
inclusion bodies with IMV particles occluded inside. The highly stable
proteinaceous bodies help to retain viral infectivity outside of host cells.
Considering the large number of gene products and multiple forms of
infectious progeny, VV must regulate a complex assembly process. By taking
advantage of such post-translational modifications as myristylation, acylation,
phosphorylation, glycosylation, and ADP-ribosylation, VV is capable to modulate
not only gene expression but also proper configuration, localization, and
interactions of proteins. During the morphogenic transition from IV to IMV, one of
the essential modifications VV undergoes is proteolytic processing.
PROTEOLYSIS IN VIRUS REPLICATION
Proteolysis is defined as hydrolysis selectively occurring at specific residues in a
polypeptide catalyzed by an enzyme called a proteinase (also called an
endopeptidase). Due to the characteristics of the proteinase, which is capable of
recognizing and cleaving specific peptide bonds in substrates, proteolytic
processing is often considered a limited process, whereas protein degradation
refers to non-specific and extensive cleavage of peptide bonds in the substrate.
Like other post-translational modifications, proteolysis often has a role in obtaining
6
and regulating protein properties such as enzymatic activity, interactions between
proteins, and assembly. Protein hydrolysis results in stimulating conformational
changes in the tertiary structures, bringing functional residues in proximity to one
another, which fit the active pocket of enzymes or other protein complexes. The
high energy requirement for reconstruction of broken peptide bonds and the lack
of a biological system identified to repair proteolysis suggest that proteolytic
processing may be a unidirectional mechanism for a variety of biological
processes such as food digestion, signal transduction, hormone/growth factor
production, complement immune cascade and pathogen eradication (Neurath,
1989, Horl and Heidland, 1982, Reich et al. 1975).
Proteolytic processing of viral proteins is common to most, if not all viruses,
including poliovirus, African swine fever virus, human immunodeficiency virus,
herpesvirus, and adenovirus (Pallanch et al. 1984, Lopez-Otin et al. 1989, Tozser
and Oroszlan, 2003, Flynn et al. 1997, Webster et al. 1994). The proteinases can
be provided by the host cell, the virus, or both. Since cellular proteinases
generally participate in signal transduction and intracellular trafficking of proteins
via secretory pathways, viruses take advantage of cleavage reactions at secretary
compartments, where signal peptides of viral envelope and membrane proteins
are proteolytically processed and transported along with the cellular pathways.
Moreover, viral proteins possess the capacity of utilizing host-driven posttranslational modifications such as acylation and glycosylation, which is observed
for the E1 and E2 golycoproteins of Sindbis virus (Schlesinger and Schlesinger,
1990).
7
Depending on the role of proteolysis during the viral replication cycle,
there are two types of proteolytic reactions, categorized as formative and
morphogenic (Byrd and Hruby, 2006). Formative proteolysis refers to the
processing of viral polyproteins into structural and nonstructural proteins,
commonly observed in RNA viruses such as picornavirus, retrovirus and
coronavirus. (Kay and Dunn, 1990) In RNA viruses, whose genomic RNAs are of
the same polarity as the viral mRNAs, polyprotein processing acts as a
mechanism of gene expression in accordance with eukaryotic translation systems
of monocistronic mRNAs (Kozak, 1983). In addition, the synthesis of a polyprotein
in small RNA viruses can maximize “the genetic economy”, yielding several
proteins from a single transcription/translation event. Once the cleavage of the
precursor protein occurs, the resultant proteins often become activated and
localized to the appropriate compartment of the cell or to the assembling virus
particles. (Dougherty and Semler, 1993) Tobacco Etch Virus (TEV), a plant
potyvirus, encodes a NIa protease, which undergoes a formative cleavage
reaction. (Dougherty and Parks, 1991) The NIa protease is a 49kDa single
polyprotein consisting of a 27kDa C-terminal protease domain and a 21kDa Nterminal genome-linked domain (VPg). The VPg domain peptide contains a
nuclear localization signal that directly transports VPg domains to the nucleus and
covalently binds to the 5’-terminus of viral genomic RNA through a
phosphodiester linkage at the hydroxyl group of Tyr-62. The NIa protease
homologous to picornavirus 3C protease contains a chemotrypsin-like fold with a
cysteine residue in the nucleophilic active site and catalyzes a cis- or transcleavage processing.
8
Morphogenic proteolysis refers to the cleavage of virus structural
precursor proteins assembled in previrions, of which the products typically
become constituents of mature virions. The cleavage reaction commences in
combination with virus assembly and is often required for the acquisition of
infectivity. Both RNA and DNA viruses such as retrovirus, picornavirus, adenovirus
and bacteriophage T4 are reported to utilize morphogenic proteolysis. Despite
little information regarding morphogenic proteolysis, several functions have been
suggested in a variety of viruses. Both avian retrovirus and HIV retrovirus have
demonstrated proteolytic processing of the retroviral Gag polyprotein, which
produces major structural proteins participating in virius assembly and
dissemination (Craven, 1996). Absence of an active proteinase led to the failure of
the Gag cleavage reaction and the incorrect dimerizations of genomic RNA in
assembling premature virus particles and resulted in the acquisition of noninfectious virus particles (Navia and McKeever, 1990, Stewart et al. 1990).
Adenovirus proteolysis catalyzed by the L3 cysteine proteinase is required for the
proper disassembly of the virus particles at the initial stage of infection (Freimuth
and Anderson, 1993) whereas the cleavage reaction in picornavirus to process
VP0 into VP2 and VP4 takes place to mediate correct aggregation of genomic
RNA with capsid components (Lee et al. 1993). Both viruses demonstrate that
cleavage of structural proteins plays an important role in virion assembly. Similarly,
bacteriophage T4 undergoes proteolysis to promote unidirectional DNA genome
packaging (Hersko and Fry, 1975).
9
VACCINIA VIRUS PROTEOLYSIS
Vaccinia virus (VV) utilizes both types of proteolytic processing. VV growth
factor (VVGF) and the VV hemagglutinin (HA) proteins are examples of proteins
that undergo formative proteolysis. They appear to have signal peptides at the Nterminus, which are removed post synthesis and most likely processed by host
cell proteinases (Chang et al. 1988, Shida and Matsumoto, 1983, Stroobant et al.
1985). Purification and microsequencing analysis of VVGF showed it to be a
processed protein. The VVGF precursor has homology to epidermal growth factor
(EGF) and its signal sequence and transmembrane domain are removed through
proteolysis. Isolated HA mutant collections have demonstrated that the mutant
proteins were accumulated on rough endoplasmic reticulum (RER) and the outer
nuclear envelope, which has morphogenic continuity with the RER and displayed
a molecular weight difference compared to wild type HA. These properties were
explained by the fact that the proteolytic cleavage at the N-terminus caused the
disassociation of N-terminal peptides attached in the RER lumen and C-terminal
peptides projected into the cytoplasm, followed by the transportation of the Cterminal peptides toward the Golgi.
Likewise, a number of VV structural proteins, including A10L (p4a), A3L
(p4b), L4R (p25K), A17L (p21K), G7L and A12L (p17K), have been shown to
undergo morphogenic processing to produce cleavage products that are localized
to the core after virion assembly (VanSlyke et al. 1991a, VanSlyke et al. 1991b,
Whitehead and Hruby, 1994a, Mercer and Traktman, 2005, Rodriguez et al. 1993).
Through N-terminal sequencing of the cleavage products, the conserved Ala-Gly-
10
X (X: any amino acid) cleavage motif was identified. Most cleavages were
observed at AG/A sites in A3L, L4R, A17L, and A12L whereas the AG/S and AG/T
residues were utilized for A10L processing. Similar to A10L, the G7L gene product
contains AG/F and AG/L motifs, which are likely involved in proteolytic processing
(Mercer and Traktman, 2005). Studies of these core and membrane proteins, as
representative proteins of VV morphogenic processing have determined the
essentiality of their proteolytic processing for virus replication and infectivity. They
also provided more details of the participation of VV proteolysis in virus
morphogenesis such as the formation of a defined core and organization of virion
membranes. Studies with temperature-sensitive (ts) and inducible mutant viruses
revealed several DNA-binding proteins such as L4R and F17R phosphoprotein.
Both are required for the structural maturation process involved in the correct
DNA packaging and efficient DNA transcription and nucleoid condensation
(Wilcock and Smith, 1996, Zhang and Moss, 1991), respectively. One of the most
abundant core proteins, A10L is cleaved into a 60kDa (4a) and a 23kDa peptide,
which are both incorporated into the mature core. The repression of A10L
expression leads to aberrant IV formation or IMV with irregular shaped electrondense cores. The observation that A10L participates in the uptake of viral DNA
into the IV structure strongly indicates that it might have a role as a nucleoprotein
complex assembled into the IV particles (Heljasavaara et al. 2001). The A17L
gene product, a virion-membrane protein, is associated with viral crescents in wild
type virus infections but no characterized viral membranes are detected in the
absence of A17L suggesting it contributes to the generation of viral nascent
membranes at an early stage of IV formation (Wolffe et al. 1996). The G7L
11
phosphoprotein is also known to be processed at the AG/F and AG/L sites,
producing a 16kDa cleavage product. The repression of G7L demonstrated similar
phenotypic defects observed from the absence of A14L and H5R core proteins
showing a disassociation of virosomes from the crescent membranes (Mercer and
Traktman, 2005). Therefore, the VV proteins involved in morphogenic processing
play an essential role in VV morphogenesis, especially during the transition of IV
into IMV.
VV CONTEXTUAL PROTEOLYTIC PROCESSING
The significant relevance between proteolytic processing and virus
assembly, which has critical effects on acquisition of infectivity, raises the
question of how the viral proteolytic processing is regulated. Possible regulators
could be specific activators/repressors, differential compartmentalization of
enzymes and substrates, and requirements for processing in sequence. For
example, adenovirus has a disulfide-bond linked peptide produced from the pVI
structural protein, which plays a role in turning on the active L3 protease function.
Retroviruses regulate their protease activity by forming a dimer after the relatively
acidic extracellular environmental signal (Pettit et al. 1991). Sindbis virus
promotes inactivation of the viral protease by auto-catalysis right after the
assembly of the nucleocapsid, resulting in a conformational change, which
displaces the C-terminal region of the protein in the active pocket, preventing the
substrates from binding and being cleaved (Choi et al. 1991).
Irrespective of the type of regulation of proteinase activity, viruses are
12
capable to modulate a cascade of cleavage reactions in polyproteins by giving
rise to structural conformational changes. Africa swine fever virus (ASFV), a large
double-stranded DNA icosahedral virus, has shown similar characteristics of
replication in the cytoplasm, and similar genetic structures to VV. ASFV encodes
two polyproteins, pp220 and pp62, which are subsequentially processed into six
different structural proteins in the core shell. The cleavage takes place after the
second glycine in the consensus motif Gly-Gly-X (X:any amino acids) and is
catalyzed by the viral protease, pS273R cysteine proteinase. The proteinase
activity on pp62, which contains two GG-X residues, shows that an N-terminal
cleavage occurs first. The first cleavage at an N-terminus releases the final
cleavage product, p15 along with the intermediate form, pp46, which is cleaved
at the second GG-X site, producing p35 and p8. However, the cleavage sitemutated pp62 was able to show partial cleavages, indicating that the GG-X motif
is a recognition site but not an absolute requirement for proteolysis. Rather, the
first cleavage induces conformational change allowing subsequent interaction
with additional proteins or further processing (Rubio et al. 2003).
Similarly, VV proteolysis is observed at conserved Ala-Gly-X (X:any
amino acids) residues and is mainly catalyzed by the I7L cysteine proteinase
(Byrd et al. 2002). However, VV core precursor proteins demonstrated a delayed
processing of precursors to be the final products requiring up to 45 min, which is
relatively slow, compared to other proteolysis and cleavage reactions. Treatment
with cycloheximide, an inhibitor of protein synthesis caused a complete
abrogation of proteolytic maturation. This requirement of the timing and de novo
13
protein synthesis strongly suggests that the viral proteolysis takes place within
the context of virion assembly, referred as “a contextual processing”.
As an attempt to identify which proteins undergo contextual processing,
the entire VV proteome was searched for the existence of AG/X motifs. There
were 82 occurrences of an AG/X motif, most of which are not cleaved. Only 18
motifs showed the same residues as the core proteins, AG/A, S, T, which are
actively utilized for proteolysis. Of these 18 tripeptides, 5 gene products, which
contain the AG/A residues, were selected and the proteolytic processing of each
gene product and the site utilizations were carefully examined (Whitehead and
Hruby, 1994a). A DNA polymerase (DNAP, E9L), a host range protein (HR, K1L),
and an outer membrane protein (p37, F13L) were not processed while only p21K
(A17L) and p17K (A12L) were reported to produce a 21kDa and a 17kDa peptide,
respectively, both of which are localized to core of mature virions. N-terminal
microsequencing of each cleavage product confirmed that the AG/A sites serve
as an active cleavage motif. The fact that each of these proteins has the same
AG/A sites but only two proteins were cleaved at the residues suggests the more
contextually constrained requirements for proteolysis other than the consensus
motifs. That is, proteins destined for this morphogenic cleavage pathway contain
an active AG/X motif, are late proteins (DNAP is an early protein and HR is an
intermediate gene product), and are proteins incorporated within the core of
assembling virions (p37 is a late protein but localized to virion membrane.)
(Whitehead and Hruby, 1994a).
14
VACCINIA VIRUS PROTEASES
The complete genomic sequence of VV gave rise to the discovery of two
possible viral proteases responsible for the viral proteolytic processing. I7L, a
47kDa cysteine protease, has a conserved active site region with homology to
the adenovirus and ASFV cysteine proteases and likely belongs to a new family
of SUMO-1 related enzymes (Byrd et al. 2002). The nucleophilic cysteine residue
is activated by the imidazol group of the catalytic histidine residue modulating the
cleavage reactions (Byrd et al. 2003). The I7L protease activity has been
analyzed in vivo and in vitro to show that I7L is the core protein protease involved
in the proteolysis of the major core proteins such as p4a, p4b, p25K and the
p21K membrane protein at a conserved AG/X tripeptide, as indicated Fig 1.2.
The absence of I7L resulted in morphological defects such as the irregular shape
of dense particles and poorly formed cores inside of IMV particles suggesting a
requirement for I7L protease activity in virus assembly (Byrd and Hruby, 2005,
Ansarah-Sobrinho and Moss, 2004).
Another VV encoded proteinase is the gene product of the G1L ORF.
Amino acid alignment of the G1L ORF suggests that it belongs to a group of
metalloendopeptidases, which contains a conserved HXXEH zinc-binding motif.
(Becker and Roth, 1992) The HXXEH motif in G1L ORF exists as an inverted
version
of
the
motif,
HXXEHXnE,
found
in
a
variety
of
matrix
metalloendopeptidases (MMP) and shows structural similarity to the β-subunit of
the yeast mitochondrial processing peptidase. (Honeychurch et al. 2006)
Previous studies of G1L have suggested that this putative virus-encoded MMP is
15
capable of carrying out cleavage at the AG/S residues in p25K, producing 25K’
that is observed as another processed form of p25K, whereas the AG/A site in
p25K is utilized by the I7L protease (Whitehead and Hruby, 1994b). Mutant
viruses lacking G1L showed the virus particles with incomplete core
condensations as well as arrested virus replications without disrupting the
proteolysis of the core proteins (Hedengren-Olcott et al. 2004). This strongly
suggests that the G1L protease participates in the structural maturation stage of
IV to IMV just like I7L but at a later stage. However, the identification of other
substrates and the specific function of G1L are still under investigation.
VACCINIA VIRUS A12L CORE PROTEIN
The VV A12L ORF encodes a late protein of 192 amino acids with an
apparent molecular weight of 25 kDa. Previously, it was demonstrated that the
precursor protein (p17K) is processed into the molecular weight of a 17kDa
product (17K) apparently at an N-terminal AG/A site similar to p4b and p25K and
that A12L processing is sensitive to rifampicin (Whitehead and Hruby, 1994a).
However, unlike these two core proteins, of which only the mature peptides 4b
and 25K are localized to the virion, both p17K and 17K are observed in the core
of mature virions. This suggests that either A12L maturation is an incomplete
processing event or both the precursor and the cleavage product may have an
independent function in mature core formation.
Despite of the fact that A12L undergoes proteolysis at an N-terminal AG/A
site, A12L displays distinct characteristics as follows. First of all, A12L protein
16
contains multiple AG/X sites ([1] AG/A and [2] AG/K sites), which have not been
analyzed to determine their utilizations as cleavage sites. Second, the
localization of the A12L precursor as well as its cleavage products in virion
particles indicates that the A12L protein might be subjected to contextual
processing constraints. Third, the common characteristics shared with the other
core proteins suggest that A12L is likely involved in the developmental stage of
IV to IMV but the possible incomplete cleavage processing may also indicate
different requirements and regulations for the A12L proteolysis such as the
participation of G1L. Fourth, the net basic charge of A12L shows the possibility of
its interaction with viral DNA and proteins, providing a clue to their biological
function. Thus, the characterization of A12L proteolytic maturation processing as
a substrate of VV proteolysis will contribute to a better understanding of the
overall regulation of VV proteolytic processing and its participation in virus
assembly. Therefore, we attempted to investigate the proteolytic maturation of
the A12L protein by answering the following questions; which protease among
I7L, G1L or cellular proteases is responsible for A12L processing; are there any
other AG/X tripeptides utilized as cleavage motif; how is the processing
regulated; what are the functions of the precursor and the cleavage product. In
addition, the essentiality of the A12L protein and its cleavage reaction in virus
replication has not been determined. Thus, the biological function of A12L in
virion morphogenesis needs to be elucidated. Through the course of the studies
presented here, we were able to demonstrate not only the properties of A12L
proteolytic processing but also the participation/function of the A12L protein in VV
replication.
17
Figure 1.1. Vaccinia virus life cycle.
18
Figure 1.1 Vaccinia virus life cycle.
The VV life cycle begins with the attachment of virus to cell surfaces and
ends with exiting from the cell. VV undergoes three stages of gene
expressions represented by dotted arrows (blue), which are regulated in a
temporal fashion. Virus assembly initiates with the formation of cytoplasmic
foci called virus factories or virosomes (viroplasm), which are surrounded by
crescent-shaped membranes (indicated by a red curve), forming the first
characterized virus particle referred as immature virus (IV). The IV undergoes
different post-translational modifications and evolves into the characteristic of
brick-shaped intracellular mature virus (IMV). The IMV buds through the
trans-Golgi network to obtain an extra membrane forming intracellular
enveloped virus (IEV), which then fuses with the plasma membrane resulting
in the loss of the outer membrane. If IEV is released from the cell, the virion
is named extracellular enveloped virus (EEV) or if retained on cell surface, it
is referred as cell-associated enveloped virus (CEV).
19
20
Figure 1.2 Diagram of VV virion-associated proteins and their cleavage
products.
This diagram provides information of six major virion-associated proteins,
which undergo VV proteolytic processing: A10L, A3L, L4R, G7L, A17L and
A12L. Black bars indicate cleavage residues utilized to produce major
cleavage products, which are demonstrated with dotted lines. Orange bars
represent the AG/X residues not utilized for cleavage events. Most proteins
such as p4b, p25K, p21K and p17K are processed at an N-terminal AG/A
site. P4a proteolysis occurs at AG/S and AG/T sites, while G7L undergoes
cleavage at AG/F and AG/L sites. However, the AG/L in p4b and the AG/N in
p4a are not utilized as cleavage sites. P25K is cleaved at an AG/S site
located near the N-terminus, yielding 25K’.
CHAPTER 2.
CHARACTERIZATION OF A12L PROTEOLYSIS AND
ITS PARTICIPATION IN VIRUS ASSEMBLY.
21
ABSTRACT
Vaccinia virus (VV), a dsDNA virus undergoes a proteolytic processing to
evolve from immature virus particles (IV) into intracellular mature virus particles
(IMV). Most of structural core protein precursors such as p4a, p4b and p25K are
assembled into previrions and then proteolytically processed yielding the
cleavage products, 4a, 4b and 25K, which become components of a mature virus
particle. The structural rearrangement that occurs via VV proteolysis is referred to
as morphogenic proteolytic processing, and observed at a conserved cleavage
motif, Ala-Gly-X (where X is any amino acid) and is catalyzed by a VV encoded
proteinase, the I7L gene product. The VV A12L gene product, a 25kDa protein
synthesized at late times during infection, is cleaved at an N-terminal AG/A site,
resulting in a 17kDa cleavage product. Both of the A12L full-length protein and
its cleavage product are localized to mature virions. The open reading frame
(ORF) of A12L contains two more putative cleavage sites (Ala-Gly-Lys) located at
internal and C-terminal regions, although cleavage at these sites has not been
determined. Thus, we attempted to examine the in vivo processing of A12L by:
determining the kinetics of the proteolysis, the responsible viral protease, and the
function of the A12L protein and its cleavage events. Surprisingly, the 25kDa
A12L precursor was cleaved into multiple peptides and the I7L cysteine
proteinase catalyzes the AG/A cleavage events. Additional cleavage events were
observed at both the N-terminus and C-terminus but no cleavage occurred at the
two AG/K sites. An immunoprecipitation experiment in concert with mass
spectrometry and N-terminal sequencing analyses led to the identification of VV
core and membrane proteins, which may be associated with the A12L protein.
22
INTRODUCTION
Vaccinia virus (VV), the prototype member of the Poxviridae family has a
large double-stranded DNA genome. Replication and viral assembly occur
entirely in the cytoplasm of host cells, in particular, in areas referred as
viroplasms or virosomes. Virus assembly initiates at virosomes surrounded by
crescent membranes, which subsequently engulf granular materials forming
spherical-shaped particles named immature virions (IV). The IVs transform into
brick-shaped structures referred to as intracellular mature virions (IMV) where
viral DNA becomes condensed and packaged in an electron dense area and is
covered by a viral envelope membrane. A portion of IMVs are enwrapped by a
membrane cisternae derived from the trans-Golgi network and result in the
formation of intracellular enveloped virus (IEV), which then become fused with
the plasma membrane. If the IEVs remain associated with the cells, they are
referred to as cell associated enveloped virus (CEV), or if they bud through the
plasma membrane spreading outside of the cells, they are considered
extracellular enveloped virus (EEV).
Despite intensive study of VV morphogenesis, the mechanism required
for the transformation of IV to IMV still remains poorly understood. The complex
morphological developments during the transition include successful DNA
replication, concatermer resolution (Delange, 1989, Merchlinsky and Moss, 1989),
and condensation/packaging of the viral genome in IV particles (Morgan, 1976).
This is followed by encapsidation of a transcription complex, formation of a
defined core, and reorganization of virion membranes (Zhang et al. 1994). In
23
addition, a variety of post-translational modifications take place during the IV to
IMV transformation. Proteolytic processing of VV structural precursor proteins is
a prerequisite for proper virus morphogenic development and acquisition of viral
infectivity. VV structural proteins synthesized during the last stage of infection are
incorporated into pre-virions, which are proteolytically processed and developed
into mature virions.
Most of the cleavage events of virion-associated proteins take place after
the second Gly residue of an Ala-Gly-X (AG/X) conserved motif producing mature
virion components as shown in Table 2.1. Most of the precursors contain acidic
residues upstream and basic residues downstream of the cleavage site, which
are located within the N-terminal 60 amino acid residues and catalyzed by I7L, a
cysteine proteinase (Byrd et al. 2002) (Table 2.1). As an example, p4b (A3L) and
p25K (L4R) are synthesized at a late stage in the virus life cycle with molecular
weights of 66kDa and 28kDa respectively, and are proteolytically processed at an
N-terminal AG/A site to yield a 60kDa peptide, 4b and a 25kDa cleavage product,
25K respectively (Vanslyke et al. 1991a). P4a, however, a 102kDa precursor
protein undergoes cleavage events at two different AG/X motifs: an AG/S and an
AG/T located at amino acids 619 and 697 (Vanslyke et al, 1991b, Whitehead et al,
1995). Proteolysis at the AG/S and the AG/T sites leads to the release of a 62kDa
(4a) and a 23kDa C-terminal peptide. Cleavage at the N-terminal AG/A site in
A17L processes a 23kDa full-length precursor protein into a 21kDa peptide and
additional cleavage at the C-terminal AG/N site is catalyzed by the I7L core
protein proteinase (Ansarah-Sobrinho and Moss, 2004). G7L has two distinct
24
motifs, AG/F and AG/L. Mutagenesis studies have demonstrated that both of
these sites are essential for the production of infectious virus (Mercer and
Traktman, 2005). It is of interest to note that the presence of the cleavage site
consensus motif is not sufficient enough to induce VV proteolysis. Although a
partial cleavage was observed at an AG/S motif in the p25K ORF with an larger
molecular weight of 25K, referred as 25K’ (Table 2.1), the tripeptides such as
AG/L and AG/N located in the N-terminus of p4b and p4a ORF do not serve as
reaction sites. However these alternate sites do appear to be utilized for the
proteolysis of G7L and A17L .
The VV A12L protein is synthesized at a late stage with an apparent
molecular weight of 25kDa and is proteolytically processed at an N-terminal AG/A
site yielding a 17kDa polypeptide. However, the A12L ORF contains two more
putative AG/X sites (AG/K) in the internal region and C-terminus, which have not
been analyzed for the utilization of cleavage events. The participation of A12L
full-length protein and a cleavage product in mature virions suggested different
regulation/function of A12L proteolysis (Whitehead and Hruby, 1994a). Thus, the
research
on
A12L
proteolytic
processing
may
contribute
to
a
more
comprehensive understanding of the requirements/regulations for VV proteolysis
other than the conserved cleavage motif. Here, we attempted to characterize the
proteolytic processing of the A12L protein in order to determine the kinetics and
the sites selected for the cleavage reactions. We also sought to identify the
responsible protease. We were able to demonstrate possible A12L interactions
with other VV proteins, providing a clue to the biological function of the A12L
25
protein in virus assembly.
26
MATERIALS AND METHODS
Cell cultures. VV WR (Western Reserve strain) was grown on confluent
monolayers of BSC-40 cells maintained in Eagle’s minimal essential medium
(EMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen), 2
mM glutamine (Invitrogen), and 10 mM gentamicin sulfate (Invitrogen) at 37 °C in
a 95% humidified atmosphere containing 5% CO2. For infection of WR, BSC-40
cells were maintained in infection media (EMEM) supplemented with 5% FCS, 2
mM glutamine, and 10 mM gentamicin sulfate and were infected at a multiplicity
of infection (MOI) as indicated. Infected cells were harvested by centrifugation at
750x g for 10 min., and resuspended in phosphate buffered saline solution (PBS),
which contained a protease inhibitor mix tablet (Roche), followed by three cycles
of freezing and thawing to lyse the cells. After a post nuclear spin at 350x g at 4
°C, cell extracts were subjected to immunoblot or immunoprecipitation analyses.
Rifampicin-reversibility experiment. Rifampicin stock solution (10 mg/ml,
Sigma-Aldrich) was prepared in 100% Methanol (MeOH) and diluted out with
dH2O for various concentrations. BSC-40 cells were synchronously infected with
VV WR at an MOI of 5 plaque forming units (PFU)/cell and then treated with
rifampicin (150 µg/ml). The treatment with rifampicin was performed at 5 hours
post infection (hpi) for the rifampicin-reversible experiment. In order to compare
the pattern of proteolysis in the absence and presence of the drug, the VV
infected cell extracts were harvested when the drug was added and removed.
After removal of rifampicin, new infection media with and without the drug was
replaced. Infected cell pellets were re-suspended in PBS, subjected to three
27
cycles of freezing and thawing, and clarified by low speed centrifugation.
Immunoblot analysis was performed on 12% NuPAGE Bis-Tris gels (Invitrogen).
Antibody of A12L was generated by bacterial expression of A12L full-length
protein, which was fused with an N-terminal 7X His tag and affinity purified over a
Ni-NTA-agarose column. (Whitehead and Hruby, 1994a)
Kinetics of A12L processing. Confluent BSC-40 cells were synchronously
infected with VV WR at a MOI of 10 PFU/cell.
The infected cells were harvested
at various time points after infection (5, 8, 12, and 24 hpi) and resuspended in
protease inhibitor-containing PBS, followed by a post-nuclear spin as previously
described. The same amount of each sample was resolved on a 12% NuPAGE
Bis-Tris gel (Invitrogen) prior to immunoblot analysis with A12L antisera and preimmune serum was used as a control.
Pulse chase. Confluent monolayers of BSC-40 cells were synchronously
infected with VV WR at a MOI of 10 PFU/cell. At 5 hpi, [35S]-methionine (10
µCi/mL, EasyTag EXPRE35S protein labeling mixture, Perkin Elmer Life Science)
was added to the infection medium. After 1 hour, the radioactive medium was
replaced with the medium containing 100X non-radioactive methionine/cysteine
and chased for 19 hours. The infected cell extracts were used for
immunoprecipitation and analyzed by electrophoresis on a 12% NuPAGE Bis-Tris
gel. The gel was dried and exposed to a film for 72 hours.
Immunoprecipitation. Protein A-Sepharose beads (Amersham) were prepared
28
according to manufacturer’s instructions. Infected cell extracts were lysed and
diluted with RIPA buffer (Radioimmunoprecipitation buffer: 50 mM Tris [pH7.4], 1
mM NP-40, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate and
protease inhibitor cocktail tablets) and pre-cleared for an hour- incubation with rehydrated beads at 4°C. After a short spin, the supernatant was transferred to a
fresh tube and incubated with A12L antibody overnight at 4°C with shaking. Fresh
beads were added and incubated for 2-3 hours at the same temperature. The
beads were collected by a short centrifugation at 14,000x g for 40 sec, followed
by three cycles of washing with 50% PBS/RIPA buffer and the final re-suspension
in 4X sample buffer. After 5 min. of boiling, the samples were analyzed by gel
electrophoresis on a 12% NuPAGE Bis-Tris gel.
Plasmid construction and transfection. To determine the cleavage residues
and the protease responsible for A12L protein processing, three possible AG/X
sites (AG/A and two AG/K) were changed into IDI and IDR, respectively by
Quickchange site-directed mutagenesis kit (Stratagene). The open reading frame
(ORF) of both the wild-type A12L and the mutated A12L genes were placed into
the pRB21 plasmid (Blasco and Moss, 1995), which has a VV early/late synthetic
promoter. Primers for the site mutations were designed as follows: site-directed
mutation 1 (SD1) for the first AG/A mutation at the residues 55-57, 5’-CTT AAT
TCT CAA ACA GAT GTG ACT ATC GAC ATC TGT GAT ACA AAA TCA AAG AGT TCA-3’,
site-directed mutation 2 (SD2) for the middle AGK site mutation the residues 119121 into IDR, 5’-CAG ATT GTC CAA GCT GTT ACT AAT ATC GAC CGC ATA GTT TAT
GGT ACC GTC AGA GAC-3’, and site-directed mutation (SD3) for the C-terminal
29
AGK site mutation at the residues 153-155 into IDR, 5’-CTT CTA GGT ATC GAC
TCA GTT AAT ATC GAC CGC AAG AAA CCA TCT AAA AAG ATG CCT-3’. Underlined
characters indicate the mutation sites. SD1&2, SD1&3, and SD2&3 are double
site mutations generated by using each combination of the primers. In addition, a
FLAG-epitope was added to the C-terminus (FC) and N-terminus of each ORF
(FN) to discriminate this transient expression from an endogenous protein.
For transfection of the plasmids into BSC-40 cells, infection media of
EMEM was placed in new eppendorf tubes and mixed with 2 to 10 µg of DNA
and 30 µl of a transfection reagent, (DMRIE-C, Invitrogen). The mixture was
vortexed, placed at room temperature for 20 min. and loaded on 6-well plates of
~100% confluent BSC-40 cells in concert with infection of VV WR or Dts8 (IHD-J
derived I7L-termperature sensitive mutant virus, Kindly provided by Dr. Rich
Condit) at an MOI as indicated.
Two dimensional gel electrophoresis (2D gel eletrophoresis). Monolayers of
BSC-40 cells in 100mm plates were infected with VV WR at an MOI of 10
PFU/cell and harvested at 24 hpi for the immunoprecipitation with anti-A12L as
described above. The beads after the final spin were resuspended with 180 µl of
rehydration buffer (9M Urea, 4% CHAPS, 50mM DTT, 2% ampholyte, and
Bromophenol blue) for an hour at room temperature with shaking. After a short
spin, the rehydration solution was applied into the strip tray where 11cm IPG
Readystrips with a pH range of 3-10 (BioRad) were positioned overnight. The
IPG strips were transferred to a Protean IEF tray (BioRad), which was placed to
the Protean IEF cell for isoelectro-focusing. For the second dimensional gel
30
electrophoresis, the IPG strips were treated with sample preparation buffer
(0.0625M Tris [pH 6.8], 5% β-mercaptoethanol, and 2% SDS), followed by
treatment with Equilibration buffer (EB) I and II, which contained 200mg of DTT
and 250mg of Iodoacetamide respectively in 10mL of EB (6M Urea, 2% SDS,
0.05M Tris [pH 8.8], and 20% glycerol). Then, the IPG strips were rinsed with 1X
Running buffer and loaded on precast Criterion gels (BioRad) for separation on
the basis of molecular weight. The gels were either stained with Coomassie R250 solution (0.1% Coomassie R-250, 40% MeOH, and 1% Acetic acid [HoAC])
or transferred to PVDF membrane, followed by the Coommassie stain R-250.
Mass spectrometry of A12L-derived products. The BSC-40 infected cell
extracts with VV WR at an MOI of 5 PFU/cell were subjected to
immunoprecipitation experiment with anti-A12L as described above. The
immunoprecipitates of A12L protein were resolved on 2D gel, followed by staining
with Coomassie R-250 and de-staining until protein bands could be easily
visualized. Protein bands of interest were excised in as small of piece of gel as
possible. The gel slices were then dehydrated with acetonitrile (AcN) and rehydrated with 50 mM ammonium bicarbonate. This procedure was repeated and
the final dehydration was dried under a vacuum. To each tube 10-40 µL of 1
µg/µL Promega trypsin in 10 mM Tris-HCl, pH=8.0 was added. After the enzyme
solution was fully absorbed, the excess trypsin solution was removed and
replaced with 40 µL of 10 mM Tris-HCl, pH=8.0. Each sample was incubated at
37ºC for 12-16 hours. The peptides were then extracted from the gel by vortexing
with 40-80 µL of 80% AcN/5% TFA. The extraction fluid was placed in a new tube
31
and concentrated to 10-15 µL. The tryptic peptides were injected onto an HPLC
system with a C18 column system (Jupiter, 0.2 x 10 mm, 300 Å) followed by liquid
chromatography electrospray ionization quadrupole ion trap (LC-ESI-QIT) mass
spectrometry (Finnigan LCQ). HPLC was performed with a gradient from 90%
Buffer A (0.1% TFA in water) to 90% Buffer B (0.01% TFA and 5% water in
acetonitrile) over 80 min (Yoder et al. 2006). The LC-ESI-QIT MS data was
converted into Sequest DTA files and searched with the Mascot program.
Mascot (Matrix Science, London, UK) software was used for the protein
identification.
The uninterpreted tandem mass spectral data were searched
against the MSDB database, a composite, non-identical protein sequence
database built from a number of primary source databases (Matrix Science).
Differential centrifugation for subcellular fractionation. Confluent BSC-40
cells were infected with VV WR at a MOI of 10 PFU/cell and harvested as
described. From 1mL of total cell lysates, 100µl was used as total cell extracts
while the rest of the lysate was centrifuged at 700x g for 10 min. to pellet the
nuclei. Subsequent centrifugation at 20,000xg for 30 min of the supernatant
separated the soluble cytosolic fraction from the insoluble cytosolic fraction. Each
pellet of nuclei and insoluble fraction was resuspended in 900µl of PBS
(Grosenbach et al. 2000).
32
RESULTS
Multiple cleavage products of A12L protein in vivo
Previous work by Whitehead and Hruby (Whitehead and Hruby, 1994a)
demonstrated that both the A12L precursor, p17K, and the AG/A cleavage
product, 17K, were present in the core of assembling virions. To determine if any
other A12L-derived protein species were evident within the cytoplasm of VVinfected cells, cytoplasmic extracts were prepared and subjected to immunoblot
analysis using A12L antisera (anti-A12L) directed against the entire A12L protein.
Surprisingly, not only were the 25kDa (p17K) and the 17kDa (17K) proteins
detected, but also five other peptides with apparent molecular weights of 21, 18,
15, 13 and 11kDa were observed (Fig. 2.1A). Pre-immune sera of A12L did not
cross-react with any of these peptides, suggesting that all of the proteins are
indeed A12L-derived products (data not shown).
In order to determine if proteolysis was the reason for the production of a
number of A12L-derived peptides, we examined the pattern of A12L protein
processing in the presence and absence of rifampicin. Rifampicin is an antibiotic
known to reversibly block the assembly of VV by disrupting the viral membrane
biogenesis and arresting maturational events of the structural core proteins, such
as p4a and p4b (Katz and Moss, 1970). VV-infected cells were incubated with
rifampicin at various concentrations from 100 µg/ml to 400 µg/ml for 24 hours
(Fig. 2.1B). Using p4b as a positive control, we were able to show that cleavage
was suppressed at concentrations of 100~200 µg/ml of rifampicin, while
proteolysis was observed in the absence of rifampicin (Rif) treatment. Drug
33
concentrations of more than 200 µg/ml inhibited the expression of the precursor
proteins, p4b and p17K. P17K was expressed in the presence and absence of
rifampicin, but the smaller products were detected only in the absence of the drug,
similar to the p4b processing. Next we performed a rifampicin-reversibility
experiment to confirm that the A12L protein undergoes proteolytic processing,
which produces multiple peptides. The hypothesis that the rifampicin-arrested
proteolysis of A12L would be re-initiated by the removal of the drug has been
proposed from other core protein processing experiments. Infected cells were
treated with rifampicin at 5 hpi to allow the A12L precursor to be expressed and
VV proteolysis to be suppressed for the next 14 hours (Fig. 2.1C). Rifampicininhibition of VV cleavage processing resulted in no production of the A12Lderived peptides. The removal of rifampicin, however, allowed the A12L-derived
peptides to be produced whereas the continuous presence of rifampicin
completely suppressed the proteolysis of A12L. In order to rule out the possibility
of protein degradation, all the cell lysates were resuspended in PBS with a
protein inhibitor cocktail tablet and the same amount of proteins were loaded for
the immunoblot analysis. Thus, it is concluded that the A12L protein is
proteolytically processed into six peptides, including 17K, in a similar
morphogenesis-associated manner to other VV core proteins.
Kinetic analysis of A12L
For the kinetic analysis of A12L protein processing, cell extracts were
prepared at various times post infection and equal amounts of the cell lysates
were loaded for the immunoblot analysis (Fig. 2.2A). The 25 kDa precursor of
34
A12L was first detected at 5 hpi, demonstrating that the A12L protein is a late
gene product. Over time the amount of the 25 kDa species accumulated
throughout from 5 to 24 hpi. The 21, 18, 15, 13, and 11 kDa bands were first
detected at 8 hpi and accumulated from 8 to 24 hpi. Unlike these other peptides,
the 17 kDa peptide began to appear at 12 till 24 hpi. Although the A12L full-length
protein is being expressed at 5 hpi, its processing appears to be initiated at 8 hpi
and reaches a steady-state at 12 to 24 hpi. This is albeit slow compared to the
processing of other core proteins, which takes 4 to 6 hpi (Vanslyke et al. 1991b).
The slow kinetics of the A12L cleavage event may be attributed to the
possibilities of either inefficient processing or different regulation of the A12L
proteolysis. As expected from the previous pulse-chase experiment of A12L, the
full-length protein by itself may be required for mature virions or once the
quantitative requirement of the intermediate and final peptides is met, the A12L
proteolytic processing may be arrested. Moreover, the fact that the 25 kDa
precursor is processed into peptides in a range of sizes from 11 to 21 kDa
suggests that the cleavage reactions are occurring not only at the AG/A site, but
also at other residues such as the two AGK sites located internal and C-terminus
of the A12L protein.
To examine further characteristics of A12L processing, a pulse-chase
labeling experiment was conducted in concert with immunoprecipitation (Fig.
2.2B). Using cells alone as a negative control, the 25 kDa full-length A12L protein
was chased for 19 hours into four peptides with apparent molecular weights of 25,
21, 18, and 11 kDa. P17K remained relatively faint while the 21, 18, and 11 kDa
35
species became more evident after 19 hours of chase. The absence of these four
peptides in the rifampicin-treated cells confirmed that all of these peptides are
cleavage products. Importantly, the precursor remained after the chase
suggesting that the cleavage reaction of the A12L protein did not proceed to
completion. In agreement with the kinetic analysis, the proteolysis of A12L was
halted when a steady-state mixture of intermediates was formed.
Predicted characterization of A12L protein processing
The multiple numbers and the molecular sizes of the A12L cleavage
products together with the slow kinetics suggested cryptic proteolysis events
occurring at AG/K sites. The amino acid sequences of the A12L proteins encoded
by several representative orthopoxviruses shows a highly conserved alignment
(>95% identity), indicating that A12L may be essential for virus replication.
Moreover, both the N-terminal AG/A and the two AG/K motifs are conserved,
suggesting that these motifs are possibly required for protein function and the
cleavage reaction. As an attempt to identify the cleavage motifs, we considered
the possible schematic cleavage products by utilizing different combinations of all
three AG/X sites. The relative position of the three AG/X motifs within the A12L
ORF is shown in Table 2.2. The molecular sizes of the predicted cleavage
products and their calculated isoelectric points (pI’s) for both complete and
incomplete processing of the A12L precursor are also indicated. If all three sites
were utilized and the processing proceeds to completion, four small proteins with
molecular weights of 6.5, 6, 4.4, and 3.6 kDa would be produced. However, the
numbers of peptides detected during the previous immunoblot analysis implies
36
that the A12L protein is not fully processed at all of the AG/X sites. Rather, it
appears that partial cleavage at AG/X sites in concert with redundant processing
might result in production of multiple cleavage products.
Of note, for the three major core protein precursors, p4a, p4b, and p25K,
the portion of the protein that is removed by proteolysis is acidic (pI’s of 4.04,
4.08, and 3.26, respectively). Among the potential A12L fragments, only the 6
kDa (pI 5.9) and the 3.6 kDa (pI 4.8) have similar characteristics. Since the 6 kDa
protein is not detected after 17K production, the 3.6 kDa peptide might be
designed to be cleaved off. Moreover, the predicted molecular weight of the A12L
protein is approximately 21 kDa, however it runs at an apparent molecular weight
of 25 kDa.
All of the A12L-derived cleavage products also run higher than
predicted in this gel system.
AG/A utilization and C-terminal proteolysis.
In order to demonstrate the utilization of each AG/X site in the A12L ORF,
we constructed A12L expression plasmids, which contained AG/A and AG/K site
mutations into ID/I and ID/R, respectively (Fig. 2.3A). In addition, a FLAG epitope
was attached at the C-terminus of the A12L ORF to discriminate the mutated
expression from the wild-type endogenous protein. To examine the capability of a
single site as a cleavage residue, different combinations of two sites were chosen
as follows; N-terminal AG/A and middle AG/K sites (SD1&2), N-terminal AG/A
and C-terminal AG/K sites (SD1&3), and middle and C-terminal AG/K sites
(SD2&3). Under the assumption that each AG/X site is being utilized, there would
37
be peptides corresponding to the sizes of 15, 8, and 4kDa, resulting from Nterminal AG/A, middle AG/K and C-terminal AG/K cleavages respectively.
Although all of the A12L constructs with double mutations demonstrated the fulllength proteins, a 17K protein was observed only in the expression of the SD2&3
plasmid, which contained the double AG/K site mutations. This result suggests a
cleavage event only at the AG/A site without the utilization of AG/K residues.
Consistently, none of the A12L mutant constructs conjugated with a FLAG
epitope at the N-terminus displayed a 17K band, due to the N-terminal AG/A site
proteolysis. (Fig. 2.3B) However, the N-terminal AG/A site mutated A12L ORF
with a FLAG epitope at the N-terminus demonstrated another cleavage product
corresponding to a 21kDa peptide, suggesting C-terminal proteolysis. The
absence of a 21kDa signal in intact A12L with a FLAG at the N-terminus may be
explained by the complete AG/A site cleavage prior to the C-terminal processing.
Here, we were able to report only the AG/A site selection as an active
cleavage residue, ruling out the possibility of AG/K utilization. Instead, possible
proteolysis was discovered to take place at the C-terminus, yielding a 21kDa
species. In addition, we were not able to detect the other A12L cleavage products
in this transient expression experiment. Possible reasons are that cleavage
events, which occur near the N- or C-terminus would result in the degradation of
FLAG-tagged small peptides, or the FLAG epitope interrupts protein folding,
allowing only partial cleavage.
More likely, the cleavage reactions occurred in a
cascade, which may also explain the loss of signals matching to the smaller
A12L-derived peptides. If proteolysis takes place first at the AG/A site, followed
38
by another cleavage in close proximity to the C-terminus, a FLAG epitope at
either end of A12L ORF would not detect any cleavage products.
AG/A site cleavage by I7L, the VV proteinase.
Since its maturation showed similar characteristics as p25K and p4b,
whose cleavages are driven by the VV I7L cysteine protease, it was likely that
A12L might be a substrate of I7L. By taking advantage of a temperature-sensitive
mutant virus of I7L, named Dts-8 (Moerdyk et al. 2006), we were able to compare
the processing of transiently expressed A12L protein with a FLAG epitope at its
C-terminus (Fig. 2.4). While the full-length protein and 17K species were
observed at the permissive temperature (31°C), the 17K species was absent at
the non-permissive temperature (39°C), suggesting that I7L is the protease
responsible for the AG/A cleavage of A12L. This result was confirmed by a
rescue experiment using plasmid borne I7L (pI7L), which permitted p17K to be
processed into 17K at the non-permissive temperature. Using as a plasmid
vector alone, pRB21 as a negative control we did not see any signal under the
permissive and non-permissive temperatures, indicating the signals are FLAGspecific. Consequently, we concluded that the I7L protease is responsible for the
AG/A site cleavage. However, it has not been determined whether I7L is also
responsible for the production of other peptides than 17K.
Priority of N-terminal cleavage of A12L.
The transient expression experiment of the A12L with a FLAG epitope
and pI7L showed not only 17K but also some faint signal at the approximate
39
molecular weight of 21kDa. In order to determine if a 21kDa species is another
cleavage product of A12L, we repeated the transient expression experiment
transfecting A12L with a FLAG tag at the N-terminus (FN) and C-terminus (FC) in
concert with WR and Dts-8 infection. As shown in Figure 2.5, both WR and Dts-8
infection at the permissive temperature demonstrated not only a 17K but also a
21kDa species. The 21kDa species, however, appeared only in the expression of
A12L FC plasmid while no cleavage products were detected in the expression of
the A12L FN plasmid. This indicates that a 21kDa peptide is not a Dts-8 virusspecific signal but a cleavage product of A12L corresponding to the N-terminal
processing. The absence of a 21kDa species in the previous transient expression
experiments and its relatively weak intensity suggests that the 21kDa peptide
might exist as an intermediate cleavage product rather than a final product.
Taken together with the fact that pA12L FN did not show any cleavage product, it
was concluded that the cleavage events at the N-terminus occur prior to the Cterminal proteolysis, which results in the loss of the signal from pA12L FN
expression.
Possible association of A12L with a variety of VV proteins.
In order to identify the cleavage residues of the A12L-derived peptides,
immunoprecipitation of A12L was performed and resolved on 12% NuPAGE BisTris gel electrophoresis. Figure 2.6 shows the PVDF membrane, which A12L
immunoprecipitates were transferred onto and stained with Commassie R-250.
Four bands were detected with approximate molecular weights of 20kDa, which
appeared to have two peptides, 21 and 18 kDa together, and 15, 13, and 11kDa.
40
Surprisingly, only one of the four peptides, corresponding to 11kDa turned out to
be A12L, which was cleaved at the N-terminal AG/A site. In contrast, the ~21kDa
peptide was identified as an A17L gene product, which is a virion membrane
protein while the 14kDa peptide matched with the A14L protein. The sequence of
the A17L 21kDa peptide represents a 21kDa cleavage product (21K) of the
23kDa full-length A17L protein (p21K), being generated by the removal of the Nterminal 16 amino acids. Consistently, the gene product of A14L, a
phosphorylated membrane protein, is previously reported to interact with 21K,
forming a stable complex with A17L and A27L envelope proteins (Rodriguez et al.
1996, Rodriguez et al. 1997). The A12L association with these VV membrane
proteins may imply its associations with viral membrane biogenesis and
reorganization. Although we were able to obtain sequence of each of the three
peptides, some of them were mixed with other protein sequences and not
enough protein of the 15 kDa was obtained for N-terminal sequencing. Thus, to
identify other cleavage residues and determine more clearly which VV proteins
are interacting with the A12L protein, we took advantage of immunoprecipitation
with 2-dimensional (2D) PAGE for better resolution and mass-spectrometry (MS)
as well as N-terminal sequencing for acquisition of protein sequences.
Compared to a negative control, mock (Fig. 2.7) and antibody of A12L
alone (data not shown), A12L specific spots were separated and cut out for Nterminal sequencing. On the basis of N-terminal sequencing (Fig. 2.7 bottom
panel), a 13kDa peptide contains the amino acids (aa) at 57 to 66 residues of the
A12L gene product while a 11kDa peptide showed two mixed sequences with the
41
same sequences as the 13kDa A12L peptide and the sequences of a F17R gene
product from 11 to 19 residues. Due to N-terminal blockage of the other peptides,
we employed mass spectrometry to identify the proteins. As a result, a variety of
different VV proteins with sequence coverage from 12 to 55% were obtained,
which is above the minimum coverage (5 %) for protein identification. The A12Limmunoprecipitates were separated into 39, 28, 25, 23, 15, 13, and 11kDa, which
were identified as a gene products of A4L, L4R, A12L (full-lenth), A10L, A27L,
A12L (cleaved at AG/A) and F17R, respectively with the sequence coverage as
indicated in Fig. 2.7. It was surprising to report that the VV gene product
identified as A4L, a 39kDa core protein is associated with a 60kDa major
cleavage product of A10L (4a) whereas A27L, a 15kDa VV envelope protein,
associates with A17L. The A4L, a 39kDa protein, is essential for virus replication,
being required for the progression of IV to IMV (Williams et al. 1999). More
recently, it was established that the complex of A4L with p4a/4a participated in
early virus morphogenesis after its incorporation into mature virions and
proteolytic maturation of p4a (Risco et al. 1999). Similarly, the A17L complex with
A27L and A14L is responsible for the initial sequence of events of VV membrane
formation and transition from IV to IMV (Rodriguez et al. 1996, Rodriguez et al.
1993, Rodriguez et al. 1997). This suggests that the A12L protein may have a
role in early biogenesis of the VV membrane, having effects on virion assembly
together with other VV membrane and core proteins.
Intracellular localization of A12L and its cleavage products.
Since N-terminal AG/A cleavage is observed in the A12L protein, it was
42
hypothesized that the N-terminal residues might be required for localization of
A12L-derived peptides. Other core proteins such as p25K (L4R) have been
shown to be cleaved at an N-terminal AG/A site like A12L. Absence of this
cleavage in p25K resulted in impaired intraviral localization and loss of packaging
into virions. (Lee and Hruby, 1995) This is commonly observed among different
viruses, which express polypeptides and localize their cleavage products into
different subcellular locations. Thus, it was of interest to determine whether the
AG/A cleavage of A12L results in different intracellular localization of the
cleavage products from the precursor. The infected cell lysates were fractionated
by differential centrifugation to yield a nuclear fraction, a cytosolic fraction, which
includes whole virions and cytoplasmic membraneous components, and a
soluble cytoplasmic fraction (Fig. 2.8). As a control, the subcellular localization of
the L1R gene product was examined. The L1R gene product is a VV membrane
protein, which is known to be located in the nucleic and the membraneous
fraction but not found in the soluble cytosolic fraction (Grosenbach et al. 2000). In
contrast to L1R, both full-length A12L and its cleaved peptides were localized to
not only nuclear fractions but also soluble/particulate cytosolic fractions of the
total lysates. Therefore, the cleavage at the AG/A site in the A12L ORF does not
lead to different subcellular localization of cleavage products. Rather, the fulllength proteins are distributed all around the cytoplasm and then undergo
proteolytic processing, generating multiple peptides. It is also suggested that
A12L protein is not subject to contextual processing, which would demonstrate all
of the cleavage products only in the fraction that contains whole virions.
43
DISCUSSION
Investigation of the proteolytic maturation of the VV A12L core protein
yielded several unexpected results.
It is most interesting that proteolytic
processing of the VV A12L protein produces a mixture of products and does not
proceed to completion, as do the other VV core proteins previously studied.
There are two hypotheses to consider for this phenomenon.
First, perhaps
some of the multiple cleavages are “accidental”, occurring due to a quirk of
having cryptic AG/X sites within the precursor. This assumption appears unlikely
since all of the sites are well conserved with the orthopoxviruses and the viruses
have had ample time to remove the sites by mutation if cleavage was deleterious.
Furthermore, other core protein precursors have cryptic cleavage sites, (AG/S in
p25K, and AG/N in p4a) which are either not recognized or do not interfere with
the reaction proceeding to completion. Second, a more intriguing possibility is
that the incomplete processing of the A12L precursor is required to produce
multiple protein species, some of which have different functions. Certainly for
other viruses such as poliovirus, partially cleaved peptides are known to have
different functions from the fully matured products (Hellen and Wimmer, 1992). In
addition, the multiple cleavage products of A12L show both characteristics of
formative and morphogenic processing pathway, which indicate that the A12L fulllength protein and its derived peptides are involved in not only assembling
mature virions but also in producing non-structural proteins.
In contrast to the presence of multiple cleavage products in vivo, only AG/A
site cleavage is reported here, catalyzed by the I7L VV core protein proteinase.
44
Despite no observation of cleavage at the putative AG/K residues, it cannot be
ruled out that the AG/K sites may become recognizable by the proteinase after
the first cleavage. In consideration of the fact that the A12L proteolysis takes
place at an N-terminus in advance to a C-terminal cleavage, it is more convincing
to speculate that the A12L cleavage is regulated in order, of which initial blockage
may inhibit a subsequent cleavage by forming an improper structure not fully
accessible to the proteinase. However, the proteolysis at both ends of A12L
raises another possibility that the A12L proteolysis utilizes a new cleavage motif
other than the AG/X site in concert with involvement of another protease. Given
this atypical behavior it is of interest to determine the essentiality of the A12L
protein in viral replication. Therefore, a conditional A12L mutant virus
(vvtetOA12L) may need to be designed and used to address the role of A12L as
well as how important each AG/X site is to the function of A12L.
The identification of the numbers of viral proteins immunoprecipitated with
antibody of A12L is contradictory to the fact that A12L precursor proteins are
processed into the multiple peptides. This result could be explained by crossreactivity of A12L antibody. Considering the immunoblot analyses at Fig. 2, which
demonstrated the consistent pattern of detection that are regulated by rifampicin,
it would be likely that antibody of A12L precipitates virus-encoded late gene
products as the identified proteins by mass spectrometry and N-terminal
sequencing analyses are synthesized at the late stage.
Recent studies of early morphogenic processing events have provided the
45
participation of the membrane proteins such as A17L, A14L and A27L in early
development of IV particles as well as IEV particles, recruiting nascent viral
membranes to the viral foci, inducing their stable attachment to the surfaces of
viral factories, and developing envelopment of IMV particles (Traktman et al.
2000). Unlike these membraneous proteins, VV core proteins are not directly
responsible for generation of viral membranes. Instead, the association of A4L
with 4a plays a role in the correct assembly of nucleoprotein complex and
organization of IV content with the membranes while F17R (a DNA-binding
phosphoprotein), and L4R (a DNA-binding protein) are proposed to work for the
correct packaging of the viral genome and efficient transcription (Risco et al.
1999, Heljasvaaraet al. 2001, Wilcock and Smith, 1994, Zhang and Moss, 1991).
The participation of the proteins associated with A12L not only in the
development of early viral membranes but also the correct assembly of the
nucleoprotein complex and IEV development suggests that the A12L may be
involved in multiple stages of virus morphogenesis.
46
47
Table 2.1. Vaccinia virus morphogenic proteolysis.
VV has six structural precursor proteins, which undergo morphogenic
proteolysis. Both A3L and L4R are catalyzed at an AG/A site while A10L
shows the cleavage at AG/S and AG/T. A17L is cleaved at AG/A as well as
AGN, which is not utilized for A10L proteolysis. G7L utilizes AG/F and AG/L,
of which site in A3L is not catalyzed. A12L has shown to be processed at an
N-terminal AG/A just like other core proteins, while AG/K sites in residue 120
and 154 have never been determined. Thus, the consensus motif is not
enough to induce VV proteolysis. Most of cleavage events are catalyzed by
I7L cysteine proteinase whereas the responsible for the proteolysis of G7L
and A12L remains unknown. From left to right, the table shows the name of
gene product, cleavage motif (red: utilized, blue: not utilized, underlined: not
determined), the location of cleavage product, and the responsible
proteinase.
48
Table 2.2. The predicted molecular weights and pI’s of potential A12L cleavage
products.
The schematic cleavage products at each AG/X site were drawn with the
molecular weights of 6, 6.5, 3.6, and 4.4kDa. Utilizing single and double AG/X
sites, proteolytic processing of A12L were predicted as follows: cleavage at the
middle AG/K site would only produce a 12kDa and a 8kDa peptide, while
cleavages at the C-terminus AG/K site and the N-terminus AG/A site only
would introduce a 16kDa and a 15kDa product (bottom), respectively. The
utilization of both AG/A and N-terminal AG/K site would generate a 10kDa
peptide.
49
Figure 2.1. Multiple cleavage products of A12L protein.
50
Figure 2.1. Multiple cleavage products of A12L protein.
A. Multiple peptides detected by immunoblot analysis of VV-infected cells.
Mock: cells alone, WR: Western Reserve VV infected cell extracts. BSC-40
cells were infected by VV WR at an MOI of 10 PFU/cell and harvested at 24
hpi. B. Rifampicin inhibition of A12L protein processing. BSC-40 cells were
infected with VV WR at an MOI of 1 PFU/cell for 24 hours and incubated with
rifampicin at concentrations of 0, 100, 150, 200, 300, and 400 µg/ml from left
to right. As a positive control of drug induced-inhibition of VV proteolysis, p4b
processing was demonstrated. C. Rifampicin-reversibility experiment. Cells
were infected with VV at an MOI of 5 PFU/cell and treated with rifampicin
(150 µg/ml) at 5 hpi. The rifampicin was replaced with new infection media
with and without the drug at 19 hpi to determine the effects of the drug on
A12L protein processing for 12 hours. Mock (lane1): cells alone, Rif- (lane 2):
rifampicin-free cell extracts harvested at 5 hpi, Rif- (lane 3): rifampicin-free
cell extracts harvested at 19 hpi, Rif+ (lane 4): rifampicin-treated cell extracts
harvested at 19hpi, Rif+/- (lane 5): rifampicin treated cells at 5 hpi and new
media without the drug was replace at 19 hpi, Rif+/+ (lane 6): rifampicin
treated cells at 5 hpi and new media containing rifampicin was replaced at 19
hpi. Both Rif+/- and Rif+/+ were harvested at 31 hpi.
51
.
Figure 2.2. Kinetic analysis and pulse chase of A12L protein.
52
Figure 2.2. Kinetic analysis and pulse chase of A12L protein.
A. Kinetic analysis of proteolytic processing of A12L. BSC-40 cells were
infected with VV WR at an MOI of 10 PFU/cell for 30 min. and harvested at
different time courses as indicated above each lane. A 25 kDa protein
corresponds to the A12L precursor, while smaller peptides with the molecular
weights from 21 to 11 kDa are suspected to be the A12L-derived cleavage
products. B. Immunoprecipitation of pulse-chase labeled VV-infected cell
extracts. Infected cells were labeled with [35 S]-methionine for an hour at 5
hpi and chased with 100x non-radioactive methionine/cysteine. Each pulse
(P) and chase (C) of cells alone (Mock), rifampicin-treated (Rif+) and no
rifampicin-treated WR (WR) infections were analyzed. The labeled full-length
of A12L protein was processed into three peptides with apparent molecular
sizes of 21, 18, 11kDa. The precursor remained after the chase suggesting
that A12L proteolysis did not go to the completion.
53
Figure 2.3. Proteolysis of A12L.
54
Figure 2.3. Proteolysis of A12L.
A. The A12L ORF with double AG/X site mutations were placed into pRB21,
which has an early/late synthetic promoter and appended with a C-terminal
FLAG epitope (FC). The N-terminal AG/A site and internal AG/K site
mutations, the N-terminal AG/A and C-terminal AG/K site mutations, and the
internal and C-terminal AG/K site mutations were indicated as SD 1&2, SD
1&3, and SD 2&3, respectively, of which transient expression would result in
4, 8, and 15kDa cleavage product. The amino aicds, A, G, and K were
converted into I, D, and R, respectively. B. All of the plasmids contained the
same mutations as described above except a FLAG epitope in the Nterminus (FN) of A12L ORF. A12L-FN represents A12L intact ORF with a
FLAG epitope at the N-terminus while Ara-C refers to the cells transfected
with A12L FN in the presence of cytosine arabinoside (Ara-C, 40µg/mL). The
FLAG tag at the N-terminus of each mutant plasmid would represent the
products of 16, 12, and 6kDa peptides resulted from each utilization of the Cterminal AG/K, internal AG/K, and N-terminal AG/A site.
55
Figure 2.4. The AG/A site cleavage by the VV proteinase I7L.
BSC-40 cells were transfected with a plasmid containing a FLAG epitope at
C-terminus of A12L ORF (pA12L-FC) and infected with WR or Dts-8 (I7L
temperature-sensitive mutant virus). Having WR-infected cells as a positive
control, Dts-8 infection at the permissive (31°C) and non-permissive (39°C)
temperatures showed I7L participation in A12L cleavage event. pA12L:
plasmid born A12L under an early/late synthetic promoter, pRB21: vector
plasmid of pA12L. pI7L: plasmid born I7L in pRB21.
56
Figure 2.5. N-terminal proteolysis of A12L.
To determine another cleavage reaction observed by a FLAG epitope at the
C-terminus of A12L (pA12L-FC), the A12L plasmids with a FLAG epitope at
C-terminus (pA12L-FC) and N-terminus of A12L ORF (pA12L-FN) were
transfected into BSC-40 cells and infected with VV WR and Dts-8 (I7L
temperature-sensitive mutant virus) at an MOI of 5 PFU/cell. Both infections
were incubated at permissive temperature (31°C).
57
Figure 2.6. Identification of A12L-derived peptides.
BSC-40 cells were infected with WR at an MOI of 5 PFU/cell, of which cell
lysates were subjected to immunoprecipitation analyses with anti-A12L. The
immunoprecipitates were resolved in 12% gel, transferred to PVDF
membrane, followed by staining with Coomassie R-250. The four bands in
molecular weights of 20, 15, 13, and 11kDa were cut out and sent for Nterminal sequencing. The sequence data we obtained from N-terminal
sequencing is represented in the table. Only the 11kDa peptide was identified
as A12L peptide, which contained sequences after the N-terminal AG/A
cleavage event, while 13kDa and 20kDa peptides were identified as A14L
and A17L proteins. Arrows indicate the peptides, which are N-terminally
blocked or not enough protein to analyze the amino acid sequences.
58
Figure 2.7. Association of A12L with other VV proteins.
59
Figure 2.7. Association of A12L with other VV proteins.
The immunoprecipitates with anti-A12L were absorbed in IPG strips for two
dimensional gel eletrophoresis (2D-gel), which were stained with Coomassie
R-250. The distinguished spots were cut out and sent for either N-terminal
sequencing or MS analyses (LC-ESI-Q-TOF MS). The upper panel shows
the immunoprecipitates of the cells alone (Mock) while the bottom panel is
WR-infected cell lysates (WR) immunoprecipitated with anti-A12L.
Arrowheads are the A12L-derived peptides distinguished from mock (upper
panel) and antibody alone (data not shown). The table underneath the 2D gel
stains shows the summary of the total results from both analyses.
60
Figure 2.8. Subcellular localization of A12L protein.
BSC-40 cells were infected with WR at an MOI of 10 PFU/cell and the cell
extracts were separated by differential centrifugations. TCE: total cell
extracts, NP: nuclear pellet fraction, PC: particulate cytosolic fraction, which
contains whole virions and membraneous cytosolic portions, SC: soluble
cytosolic fraction. Right and left panels show each localization of A12L and
L1R, respectively. The localization of L1R, a VV membrane protein in the
nucleic fraction and particulate cytosolic fraction showed the differential
fractionation was conducted correctly.
CHAPTER 3.
ESSENTIALITY OF A12L IN VIRAL REPLICATION AND
MORPHOLOGICAL TRANSITION.
61
ABSTRACT
Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25
kDa VV A12L late gene product (p17K) undergoes proteolysis at the conserved
Ala-Gly-Ala motif. However, unlike p4b and p25k, the p17K precursor was
cleaved into multiple peptides via additional cleavage events. Furthermore, the
association of A12L with VV core and membrane proteins suggested that A12L
might have a function in the generation of immature virion (IV) membranes and
core formation of intracellular mature virions (IMV). Here, in order to test the
requirement of the A12L protein in viral replication and elucidate its biological
function, a conditional lethal mutant virus (vvtetOA12L) was constructed to
regulate A12L expression by the presence or absence of tetracycline. In the
absence of tetracycline, replication of vvtetOA12L was inhibited by 75% and this
inhibition could be overcome by transient expression of the wild-type copy of the
A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the
transfected A12L gene to rescue just like other A12L truncates, indicating that
A12L proteolysis is required for VV replication. Electron microscopy analysis of
the A12L deficient virus infection demonstrated the aberrant viral assembly,
which displayed albeit larger aggregates of virus factories, viroplasm detached
from the viral membrane and disrupted core formation inside of IMV. Taken
together, we concluded that the VV A12L gene product and its cleavage products
are required during the viral replication cycle.
62
INTRODUCTION
Proteolytic processing in vaccinia virus (VV) plays an important role in
morphogenic transitions during the virus replication cycle. To date, six VVencoded, proteolytically processed proteins have been reported. They are the
gene products of A10L (p4a), A3L (p4b), L4R (p25K), A17L (p21K), G7L, and
A12L (p17K) (Vanslyke et al. 1991b, Whitehead et al. 1995, Vanslyke et al.,
1991a, Whitehead and Hruby, 1994a, Mercer and Traktman, 2005). Extensive
studies of the structural proteins have provided more specific mechanisms of VV
proteolysis in terms of the transformation of immature virions (IV) into intracellular
mature virions (IMV).
One of the VV major core proteins, A10L has been shown to be essential
through a IPTG (isopropyl β-D-thiogalactopyranoside)-regulated mutant virus,
with more than one log reduction in virus yield in the absence of the inducer
(Heljasvaara et al. 2001). Aside from the expression of A10L, VV protein
synthesis was the same both in the presence and absence of IPTG while little
processing of p4b and p25K occurred in the absence of the inducer. The absence
of A10L resulted in defective virus morphology such as IV-like particles, which
lacked dense viroplasm and consequently produced the irregular-shaped virus
particles. Regardless of the disturbed IV contents, the formation of the viral
membrane and envelope was indistinguishable from WR infected cells. On the
basis of the electron microscopic analyses, it was suggested that p4a and its
cleavage products are required for VV proteolysis and the correct organization of
the nucleocomplex within the IVs (Heljasvaara et al. 2001, Risco et al. 1999).
63
L4R (p25K), a DNA binding protein is essential for virus replication. A conditional
mutant virus of p25K demonstrated approximately 80% of the wild-type level of
mature virus particle formation such as intracellular mature virion (IMV) or
extracellular enveloped virion (EEV) but a two log-reduction in infectivity,
indicating that p25K is required for acquisition of viral infectivity not for the virus
particle formation. This inconsistent result implied that p25K might be involved in
early stage of infection such as early transcription or unpackaging viral DNA and
core (Wilcock and Smith, 1994). The absence of p25K led to a defective
phenotype with non-associated viroplasm and surrounding viral membranes but
no blockage of proteolysis of p4a and p4b, suggesting an important role for p25K
in correct incorporation of viral DNA and cores with immature virus membrane
but not for VV proteolysis.
Both the G7L and A17L gene products are essential for virus replication
and are involved in the early development of IV membranes in contrast to A10L
and L4R, which play a role in the correct IV content and its association with the
membrane. G7L, a phosphoprotein associated with the A30L and H5R proteins,
is responsible for the correct recruitment and attachment of crescent-shaped
membranes to viroplasms (Szajner et al. 2003). The absence of G7L
subsequently led to defective IV formation, which showed tubular elements apart
from the granular virosomes and instead displayed empty or multiple wrapped IV
particles, eventually affecting the integrity of the virosomal matrix in corporation
of A30L and H5R (Mercer and Traktman, 2005, Szajner et al. 2004). The A17L
mutant virus under non-permissive conditions produced large aggregates of
64
accumulated electron-dense materials, and numerous vesicles/tubules engulfing
viroplasms, demonstrating that A17L is an essential component for generation of
IV and IMV membranes (Rodriguez et al. 1996, Wolffe et al. 1996, Mercer and
Traktman, 2005). A17L (p21K) and its cleavage product (21K) co-localized with
GTPase Rab1, a marker of intermediate compartment (IC) membranes, the origin
of viral membrane (Krinjse-Locker et al. 1996), suggesting the A17L participation
in very early stage of the membrane biogenesis. Another viral membrane
component, A14L, was localized into virosomes only in the presence of A17L,
which also associated with A27L, an envelope protein required for IMV transport
out of virus factories (Rodriguez et al. 1996, Sanderson et al. 2000) Thus, A17L
protein participated in multiple steps of VV morphogenesis from biogenesis of
viral membranes to the formation and envelopment of IMV particles. Despite the
fact that most of the VV structural precursor proteins that undergo proteolytic
maturation play a role in the organization of the first recognized membrane and
the formation of viral genome content, the essentiality and biological role of the
A12L gene products have not been analyzed.
VV A12L is a late gene product, which is proteolytically processed from a
25kDa precursor into a 17kDa cleavage product (Whitehead and Hruby, 1994a).
Its proteolysis is similar to the processing of the other VV core proteins in that the
cleavage is regulated by rifampicin, takes place at the conserved recognition
motif, Ala-Gly-X (X: any amino acid), is catalyzed by the I7L VV core protein
proteinase, and is associated with mature virions. On the other hand, there is
also an evidence to distinguish A12L proteolysis from other cleavage events.
65
First of all, a 25kDa full-length protein is processed into more than one cleavage
product, yielding a total of six peptides. Second, the proteolysis of A12L does not
proceed to completion, rather it is halted after a mixture of intermediate and final
products is obtained, suggesting an incomplete but redundant cleavage
processing of A12L. Third, A12L maturation takes relatively longer in comparison
to p4b and p25K, which are also cleaved at an N-terminal AG/A residue. Despite
the same cleavage motif located within the N-terminal 60 residues, the kinetics of
A12L proteolysis is more similar to that of p4a maturation, which takes 8 to 12
hours after infection. Thus, A12L cleavage event may introduce different
requirements or participants for the regulation of VV proteolysis.
To investigate the requirement of the A12L protein and elucidate its
function in virion-morphogenesis, we constructed a conditional mutant virus of
A12L, in which protein expression can be regulated by tetracycline (Tet)
(Hedengren-Olcott and Hruby, 2004). The Tet-regulated genetic system is
introduced in Table 3. 1. By placing the Tet operator (TetO) in front of the A12L
open reading frame (ORF), A12L gene expression can be suppressed by the
presence of the Tet repressor (TetR), which binds to the TetO and consequently
blocks the transcription of A12L. In the presence of Tet, however, Tet binds to
TetR and leaves TetO available for transcription. For this system, the recombinant
virus contains the TetO and we used T-REx 293 cells (Invitrogen), which
constitutively express the TetR. In order to construct the mutant virus, we
designed a plasmid, which contains the Neomycin selective marker (NeoR) as
well as two homologous regions to A12L and its neighbor gene, A13L, with the
66
TetO placed between A12L and A13L. Two rounds of virus homologous
recombination resulted in generating an A12L mutant virus, referred to as
vvtetOA12L.
Here, we report that the absence of A12L results in approximately one log
reduction of virus replication in concert with phenotypic defects. In addition,
plasmid bourn A12L with an AG/A site mutation, which would prevent A12L
proteolysis, failed to rescue the lack of A12L demonstrating that cleavage of A12L
is essential for virus replication as well as formation of mature virions.
67
METHODS AND MATERIALS
Cell cultures. VV WR (Western Reserve strain) was grown and titered on
confluent monolayers of BSC-40 cells maintained in Eagle’s minimal essential
medium (EMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS,
Invitrogen), 2 mM glutamine (Invitrogen), and 10 mM gentamicin sulfate
(Invitrogen) at 37 °C in a 95% humidified atmosphere containing 5% CO2. For
infection of the conditional mutant virus of A12L (vvtetOA12L), T-REx 293 cells
(Invitrogen) were grown in Dulbecco’s modified Eagle’s medium (D-MEM,
Invitrogen) supplemented with 10% Tet system approved fetal bovine serum (BD
Biosciences), 2 mM Glutamax (Invitrogen), and 1% penicillin–streptomycin
(Invitrogen), and incubated as described above. Blasticidin (5 µg/ml, Invitrogen)
was added to the D-MEM growth media for selection of the pcDNA6/TR plasmid
(Yao et al. 1998), which expresses the tetracycline repressor (TetR).
Virus infections and titers. BSC-40 cells in infection media of EMEM
supplemented with 5% FCS, 2 mM glutamine, and 10 mM gentamicin sulfate
were infected with VV WR at an multiplicity of infection (MOI) as indicated.
Infected cells were harvested by centrifugation at 750x g for 10 min., followed by
a post nuclear spin at 350x g at 4 °C. The mutant virus (vvtetOA12L) was grown
on T-REx 293 cells as previously described. When T-REx 293 cells were
approximately 80% confluent, vvtetOA12L virus in phosphate-buffered saline
(PBS) at an MOI of 1 plaque forming unit (PFU)/cell were placed on the cells for
30 min at room temperature. The infection D-MEM containing 5% of Tetapproved FBS, L-glutamax (10 mM), penicillin–streptomycin (10 mM) was then
68
added. Tetracycline (10-30 µg/ml, Sigma–Aldrich) was placed in infection D-MEM
media for induction of the A12L protein. Cell extracts were harvested at 24-48
hours post infection (hpi) by centrifugation (750x g) for 5 min at 4°C, followed by
three cycles of freezing and thawing to lyse the cells. Virus titers were conducted
on BSC-40 cells, incubated at 37°C for 40 hours, and stained with 0.1% crystal
violet solution in 30% ethanol.
Construction of conditional mutant A12L virus (vvtetOA12L). VV WR was
used for the construction of the conditional mutant A12L virus (vvtetOA12L). The
tetracycline operator (TetO) was inserted in front of the A12L ORF by virtue of
two-step polymerase chain reaction (PCR) and amplified with 215 nucleotides
(nts) upstream of the A12L ORF and 213 nts downstream of the A13L ORF. The
PCR products were cloned into the p7.5:NEO vector (Franke et al. 1985),
resulting in the construction of the p7.5:TetOA12L:NEO plasmid. Transfection of
the p7.5:TetOA12L:NEO plasmid in concert with VV WR infection induced the
first recombination (Table 3.1). The Neomycin resistance gene (NEOR) in the
p7.5:TetOA12L:NEO plasmid was used as a transient selective marker in the
presence of Geneticin G418 sulfate (Invitrogen). The second recombination of
NEOR-containing viruses occurred in the absence of Geneticin G418 sulfate,
producing a wild type virus and an A12L mutant virus (vvtetOA12L) containing
TetO without NEOR. Plaque purifications were performed in concert with PCR
screens using the primers specific for TetO and 3’ end of A12L ORF to identify
pure vvtetOA12L isolates. Experimental infections of vvtetOA12L were carried
out in T-REx 293 cell line, which constitutively expresses the Tetracycline
69
repressor (TetR).
Transfection and marker rescue. In order to rescue the absence of A12L by
plasmid-bourn intact A12L (pA12L), full-length of A12L ORF was placed right
after an early/late synthetic promoter in pRB21 (Blasco, R. 1995). The same ORF
were placed in TOPO TA cloning vector (Invitrogen) to have its native promoter,
which contained upstream 233 nucleotides (p233-A12L). To place A12L ORF in
both pRB21 and TOPO vector, two different sets of primers were designed;
pA12L-forward:
pA12L-reverse:
5’-CACTCCATGGATGGCGGATAAAAAAAATTTAGCC
5’-CAGGATCCTTAATACATTCCCATATCCAGACAAC;
and
p233-
forward: 5’-ATGGCGGATAAAAAAAATTTAGCC and A12L-reverse: 5’-TTAATACA
TTCCCATATCCAGACAAAATTCG. In order to construct A12L with abrogated
cleavage at an N-terminal AG/A site (AG/A), the AG/A sites were altered into ID/I
by site-directed mutagenesis kit (Stratagene) with a specific primer, which has the
changed sequences at the residues 55-57 (underlined), 5’-CTTAATTCTCAA
ACAGATGTGACTATCGACATCTGTGATACAAAATCAAAGAGTTCA-3’
and
was
inserted in pRB21 vector.
To construct all of the A12L truncates, different forward primers were
designed as follows. For the construction of 17K, we used a 17K-forward primer,
5’-ATGGCATGTGATACAAAATCAAAGAGTTC and the A12L reverse primer,
while 2K and 3K were generated by a A12L forward primer, 5’- ATGGCG
GATAAA AAA AATTTAGCC and reverse primers covering nucleotides at middle
and C-terminal AG/K: 2K-reverse: 5’-TTAACCAGCATTAGTAACAGCTTGGAC
and 3K-reverse: 5’-TTACCCAGCATTAACTGAGTCGATACCTAG. To make A12L
70
truncates cleaved at an N-terminal AG/A site and at a middle AG/K (A2K) or Cterminal AG/K (A3K), we used the 17K-forward primer, and the 2K and 3K
reverse primers. The PCR products were conjugated with the 233nts native
promoter by performing another PCR and placed into TOPO TA cloning vector
(Invitrogen).
For transfection of plasmids into T-REx 293 cells, infection media of DMEM medium was placed in new eppendorf tubes and mixed with 2 to 10 µg of
DNA and 30 µl of the transfection reagent, DMRIE-C (Invitrogen). After vortexing
the mixture, it was placed at room temperature for 20 min. and loaded on 6-well
plates of ~60% confluent T-REx 293 cells. The cells were incubated at 37 °C for
5-6 hours and infected by vvtetOA12L at an MOI of 1 PFU/cell for 24 hours. Virus
titers were determined as described earlier.
Electron microscopy. T-REx 293 cells were infected at an MOI of 1 PFU/cell
with vvtetOA12L and harvested at 24 hpi by centrifugation (270x g) at 4°C. The
cell extracts were resuspended with 1X PBS, followed by incubation with fixative
buffer (2% glutaraldehyde, 1.25% paraformaldehyde in 0.1 M cacodylate buffer
[pH7.3]) for 2 hours at room temperature. Postfixation, ultrathin section, and
staining were performed as described (Hedengren-Olcott et al. 2004).
71
RESULTS
Tet-regulated conditional mutant virus of A12L.
To examine the regulation of a conditional mutant virus of A12L
(vvtetOA12L), we infected T-REx 293 cells with vvtetOA12L at various
concentrations of Tet from 0 to 40 µg/mL (Fig. 3.1A). Virus yield increased as the
concentration of Tet increased from 0 to 30 µg/mL. This increased virus yield
demonstrates that vvtetOA12L replicates in a Tet-dependent manner. Setting the
optimal concentration of Tet at 30 µg/mL, we performed a one-step growth curve
of vvtetOA12L with the cell extracts harvested at different time points after
infection. Figure. 3.1B shows the initial drop of virus yield at 5 hours post
infection (hpi), when the A12L protein begins to be expressed as a late gene
product. The maximum viral yields of vvtetOA12L in the presence of Tet was
obtained at 24 hpi with approximately one log difference, which is attributed to
the expression of the A12L protein and its essentiality in virus replication.
Essentiality of A12L protein in VV replication.
The sequence alignment of the A12L ORF with other representative
orthopoxviruses such as cowpox, variola, and ectromelia viruses has shown
highly conserved sequence alignment with more than 95 % identity. Thus, it is
expected that A12L may be essential for virus replication. Moreover, both the Nterminal AG/A and the middle AG/K motifs are highly conserved while the Cterminal AG/K residue is relatively less conserved. This may indicate that these
motifs are possibly required for maintaining the protein function and proteolytic
maturation.
72
To determine the essentiality of the A12L protein in viral replication, a
conditional A12L mutant virus (vvtetOA12L) was used to address the requirement
of the A12L protein and the AG/A site cleavage for viral replication. To begin with,
A12L protein expression was confirmed by immunoblot analysis with A12L
specific bands obtained only in the presence of Tet (data not shown). Infection of
vvtetOA12L was carried out in T-REx 293 cells and titered on BSC-40 cells.
Approximately 75 % reduction of virus titer was observed in the absence of Tet
(Fig 3. 2), suggesting that A12L is required for VV replication. Confirmation that
the defect in replication was due to the shut-off of A12L expression was obtained
by marker rescue experiments. Plasmid-borne A12L under the control of either its
native promoter, which includes 233 nucleotides upstream of the A12L ORF
(p233-A12L), or an early/late synthetic promoter in pRB21 vector (pA12L)
provided almost 100 % rescue in virus yield. This rescue experiment established
the essentiality of A12L expression in viral replication despite the leakiness of
vvtetOA12L. Another rescue experiment of A12L expression with the AG/A site
mutation (AG/A) failed to complement the absence of A12L protein, resulting in a
virus yield, which is similar to the titer of vvtetOA12L infection in the absence of
Tet. Therefore, it is suggested that cleavage at the AG/A site plays an essential
role in A12L functionality. In comparison to the AG/A mutant A12L plasmid, the Nterminal AG/A cleavage product, 17K, showed even lower levels of rescue,
implying that the proper removal of the N-terminal residues at the AG/A site is
required for virus replication.
As an attempt to discover the final cleavage product of A12L, we
73
constructed possible truncates of A12L proteins by utilizing all three AG/X sites
and examined the virus replication under the transient expression of each
truncate (Fig 3. 2). Since all the truncates of A12L failed to rescue the lack of
A12L, it is hypothesized that A12L may not be proteolytically maturated into one
final cleavage product, rather required to have a mixture of different sizes of
products, which are all essential for VV replication. The failure of two AG/K
mutations of A12L ORF also indicated that all three AG/X residues play an
important role in maintaining the biological function of A12L proteins (data not
shown).
Morphology defects in the absence of A12L expression.
In order to study the phenotypic effects of A12L repression in the mutant
virus, T-REx 293 cells were infected with vvtetOA12L in the presence and
absence of Tet (Fig. 3.3). In the presence of Tet, vvtetOA12L was able to
assemble into mature virions as wild type VV does, producing oval particles with
condensed cores (Fig. 3.3). In the absence of Tet, vvtetOA12L displayed several
phenotypic defects. The A12L deficiency caused accumulated granules of
electron-dense areas including viral DNA and protein-rich aggregates while
crescent membranes were formed. Some immature virus particles (IV) were
devoid of the internal materials or contained small IV contents surrounded by
irregular-shaped membranes. This indicated that the absence of A12L might
delay or interrupt recruitment of viral membrane to engulf and adhere to the viral
materials, which eventually led to the abrogated formation of spherical
membranes. A portion of the abnormal IV particles was able to mature into IMV,
74
however the core did not form the characteristic of the bi-concave core shape.
Rather the cores of the IMV retained a round shape, which appeared to lose the
center-compressed concave structure. Thus, we concluded that the lack of A12L
led to not only the defects in the association of the viroplasm with crescentshaped membranes but also the formation of spherical IV membranes and
subsequent disruption of interior cores of the IMV.
Morphology defects by abrogated AG/A cleavage of A12L.
The morphogenic defects of the mutant virus under the restrictive
conditions could be overcome by the transient expression of plasmid borne A12L
(Fig. 3.4). Consistent with the rescue experiment, plasmid born A12L was able to
form regular IV particles, which had electron-dense viral materials inside and
associated with the spherical membrane tightly. In addition, a condensed core
was observed together with the development of the inner layer, which established
the biconcave characteristics of IMV particles. The AG/A site mutant A12L,
however, failed to produce fully matured IMV particles. The transient expression
of AG/A site mutant A12L demonstrated similar phenotypic deformities as the
absence of A12L, producing the irregular shaped IV-like particles with little viral
material. Relatively more regular IV particles, however, were displayed while the
core appeared to be condensed but failed to form a concaved layer inside of the
IMV particles. Similarly, IMV particles retained round boundary membranes and
abnormal inner layers. This can be explained by the fact that the impaired
cleavage at an N-terminal AG/A site might lead to the correct assembly of IV
contents and its close adhesion to the IV membrane but not to the proper core
75
condensation in concert with a concave inner core layer.
76
DISCUSSION
The VV A12L protein, which has a highly conserved sequence alignment
with other orthopoxviruses, plays an essential role in virus replication
demonstrating more than 70% reduction in virus yield in the absence of A12L.
Marker rescue experiments with vvtetOA12L infected cells not only confirmed the
essentiality of A12L but also demonstrated that the mutation of AG/A site in the
A12L protein eliminated its complementing functionality, strongly suggesting that
the proteolytic processing of A12L is essential, possibly due to the impaired
morphological transition of IV to IMV as indicated by electron microscope
analysis.
In addition, the failure of each of the A12L truncates to rescue the lack of
A12L protein provides several clues regarding its proteolysis. First of all, A12L
proteolysis does not simply produce one major cleavage product, rather it yields
a stable mixtures of intermediate and final peptides, both of which have an
essential role in virus replication. Second, the fact that A12L truncates such as
17K, 2K, and 3K were not able to rescue despite relatively more coverage of the
A12L ORF indicates that the interruption of the first cleavage at an N-terminus or
C-terminus may block subsequent proteolysis. In agreement with previous
studies of A12L proteolytic processing, we have shown that the cleavage events
of A12L take place in order in concert with conformational changes, which allow
the very next cleavage event. Third, the virus yield obtained by the transfection of
AG/A site mutated A12L (AG/A) displayed higher virus titers than 17K, suggesting
that the N-terminal residues of the AG/A may have a critical role in A12L
77
maturation. This could explain the lower virus titer in the cells transfected with
A2K and A3K. The AG/A site mutated A12L might inhibit further cleavage
processing but may also lead to N- and C-terminal cleavages producing ~21 kDa
peptides, which could explain the relatively higher rescue than 17K.
The absence of A12L resulted in the phenotypic defects of arresting the
development of IV particles prior to acquisition of the typical biconcave-core
shape. It is of interest to report that these defects were observed throughout the
virus life cycle from the early stage of IV formation to the very last stage to
become fully matured IMV. The IV particles generated in the absence of A12L
were filled with small electron-dense viral DNA or surrounded by irregular-shaped
membranes, suggesting A12L participation in formation of spherical membranes
and its tight association with IV contents. In addition, the A12L deficiency
displayed aberrant core condensation together with more round shaped-inner
layers of IMV. This phenotypic defect is similar to that of the A10L deficient virus
infection, which suggested A10L-induced DNA uptake into spherical IV particles
and formation of nucleoprotein complex within the IV (Heljzsvaara et al. 2001).
The empty and partially electron-dense materials observed by A12L deficient
virus infection provides supportive evidence of possible incorporation of A12L
with A10L (23kDa cleavage product) previously reported, implying that the A12L
protein together with the C-terminal cleavage product of A10L might be
responsible for the correct organization of the IV contents. The absence of the
association between A12L and A10L might allow only partial uptake of the dense
viral materials into virus particles. In addition, the abrogated biconcave IMV
78
particle with inner layer was obtained by the lack of both proteins, extending a
role of the A12L into the formation of a center-compressed core in IMV particles.
It is worthwhile to note that the aggregated viral foci and IV-like particles
were reported by A17L and A14L deficient viruses, which have a role in the
recruitment of viral membrane adjacent to viroplasmic materials and the
attachment of virus membranes with electron-dense viral materials (Wolffe et al.
1996, Rodriguez et al. 1998). In terms of the generation of viral membranes,
A12L deficient virus introduced neither the absence of viral membrane nor
unfinished or interrupted IV membranes, which were observed by the lack of
A17L and A14L, respectively. On the basis of the possible association of A12L
with A17L and A14L previously suggested, it can be speculated that A12L with
these two proteins might not be responsible for the generation of the crescent
membranes but instead for their correct positions and linkage to viroplasm. Thus,
this electron microscopy analysis confirms that the A12L protein is required for
virus replication and complete virion maturation. The similar phenotypic defect of
each mutant virus of A12L and its potentially-associated proteins may highlight
the participation of VV proteolysis in the correct assembly of nucleoprotein
complex in IV particles, the capability to maintain the stable spherical shape of IV,
proper condensation of the core and its layer into center-concaved IMV formation.
Therefore, additional characterization of the vvtetOA12L mutant virus would be
interesting in order to establish the more specific biological function of the A12L
protein regarding its contribution during VV morphogenic transitions.
79
Table 3.1. Schematic diagram of a conditional mutant virus of A12L.
80
Table 3.1. Schematic diagram of a conditional mutant A12L virus.
To investigate the essentiality of A12L in virus replication and its biological
function, a conditional mutant A12L virus (vvtetOA12L) was constructed by
taking advantage of the viral homologous recombination. The Tet operator
(TetO) flanked with A12L and A13L was placed in the p7.5:NEO plasmid,
which contains the Neomycin-resistance gene (NeoR) as a selective marker.
The resulting p7.5:TetOA12L:NEO plasmid was subjected to another
homologous recombination and screened for the vvtetOA12L mutant virus.
The vvtetOA12L mutant virus can be regulated through the absence or
presence of Tet. In the absence of Tet, the tetracycline repressor (TetR)
expressed from T-REx 293 cells occupies the TetO blocking the transcription
of A12L. The addition of Tet, however, binds to the tetracycline repressor
(TetR) leaving the promoter of A12L available for transcription.
81
Figure 3.1. Tet-dependent replication of vvtetOA12L and
one-step growth curve.
82
Figure 3.1. Tet-dependent replication of vvtetOA12L and one-step growth
curve. .
A. Tet-dependent replication of vvtetOA12L. T-REx 293 cells were infected
with vvtetOA12L at a MOI of 1 PFU/cell in the presence of tetracycline (Tet)
at various concentrations of 0, 10, 20, 30, and 40 µg/mL. The infected cell
extracts harvested at 24 hpi were titered on BSC 40 cells to determine the
virus yields. As the concentration of Tet increased, the virus yield increased
showing that virus replication of vvtetOA12L is Tet-dependent. B. One-step
growth curve. T-REx 293 cells were infected with vvtetOA12L in the presence
and absence of Tet (30 µg/mL) and harvested at 3, 5, 8, 12, and 24 hpi. The
reduction of virus titer was observed at the time when A12L protein
expression was initiated and reached maximum levels at 24 hpi.
Approximately one-log reduction of vvtetOA12L in non-permissive condition
showed the requirement of A12L protein in VV replication. Each virus titer
(PFU/ml) was scaled in log phase.
83
The construction of A12L truncates cleaved at all three AG/X sites
Figure 3.2. Essentiality of A12L protein in VV replication.
84
Figure 3.2. Essentiality of A12L protein in VV replication.
The upper panel is a diagram of the constructed A12L truncates. Under the
assumption that all of three AG/X sites in A12L ORF are utilized, A12L
truncates were constructed. 17K is the cleavage product at an N-terminal
AG/A while 2K and 3K are the cleavage products at an internal (2) and Cterminal AG/K (3) sites, respectively. A2K and A3K are the peptides
processed at an N-terminal AG/A, followed by an additional cleavage event
at an internal and C-terminal AG/K residue. All of the A12L truncates were
placed under the native promoter, which contains upstream 233 nts. Bottom
panel shows the virus rescue experiment of vvtetOA12L with plasmid born
A12L and A12L truncates. T-REx 293 cells were infected with vvtetOA12L in
the presence/absence of Tet (Tet+/-) to determine the essentiality of the A12L
protein in viral replication. The lack of A12L was complemented by the
transient expression of plasmid born A12L under the control of an early/late
synthetic promoter (pA12L) or the native promoter (233 nucleotide upstream
of A12L ORF, p233-A12L). In addition, the A12L truncates generated by
different combination of three AG/X sites failed to rescue the absence of
A12L protein. pA12L: A12L ORF under the control of the early/late synthetic
promoter; p233-A12L: plasmid born A12L under the native promoter; pRB21:
vector plasmid alone; AG/A: plasmid born A12L with N-terminal AG/A site
mutation into ID/I. A12L truncates were indicated above. Each virus titer
(PFU/ml) was scaled in log phase.
85
86
Figure 3.3. Morphology defects in the absence of A12L expression.
To investigate a role of A12L protein in virus assembly, T-REx 293 cells were
infected by vvtetOA12L in the presence (left panels: a, c, e) and the absence
of Tet (right panels: b, d, f). In the presence of Tet (a and c), spherical IV
particles were demonstrated, which evolved into the biconcave IMV particles.
An arrow shows a virus particle, which initiated developing the inner layer of
the core. The inner layer of the core is localized along with the outer
membrane (panel e). In the absence of Tet (panel b and d), mostly IV-like
particles (IV*) were observed with accumulated electron-dense viral materials
(V). IV-like particles contained little of viral dense materials in the
membranes, which also formed not round but irregular-shape. Arrowheads in
the panel b indicate IV particles with a gap between the viral materials and
the surrounding membranes and c in the panel d indicates a crescent
membrane. Some of IV particles were developed into IMV-like particles
(IMV*), of which cores showed abrogated condensation along with abnormalshaped layer as indicated by arrows at the panel f. Bars indicate 0.5µm.
87
88
Figure 3.4. Morphology defects by abrogated AG/A cleavage of A12L.
In order to examine VV morphology by rescuing the absence of A12L, we
transfected plasmid born A12L under the control of an early/late synthetic
promoter (pA12L) and infected with vvtetOA12L in the absence of Tet. The
transient expression of A12L induced regular IV and IMV particles as
observed in the panel a and b. However, the transfection of A12L plasmid,
which contained AG/A mutation into ID/I (AG/A) displayed defective
phenotypes the same as to the absence of A12L protein (panel c, d, and e).
Panel c shows IV like particles, which are more filled up with densematerials, while panel d and e demonstrate the aberrant layers of the cores.
IMV* represents defective IMV-like particles. Bars indicate 0.2 or 0.5µm.
CHAPTER 4.
PARTICIPATION OF A12L IN VACCINIA VIRUS ASSEMBLY
SUBSEQUENT TO D13L INVOLVEMENT.
89
ABSTRACT
The vaccinia virus (VV) A12L gene product, a 25kDa core protein has
been shown to be associated with VV-encoded core proteins (A10L, L4R, and
F17R) as well as membrane proteins (A17L, A4L, and A27L), suggesting the
possible participation of A12L protein in the organization of immature virions and
intracellular mature virions. In addition, the phenotypic defects in the absence of
A12L protein implies that A12L might have a role in recruiting VV proteins to form
the rigid connection between the core and the surrounding membrane. In order to
investigate more details regarding to the biological function of A12L in virus
assembly, we attempted to compare the protein synthesis and subcellular
localization of A12L with D13L gene product, which is an internal scaffolding
protein responsible for development of viral crescent membranes. By screening
rifampicin-resistant viruses (RifR), we were able to obtain the D13L mutant
viruses conferring rifampicin-resisant loci at both terminus and internal region of
D13L open reading frame. The RifR viruses showed stable expression of D13L
and A12L proteins in the presence of the drug, which were attributed to the
release out of rifampicin-inhibited VV replication as well as morphogenic
development. Thus, we concluded that the stable expression of D13L appears to
be a prerequisite for not only the expression and proteolysis of A12L but also its
participation in the viral morphogenic transition.
90
INTRODUCTION
The assembly and replication of vaccinia virus (VV) has been shown to
be reversibly blocked at a discrete step in the virus life cycle by treatment with
the macrolide antibiotic rifampicin (Rif) (Moss et al. 1969b.). Rifampicin
irreversibly binds to the β-subunit of the Escherichia coli RNA polymerase to
inhibit bacterial transcription (Boothroyd et al. 1983.). Treatment of VV with
rifampicin, however, leads to viral morphogenic defects including irregularshaped crescent membranes surrounding viroplasm, referred to as rifampicin
bodies or membrane-limited domains (Szjaner et al. 2005). In addition, two more
unique virus morphologies were observed: more electron-dense materials with
aberrant shaped crystalloid structures containing viral DNA, and punctate
cytoplasmic structures referred to as inclusion bodies (Sodeik et al. 1994). The
study done by McNulty-Kowalczyk and Paoletti discovered that rifampicin
interrupted the formation of a dense-spicule layer on nascent membrane
structures and subsequently impaired the curvature and rigidity of IV particles
(Dales et al. 1978, McNulty-Kowalczyk and Paoletti, 1993). Furthermore,
biochemical analyses of rifampicin effects on VV replication and proteolysis
demonstrated that rifampicin treatment resulted in the failure of viral DNA to
package into mature virions and led to the abrogated proteolysis of the major
core protein precursors (Moss et al. 1969a, Katz and Moss, 1970). In contrast,
rifampicin treatment had little effect on the synthesis of RNA, DNA and proteins
(Pennington, 1973, Ben-Ishai et al. 1969) The removal of the drug, however, led
to recovery not only of arrested packaging of the viral genome and proteolytic
processing but also to correct virus assembly. The re-initiation of viral assembly
91
occurred rapidly upon removal of rifampicin with the generation of convexshaped membranes around rifampicin bodies and the development of normal
immature virus particles (IV) and infectious intracellular mature virus particles
(IMV) (Grimley et al. 1970). In agreement with the normal protein synthesis in the
presence of rifampicin, the fast reversibility suggested that de novo synthesis of
macromolecules or protein was not required for the release of the rifampicininduced inhibition (Moss et al. 1969a, Moss et al. 1969b).
The rifampicin target is the gene product of the D13L open reading frame
(ORF), which is synthesized as a 65kDa late structural protein named L65
(Tartaglia and Paolett, 1985). Rifampicin-resistant viruses led to the identification
of mutations in the D13L gene located either at the N-terminus or C-terminus
(Baldick and Moss, 1987, Tartaglia and Paoletti, 1985, McNulty-Kowalczyk and
Paoletti, 1993). In addition, D13L deficient virus demonstrated the same
phenotypic defects as rifampicin-treated viruses, confirming that D13L is the
target gene modulated by the antibiotic (Zhang and Moss, 1992). D13L is
normally localized to the viroplasms and the crescent-shaped membranes,
predominantly on the inner surface of the viral cisternae or the concave surface
of the virions. In the presence of rifampicin, however, the distribution of D13L was
not limited to viroplasm but to inclusion bodies, showing the inhibited
translocation of D13L into the viral foci, which resulted in the subsequent
repression of the production of infectious virus particles (Miner and Hruby, 1989,
Sodeik et al. 1994). Another electron microscopic analysis of the D13L deficient
virus or its distracted localization in the presence of the drug showed little or no
92
production of viral crescent membranes, which appeared to be derived from the
membrane cisternae of the intermediate compartment (IC) (Sodeik et al. 1993,
Zhang and Moss, 1992). On the basis of this observation, it was hypothesized
that D13L may serve as an internal scaffolding protein to recruit the host IC into
the crescent-shaped viral membranes at an early stage of IV morphogenesis
(Sodeik et al. 1994).
A12L, a 25kDa late gene product demonstrated that A12L might have
associations with VV core proteins such as A10L (p4a), L4R (p25K), and F17R
(11K) and viral membrane proteins, A17L (p21K), A14L (15K), A27L (14K), and
A4L (39K). Taken together with the viral phenotypic defects of conditional lethal
mutant virus of A12L in the absence of inducer, which displayed detached
viroplasm from the membrane and irregular core formation, it is suggested that
A12L might be involved in the generation of immature virus particles (IV) and
correct core assembly by contributing to the proper protein architecture, which
promotes the association between viral crescent membranes and viroplasm in IV
particles and the core and outer membranes of IMV particles. Since both D13L
and A12L may have a similar role as matrix proteins and participate in the early
development of IV formation, it was of interest to define a more detailed function
of each protein and determine the hierarchy between the two proteins. Despite
rifampicin-regulated proteolytic processing of the A12L protein, the protein
expression as well as subcellular localization of the A12L in the presence of
rifampicin has not been determined. Thus, the comparison of each protein
synthesis and distribution in the presence and absence of rifampicin would
93
provide clues not only to the function of each protein but also the mechanism of
action of rifampicin on virus assembly.
Here, we attempted to obtain rifampicin-resistant (RifR) viruses to
characterize the subcellular localization of D13L and A12L and to determine their
association with viral crescent membranes. Using WR-infected cells in the
presence and absence of rifampicin, we compared rifampicin-induced protein
expression of D13L and A12L and established that A12L participation in virus
assembly may take place subsequent to the involvement of D13L.
94
MATERIALS AND METHODS
Cell cultures. VV WR (Western Reserve strain) was grown on confluent
monolayers of BSC-40 cells maintained in Eagle’s minimal essential medium
(EMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen), 2
mM glutamine (Invitrogen), and 10 mM gentamicin sulfate (Invitrogen) at 37 °C in
a 95% humidified atmosphere containing 5% CO2. For infection, BSC-40 cells
were maintained in infection media (EMEM) supplemented with 5% FCS, 2 mM
glutamine, and 10 mM gentamicin sulfate and infected with VV WR and
rifampicin-resistant viruses (RifR) at an multiplicity of infection (MOI) of 1 plaque
forming unit (PFU)/cell.
Rifampicin-resistant (RifR) viruses. In order to obtain RifR viruses, confluent
BSC-40 cells were infected with VV Western Reserve strain (VV WR) at an MOI
of 1 PFU/cell in the presence of 100 µg/ml of rifampicin followed by two more
series of infection with the previous cell extracts in a higher concentration of
rifampicin (150 and 200 µg/ml). Rifampicin stock solution (1000x, Sigma-Aldrich)
was stored in dimethyl sulfoxide at –20°C and diluted out with dH2O for various
concentrations. After a total of three series of infections in the presence of the
drug, the infected cell lysates were titered and screened for mutations through
polymerase-chain reaction (PCR) with D13L specific primers. In order to screen
the full sequences of D13L, primers were designed to have the nucleotides (nts)
from the 3’ region of A1L to the internal region of D13L (837 nts) and from the
internal region of D13L (812 nts) to the 5’ region of D12L: A1L forward, 5’TCTCTACGGAGTTTATTGTAA; D13L internal primer (IP-reverse), 5’- TGCATCT
95
ATATATCTTTTTC; D13L-IP forward, TTCGTATCCAGGGTACTCACAAG; D12Lreverse, 5’- ATGGATGAAATTGTAAAAAATATCCG. After screening, the resistant
viruses were subjected to three rounds of plaque purification prior to the virus
purification.
Protein expression of D13L and A12L. Confluent BSC-40 cells were infected
with VV WR and RifR viruses at an MOI of 1 PFU/cell and treated with rifampicin
(100 µg/mL) at the time of infection. The infected cells were harvested at 24 hpi
by centrifugation at 750x g for 10 min. and resuspended in the protease inhibitorcontaining PBS, followed by three cycles of freezing and thawing to lyse the cells.
After a post-nuclear spin at 350x g at 4 °C, the same amount of each sample was
resolved on a 12% NuPAGE Bis-Tris gel (Invitrogen) prior to immunoblot analysis
with D13L and A12L antisera. D13L antisera (anti-D13L) were developed by over
expression of L65 fusion protein with trpE and injection of the purified protein into
New Zealand white rabbits. (Miner and Hruby, 1989). A12L antisera (anti-A12L)
were generated by expression of the fusion protein, A12L with a 7x his tag at an
N-terminus, followed by purification and injection into New Zealand white rabbit
as described (Whitehead and Hruby, 1994a).
96
RESULTS
Identification of new rifampicin-resistant loci in the D13L ORF.
In an attempt to obtain rifampicin-resistant (RifR) viruses, we performed
plaque purification in the presence of the drug and screened for D13L mutations.
Surprisingly the mutations were mapped to not only the N- and C-terminus but
also to an internal region of the D13L ORF. Figure 4.1 shows all of the mutant loci
conferring rifampicin-resistance discovered from previously reported RifR viruses
and from newly identified resistant viruses. Due to all the amino acid (aa)
substitutions in D13L located to both termini of the D13L ORF, it was suggested
that the D13L protein might form a head-tail structure, which can be modulated
by rifampicin. Thus, it is of interest to report here that a single aa change in an
internal ORF such as K into T at residue 159 and S into L at residue 227 provides
the drug resistance. This could be explained by the possible participation of the
internal amino acids in formation of either the head-tail interaction of D13L or in
generation of another conformational structure, which prevents D13L from
interacting with rifampicin. By taking advantage of these four different RifR viruses,
we determined the sensitivity of each resistant virus to different concentrations of
rifampicin and compare protein stability of D13L and A12L.
In order to confirm that all of the RifR viruses are resistant to rifampicin,
each virus including WR was infected in the presence of rifampicin in a range of
concentrations at 0, 50, 100, 200, or 300 µg/mL (Fig. 4.2). WR infection showed
more than three-log reduction of virus yield with addition of 50-300 µg/mL of the
drug while the other resistant viruses showed approximately one-log reduction in
97
titer at 50 µg/mL and a 2.5 log reduction in titer at the higher concentrations.
Compared to WR, all the RifR viruses demonstrated the most significant
resistance to rifampicin at 50 µg/mL and at least one-log higher virus yield at 100300 µg/mL of rifampicin. Thus, we reported here that the internal mutations in
D13L could provide the same resistance against rifampicin as the other resistant
viruses with the mutations at an N- or C-terminus of D13L ORF.
Protein expression of D13L and A12L in the presence of rifampicin.
To determine whether the resistance to rifampicin observed in the RifR
viruses is attributed to protein instability of D13L, we examined protein
expression levels by immunoblot analysis (Fig. 4.3). Equal amounts of protein
were loaded on a 12% NuPAGE Bis-Tris gel and subjected to immunoblot
analysis with antibody of D13L (anti-D13L). D13L (L65) was stably expressed
from WR in the absence of rifampicin, but relatively little of protein was
expressed in the presence of the drug (Fig. 4.3, lane 6). Each of the resistant
viruses, however, showed stable expression of D13L in the presence of
rifampicin, regardless of the location of the mutant loci. Using the same cell
extracts, the protein stability as well as the cleavage of the A12L protein was
examined with antibody of A12L (anti-A12L). Like D13L, A12L was expressed
and processed in all the RifR viruses in the presence of rifampicin (Fig. 4.3). As a
control, WR in the presence of rifampicin showed little expression of A12L and
consequently no proteolysis was observed. From this we concluded that
rifampicin repressed the synthesis of D13L and A12L, which might result in
rifampicin-induced defects of VV proteolysis. It is of interest to note that
98
considering the fact that rifampicin has not hampered VV protein synthesis, it
may be speculated that rifampicin stimulated the degradation of each A12L and
D13L protein. However, due to the high MOI of virus infection in the presence of
rifampicin (100 µg/mL) previously used, it cannot be ruled out whether the drug
by itself at a low MOI of 1 introduced the interrupted proper expression of late
gene products. The fact that all the rifampicin-resistant viruses were capable of
expressing both D13L and A12L protein even in the presence of the drug
suggests that the proper D13L expression in the presence of rifampicin appears
to be enough to either re-synthesize A12L gene product or inhibit the repression
of A12L protein.
99
DISCUSSION
The identification of the rifampicin-resistant viruses introduced a new map
of the resistance loci. It has been previously reported that all the altered amino
acids were located in either an N- or C-terminus of the D13L ORF as described
Fig. 4.1. Distribution of the resistant loci at both ends established a model of a
head-tail interaction of D13L, of which mutations would inhibit its association with
rifampicin. However, the mutant loci we identified here are located in an internal
region of D13L at amino acid residue 159 and 227. There are two possibilities
that either both substitutions of internal amino acids cause a change in a tertiary
structure, interrupting the head-tail conformation or the internal amino acids are
located in an active pocket that the drug binds to. In addition, it is of interest to
report that rifampicin-treated cell did not express D13L (Fig 4.3), which is
opposite to reports from the previous studies. Rifampicin did not hamper the
synthesis of RNA, DNA and proteins, which was not observed here. The
presence of the drug inhibited expression of D13L and A12L while all the
resistant viruses displayed the both proteins and A12L cleavage products,
suggesting that the rifampicin-induced phenotypic defects and halted proteolysis
might result from the lack of D13L protein. This inconsistent data might be
caused by different experimental procedures such as MOI and timing of
placement of rifampicin.
Consistently, the subcellular localization of A12L determined by
immunofluorescent microscopy (data not shown) demonstrated the consistent
localization of A12L observed by rifampicin-resistant viruses compared to WR-
100
induced normal distribution, implying that rifampicin, by itself cannot alter A12L
protein expression and localization unless the D13L protein is depleted or its
localization is disrupted. Therefore, it is hypothesized that D13L might have a role
in recruiting and localizing A12L protein to viral factories and stimulating proper
proteolytic maturation and localization either in a direct or indirect manner.
101
102
Figure 4.1. Rifampicin-resistant loci in D13L ORF.
Black lines represent the resistant viruses we obtained while the dotted lines
represent the previously reported D13L loci conferring rifampicin-resistance.
†
RifR 3: Q to K amino acid change at residue 27, RifR 4: K to T amino acid
†
change at residue 159, RifR 5: S to L amino acid change at residue 227, RifR
†
9: K to N amino acid change at residue 484.
: Newly identified D13L
mutant loci located in the internal region of D13L ORF. The rifampicinresistant loci had been limited to either an N-terminus or a C-terminus of
D13L, while the newly reported mutant loci are located in the internal region
(RifR4 and RifR5).
103
Figure 4.2. Sensitivity of RifR viruses to different concentrations of rifampicin.
104
Figure 4.2. Sensitivity of RifR mutant viruses to different concentrations of
rifampicin.
To determine the sensitivity of all of the rifampicin-resistant viruses (RifR),
BSC-40 cells were infected at an MOI of 1 PFU/cell in the presence of
various concentrations of rifampicin (Rif): 0, 50, 100, 200, and 300 µg/mL.
The infected cells were harvested at 24 hpi and titered. Each RifR virus
contains a different amino acid change as mentioned in Fig.41, but showed
the similar sensitivity to rifampicin. Wild-type VV WR, however, could not
replicate in the presence of rifampicin, compared to RifR viruses.
105
Figure 4.3. Protein stability of D13L and A12L in the presence of Rif.
To examine the protein stability of D13L (L65) and its effect on the A12L
proteolysis, BSC-40 cells were infected with WR and the RifR viruses in the
presence of rifampicin (100 µg/mL). The same amount of each cell lysate
was analyzed by the immunoblot analyses with anti-D13L (upper panel) and
anti-A12L (bottom panel). WR (lane 1) is the VV WR-infected cells in the
absence of rifampicin, while Q27K (lane 2), K159T (lane 3), S227L (lane 4),
K484N (lane 5), and WR (lane 6) are each RifR virus and WR-infected cells in
the presence of rifampicin.
CHAPTER 5. CONCLUSIONS.
106
As an attempt to elucidate more details of the regulation and requirement
of vaccinia virus (VV) proteolytic processing, we chose to study the A12L late
gene product. The 25kDa full-length protein A12L protein is proteolytically
processed into six multiple peptides with molecular weights of 21, 18, 17, 15, 13,
and 11kDa. Proteolysis of A12L appears to be halted once a stable mixture of
these peptides is obtained. Cleavage events are initiated with the processing at
an N-terminus AG/A site as well as at unknown locations in proximity to the Nterminus, producing 17kDa and 21kDa peptides. This is followed by a C-terminal
processing event. Since cleavage has only been demonstrated at the AG/A site
among the three AG/K motifs in the ORF, the two AG/K motifs in A12L ORF may
not participate in the initial cleavage reaction but rather in subsequent processing.
In addition, we report here that the VV I7L cysteine proteinase is responsible for
cleaving A12L at an AG/A site. Considering the incomplete cleavage observed at
each of the AG/X sites and the appearance of multiple products, we speculate
that A12L proteolysis is a redundant processing event, which appears to form a
specific tertiary structure by the first cleavage for further processing and might be
catalyzed by not only the I7L cysteine proteinase but also another VV-encoded
proteinase.
Although we were not able to identify all of the exact cleavage sites of
A12L, we demonstrated that A12L might associate with VV core (A10L, A4L, L4R,
F17R) and membrane (A17L, A14L, A27L) proteins, playing a role as a matrix
protein. These possible associations of A12L with a variety of VV proteins led to
the hypothesis that A12L may participate in connecting viral cores and
107
membranes tightly to form rigid virion particles not limited to the formation of
immature virus particles (IV) but also involved in formation of intracellular mature
virus (IMV) and IEV particles.
The construction of a conditional lethal mutant virus of A12L (vvtetOA12L)
showed that A12L protein and its cleavage processing are essential for VV
replication. The failed rescue experiment with AG/A site mutant A12L and A12L
truncates demonstrated that the removal of an N-terminal AG/A site is required
for viral replication and subsequent cleavage reactions. Processing at the AG/A
site is likely required for the development of the correct tertiary structure.
Disrupted cleavage abrogates the ability to complement the deficiency of A12L.
In terms of VV morphology, the absence of A12L resulted in the accumulation of
electron-dense materials and irregular shaped membranes of immature virions
(IV), which displayed a gap generated by incomplete association between the
viroplasm and membranes. Some of virus particles were able to form typical IV
with spherical membranes and correctly filled viral materials. However, no typical
IMV particles were observed, supporting the theory that A12L participates in
assembling correct IV content with surrounding membranes and core
condensation into biconcave-shape rather than the generation of IV membranes.
The similar morphogenic defects observed by the expression of AG/A mutated
A12L directly show the requirement of A12L proteolysis during VV morphological
transitions.
Due to the multiple associations of A12L with other virus-encoded proteins
108
involved in the early development of virus membranes, we raised the question of
whether A12L has any interaction with D13L, a scaffold protein, which
participates in the formation of crescent-shaped viral membranes. Since D13L
localization and its resistance against rifampicin have been well studied, we
screened all rifampicin-resistant viruses with D13L mutant loci, which were used
as positive controls in the presence of drug. The effects of rifampicin on A12L
expression suggested that despite the presence of rifampicin, proper D13L
expression by the resistant loci resulted in proper expression and subsequent
proteolysis of A12L protein. Therefore, we concluded that the generation of the
crescent membranes by D13L would occur prior to the participation of A12L in IV
assembly.
In summary, the study of A12L proteolysis and its biological function
provided a more comprehensive understanding of regulation and participation of
VV proteolysis. VV proteolysis of A12L may not be designed to produce one or
two major cleavage products as observed with the other proteolytic maturation of
A10L, A3L, L4R, A17L, and G7L. Rather, multiple stable cleavage products can
be obtained by sequential cleavage processing, which appears to be required for
specific conformational structures. Once a desirable set of products is obtained,
the proteolysis is halted. Besides, A12L-induced defects in virus assembly
demonstrate that proteolytic maturation of viral structural proteins as an essential
post-translational modification, assembles the correct nucleoprotein complex with
spherical-shaped membranes and compresses an inner layer of core into the
characteristics of biconcave-shaped IMV particles. Further research of each
109
biological function of all the A12L-derived cleavage products as well as the
participation of A12L associated with each protein will eventually discover more
details of VV morphogenesis developed by VV proteolysis.
110
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