Joseph Marcotrigiano
Center for Advanced Biotechnology and Medicine
Rutgers University
jmarco@cabm.rutgers.edu
June 22, 1998
Number of Publications
Non-A, Non-B hepatitis to HCV
NANB agent
Transmission
to chimps
cDNA clones
diagnostics
IFN therapy
Year
HCV: Global Epidemic 120-170 million people chronically infected
Hepatitis C: Spectrum of disease
Acute HCV infection
70%
Chronic HCV infection
20 years
Cirrhosis Chronic Hepatitis
30 years
Liver Cancer
End-stage liver disease
30%
Recovery
Treatment
(5 multicenter trials: all genotypes)
55%
Sustained Response
60%
44%
50%
40%
33%
26%
30%
16%
20%
10%
6%
0%
(n=)
IFNα
IFNα
PegIFN
24wks
48wks
48wks
IFNα +
Rbv
24wks
(912)
(505)
(231)
(1070)
IFNα +
Rbv
48wks
PegIFN +
Rbv
48wks
(1454)
(964)
Hepatitis C Virus
•Significant human pathogen
3 million people nationwide, 120 million people worldwide
•About 70-75% of people infected with HCV develop chronic infection
•No vaccine available
•Current antiviral therapy pegylated interferon alpha and ribavirin
•Sustained response in only 50% of genotype 1-infected patients
•Number one cause of liver transplantation in the developed world
GBV-B
HCV
Flaviviridae
Flavi
GBV-A
GBV-C
HGV
•  Enveloped
•  ssRNA
•  (+) sense
Pesti
4
HCV
J
5
G A/E
K
A
F
H
D
1
2
B
C
10%
HIV-1 group M
6 (7 8 9 11)
3 (10)
10%
Stuart Ray, JHU
HCV Genome Organization
Structural
IRES Proteins
Core
Envelope glycoprotein 1
Envelope glycoprotein 2
C E1
NS2
Autoprotease
E2
P
7
NS2
NS3
Protease/
RNA helicase
Nonstructural
Proteins
NS3
4
A
4B NS5A
NS4A
NS4B
NS3 protease Membrane
cofactor
protein
3 UTR
NS5B
NS5A
?
NS5B
RNA
polymerase
HCV Life Cycle
Systems to Study
HCV Replication
HCV infectious clones
Run-off site
T7/SP6
Full length HCV (H77) cDNA template!
Transcription
5 NTR
3 NTR
H77
Chimp
Replication √
Spread √
Kolykhalov Science 1997 vol. 277 pp. 570-4
HCV 2a (JFH)
JFH-1!
C
E1 E2 p7 NS2
NS3
NS4
A B
A
C
E1 E2 p7 NS2
NS3
NS4
A B
A
C
E1 E2 p7 NS2
NS3
NS4
A B
A
C
E1 E2 p7 NS2
NS3
NS4
A B
A
J6!
J6/JFH-1!
(FL)!
J6/JFH-1!
(pol-)!
SG/JFH-1!
(SG)!
Neo
EMCV
IRES
NS3
NS4
A B
A
NS5
NS5
NS5
NS5
NS5
B
B
B
B
B
Lohmann et al. Science 1999 vol. 285 pp. 110-3
Blight et al. Science 2000 vol. 290 pp. 1972-4
Wakita et al. Nat Med 2005 vol. 11 pp. 791-6
Zhong et al. PNAS 2005 vol. 102 pp. 9294-9
Lindenbach et al. Science 2005 vol. 309 pp. 623-6
Full-length 2a Genome is Infectious
hours post-transfection
24
FL
SG
Pol-
48
96
filtered media
transferred to
naive cells
Complete Replication of HCV
electroporation
Chimps
mice
“HCVcc”
Infect naïve cells
Wakita Nat Med 2005
Lindenbach Science 2005
PNAS 2006
Zhong PNAS 2005
New Therapies on the
Horizon
18
Protease and Polymerase inhibitors
Screening & structure-informed drug design
NS5B RNA polymerase
NS3-4A Ser protease/helicase
Protease inhibitor VX-950
7
Placebo
Median HCV RNA
(Log10 IU/mL)
6
5
VX-950
450 mg q8h
4
VX-950
1250 mg q12h
3
Mean 4.4 log decline
4/8 HCV RNA<30 IU/ml
2
1
VX-950
750 mg q8h
0
1
2
3
4
5
6 7 8 9 10 11 12 13 14
Study Time (in days)
Reesink HW et al. Gastroenterology (2005)
Protease inhibitors BILN2061 & VX-950: drugresistant variants with some cross-resistance
Resistance mutants selected in the replicon system
BILN 2061
D168V/A
A156V/T
VX-950
A156S
Fold-shift in potency
Text
Lu et al. (2004) Antimicr. Agents Chemother.
Lin et al. (2004) J. Biol Chem.
A Sheaffer (2004) (+)-strand RNA viruses Symp
Single point mutations confer resistance to each
polymerase inhibitor
P495L
Benzimidazole
A
B
C
M423K
Thiophene-COOH
M414T
Benzothiadiazine
S282T
2 -Me-Nucleosides
Tomei et al (2004) J. Virol.; G Migliaccio, unpublished
New Drug Targets
NS5A and NS2
NS5A General Features
C E1
E2
P
7
NS2
NS3
4
A
4B NS5A
NS5B
NS5A
•Important yet undefined role in RNA replication
–  Phosphorylated by unknown cellular kinases
–  Adaptive mutations, lethal mutations
–  Interactions with other HCV replicase proteins
–  Membranous web localization with HCV replicase components
–  Amino terminal amphipathic membrane anchor sequence
•Numerous interactions with cellular proteins
–  Signaling, lipid transport, transcription, etc.
–  Modulation of the host cell environment
NS5A Architecture
•Cytoplasmic protein
•N-terminal amphipathic helix for membrane association
•Three domains determined by limited proteolysis
•RNA binding protein (Huang L et al. Protein Expr Purif Sept 2004)
•Zinc coordination site in Domain I (Tellinghusien et al. JBC Nov, 2004)
•Crystallization construct consist of Domain I without N-terminal helix
Domain II
Domain I
1
213
250
342
Domain III
356
447
α helix
C39 C57P58C59 C80
Domain I delta Helix
Crystallization construct
Topology of NS5A Domain I
N
B2
Zn
H2
B1
B7
B4
B5
B9
B6
B8
B3
S-S bond
subdomain IA
3β strands, 1α-helix
subdomain IB
6β strands
C
Domain I Disulfide Bond With Experimental Density
Cys 142
S-S Distance = 2.03 Å
MAD Experimental Electron Density at 1σ
Cys 190
DI Non-Reduced
DI Reduced
MW
Domain I Electrophoretic Mobility Shift
non-reduced
reduced
5 mM DTT treatment in reduced samples
NS5A is RNA Binding Protein
3X
18S130nt
no 1 2 5 10 no 1 2
5Bcre
5 10 no 1
2 5 10
N/S270nt
no 1
2 5 10
5 ntrΔSL1380nt 5 ntr400nt
no 1
2 5 10 no 1
see also, Huang et al., JBC, 280(43):36417-28
2 5 10
Multiple Views of the NS5A Domain I Dimer
top
side
bottom
•buried surface area 680Å2 good shape and electrostatic
complementarity
•r.m.s.d of 0.43 Å between CA from both chains
Model of HCV NS5A Dimer
ER
Cyt
RNA
II
III
II
III
Novel HCV Inhibitors
• thiazolidinone core structure
• Found by high through-put screening against the HCV replicon
Lemm J et al. JV 2010 vol. 84 pp. 482–491.
Antiviral selectivity of BMS-824
Replicon or Virus
Cell line
EC50 (µM)
CC50 (µM)
HCV replicon
Huh-7
0.005
>50
BVDV replicon
Huh-7
1.7
>50
BVDV
MDBK
5
>150
HIV
MT2
10
23
HRV
MRC-5
>100
>100
RSV
Hep-2
14
14
CC50 = 50% cytotoxic concentration
EC50 = 50% effective concentration
Resistant mutations arise
in NS5A Domain I
Replication of HCV in the presence of low
concentration of inhibitor allows for the selection of
resistance mutations
Location of Y93 on NS5A
3
Questions Remaining about NS5A
Inhibitors
•  What is the mechanism of action?
•  Does the inhibitor bind NS5A?
•  How does the inhibitor work on other
HCV genotypes?
•  Toxicity in animals?
HCV NS5A Summary
•Novel Fold and Zn binding site - DALI, SCOP,
ProCat, SUMO databases
•Structure has provided a foundation for further functional studies
-disulfide bridge
-RNA binding surface
-existence of the dimer
-conserved surface features
HCV Genome Organization
and Protein Processing
Structural
Proteins
IRES
Signal peptidase
Signal peptide peptidase
C
E1
E2
P
7
Nonstructural
Proteins
NS2-3
autoprotease
NS2
3 UTR
NS3-4A serine protease
NS3
4
A
4B
NS5A
NS5B
• Structural proteins cleaved by cellular proteases
• Nonstructural proteins cleaved by NS2-3 autoprotease
and NS3-4A serine protease
NS2/3 Cleavage
ER
p7
NS3
NS2
Cyt
NS2pro
Protease
94
217
Helicase
NS2-3 autoprotease
• NS2/3 cleavage required for virus replication in chimpanzees
and in tissue culture
• NS2 94-217: NS3 1-181 minimal region required for autoproteas
activity (Pallaoro et al, J. Virol., 2001; Thibeault et al, JBC, 2001)
•  NS3 protease activity is not required
• Catalytic mechanism of the NS2-3 autoprotease remains controversial
• Zinc metalloprotease vs cysteine protease
• His 143, Glu 163, Cys 184 in NS2 are important for autoproteolysis
Topology of C-terminal
Domain of HCV NS2pro
H2
H1
N
b1
b2
b4
b3
C
b5
Dimeric Structure of NS2
N
N
C
C
• 2 a helices and 5 stranded, antiparallel b sheet
• Total buried surface area of about 1300 Å2
• Good electrostatic and shape complementarity
Location of His 143, Glu 163, Cys 184
Glu
His
Cys
Cys
35Å
Glu
His
Active Site of NS2pro
Pro 164
Cys
184
Glu 163
3.0 Å
4.1 Å
3.0 Å
3.2 Å
3.1 Å
3.1 Å
His 143
Leu 217
(C-term)
• Two copies per dimer
• Cysteine protease active site
• C-terminus remains bound
pro
NS2
Comparison of
with
Papain Protease
pro
NS2
Comparison of
with
Sindbis Virus Capsid
pro
NS2
Comparison of
with
Subtilisin Bound to Eglin-C
Active Site Mutant Mixing
Experiment
Glu
X
X
His
Cys
Cys
Glu
His
Active Site 1
Active Site 2
Chain A - His 143 to Ala
Chain B - Cys 184 to Ala
Chain A - Cys 184
Chain B - His 143
  no NS2-3 cleavage
  NS2-3 cleavage!
Active Site Mutant Mixing
Experiment
T7-driven expression of full-length 1a HCV DNA in Huh7.5 cells, 35S metabolic labeling
100
90
70
60
50
NS2-3
NS3
40
30
25
NS2
20
anti-NS2
anti-NS2/NS3
Mutant Mixing Experiment Using Epitope Tags
H143A
H143A
H143A
C184A
Epitope tag 1 H143A
Epitope tag 2 C184A
C184A
C184A
Active Site Mutant Mixing Experiment 2
Transfection of U2OS cells with FLAG- or HA-tagged NS2-3,
+
+
FLAG
HA
-
- wtH/C H C wtH/C H C -
40
anti-NS2
+
-
-
+
-
+
+
-
metabolic labeling
FLAG H
FLAG C
HA H
HA C
FLAG-NS2-3
HA-NS2-3
30
25
anti-FLAG
-
-
+
+
35S
20
FLAG-NS2
HA-NS2
40
FLAG-NS2-3
30
25
anti-HA
20
FLAG-NS2
40
HA-NS2-3
30
25
20
HA-NS2
Detergent Binding Sites
Interaction of NS2pro with the
ER Membrane
•  Uncharged residues interact with fatty acid tails
•  Basic residues interact with phosphate head groups
HCV
pro
NS2
Summary
• NS2 C-terminal domain contains a cysteine protease active site - no
zinc ion
• Each NS2 dimer contains a pair of composite active sites,
i.e. both monomers contribute residues to each catalytic triad
• After cleavage, the C-terminus remains bound in the active site,
blocking processing of other substrates
• HCV NS2 has a novel protease fold
• First example of a cysteine protease with composite active sites
HIV Protease
Retroviral aspartic protease - dimerization forms a single active site
HCV Entry:
Cellular Factors and Viral
Envelope Proteins
55
CD81
•  tetraspanin family
•  26 kD, 236 aa
•  expressed in all nucleated cells
•  part of the B-cell receptor complex
•  required for Plasmodium falciparum
infection of liver cells
•  natural ligand--unknown
CD81 is Essential for HCV Entry
•
•
•
•
•
•
Entry blocked by anti-CD81, soluble LEL, CD81 knockdown
HepG2 (CD81-) transduced with CD81 become permissive
Low level expression is not sufficient to support entry
Likely function as a co-receptor at a post-attachment step
Unlikely to determine tissue tropism (all nucleated cells)
Possible but not sole determinant of narrow HCV host range
Scavenger Receptor BI (SR-BI)
•  509 aa
•  heavily glycosylated
•  expressed in hepatocytes, adrenal
cortex, gonads (lower levels
elsewhere)
•  most important receptor for HDL
•  multiple other ligands (oxLDL)
Role for SR-BI in HCV entry?
•  Entry inhibited by SR-BI siRNA knockdown
•  Natural SR-BI ligands (HDL vs. oxLDL) have opposite effects
on infectivity
•  Cholesterol transfer via SR-BI is important for SR-BI s function
in entry
•  SR-BI could contribute to HCV hepatotropism.
Claudin-1 (CLDN1)
EL1
EL2
Extracellular
Intracellular
N
20-26 kD
C
•
CLDN1 and CLDN2 first isolated from
chicken liver by Furuse and
colleagues (JCB 1998)
•
•
•
24 family members
•
Nature vol 446 pgs 801-805
backbone of the tight junction strand
homotypic and heterotypic
associations within the strand and
with claudins in neighboring cells.
modular claudin composition
determines epithelial permeability and
charge selectivity
Occludin (OCLN)
EL1
EL2
Extracellular
Intracellular
N
•
Four transmembrane protein with an
intracellular domain involved in cell
signaling
•
found in tight junction of polarized
cells
•
determines epithelial permeability and
cell adhesion
•
Expression of CD81, SR-BI, CLDN1,
OCLN allows HCV entry into murine
cells
C
Ploss et al. Nature 2009 vol 457 pp 882-6
HCV Glycoproteins E1
and E2
•  Type I membrane proteins
N-terminal ectodomain and C-terminal membrane associating
helix
•  E1= fusion; E2=receptor binding
•  Intramolecular disulfide bonds - numerous highly conserved
cystein residues E2 ectodomain has 17.
•  Glycosylation
E1 4 potential sites
E2 11 potential sites
•  E2 binds to CD81 and SR-BI
Disulfide Bonded
Aggregates of E2
Patel et al. J Gen Virol, 81, 2873-2883 2000
Elution fractions
Std
Std
Elution fractions
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
-IgG
11697-
220-
-eE2
66-
11697-
Non-reducing
Production and Purification of E2
ectodomain (eE2)
-heavy chain
(reduced)
-eE2
6645-
-light chain
(reduced)
45-
Yield = 0.1 mg/L
Yield = 8-10 mg/L
Recognition of E2 Ectodomain by Patient Serum
patient 183 (genotype 1)
patient 153 (genotype 2)
patient 155 (genotype 3)
A450
healthy donor 156
% serum in well
E2 from genotype 2a is recognized by patient serum infected
with different genotypes
eE2 can inhibit HCV entry
100
80
60
eE2
hCD81
mCD81
GST
40
20
0
Concentration (ug/ml)
Human CD81 (hCD81) is a cellular receptor for HCV entry
Possible uses of the E2 ectodomain
•  Subunit vaccine to prevent HCV infection
•  A reagent to identify compounds that bind to E2
and inhibit HCV entry
•  Use of a portion of eE2 as a inhibitor – similar
to enfuvirtide or fuzeon for HIV gp41
HCV Structural Biology