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The Molecular Virology
of Hepatitis C Virus
Stephen J. Polyak, Ph.D.
Research Associate Professor
Department of Laboratory Medicine
University of Washington
Overview
• HCV Timeline
• HCV: from clinical description to virus cloning
– Hepacivirus, Genome
• HCV Lifecycle
– Entry, replication, assembly
• HCV Variability
– Genotypes and quasispecies
• New Ways to Combat HCV
– Current treatments
– Treatments in development
• Targeting the host cell
– Silymarin as a Complementary and Alternative Medicine
• Future and Limitations
HCV Research Timeline
Infectious HCV cell
culture system
Functional HCV
pseudoparticle system
Infectious cDNA clone constructed
HCV serine protease structure (NS3-4A)
2009
2005
2003
Non-infectious
replicon system
1997
1999
1996
1998 IFN-alpha and ribavirin
combination therapy
1993
Delineation of HCV genome organization and
polyprotein processing
1989
Identification of
hepatitis C virus
1975
Description of non-A, non-B hepatitis
Courtesy of Charlie Rice
HCV Identification
• 1970’s: Hepatitis A and B diagnostics
– Most transfusion associated hepatitis not caused
by HAV and HBV or other known viral agents (NEJM
292: 767; Gastro. 69:1278; JID 139:511; Vox Sang. 44:231)
• non-A, non-B hepatitis (NANBH)
– Might be a small, enveloped virus transmissible to
chimpanzees (Gastro 88:773; JID 156:636)
– Standard immunological methods failed to identify
viral antibodies or antigens
– Chiron group
• Thought that there was not enough viral antigen in
NANBH
• So they decided to try and concentrate the virus
HCV Identification
High titer NANBH chimpanzee plasma
Ultracentrifugation at 104,000g for 5 hours
Extract nuclei acid, random primed cDNA into gt11 phage
Immunoscreened with NANBH patient serum
Clone 5-1-1
Larger overlapping
Clone 81
Library Screening
 Phage with
portions of
HCV genome
Infect E.coli
Phage kills
E.coli and
leaves plaques
Screen with
HCV Patient
serum
Southern Blots with Clone 81
Infected Chimp Liver
Acute Chronic
Clone 81 probe
Placenta
Placenta
IFN- probe
Clones 5-1-1 and 81 not from host genome and DNA replication
intermediates related to these clones not detected
Northern Blots with Clone 81
2
4
12 g
Infected Chimp Liver
Uninfected Chimp Liver
Uninfected Chimp Liver
Strand 1 Clone 81 probe
Strand 2 Clone 81 probe
Viral RNA
ds 81
Clones Hybridize to RNA from infected animals, RNA is single
stranded, size approx 5-10,000 nucleotides
Western blot
Clone 5-1-1 expressed as SOD fusion protein called PS5
NANBH patient serum
Experimentally infected chimp serum
Protein from clone 5-1-1 closely associated with NANBH infections
HCV was the first virus to be isolated and characterized solely by
molecular methods, ie without culture methods
Happy 20th Birthday HCV!
April 21, 1989
GBV-B
HCV
Flavi
Flaviviridae
-enveloped
-ssRNA
-(+) sense
-HCV is related to flaviviruses (West Nile virus,
Dengue virus) and Pestiviruses
-HCV occupies a unique branch on the flavivirus
phylogenetic tree, known as hepaciviridae
GBV-A
GBV-C
HGV
Pesti
HCV Genome
-HCV genome is about 9400 nucleotides long, it is ssRNA and positive sense
-the 10 viral proteins are first made as a large polyprotein
-individual proteins are released from polyprotein by cellular and viral proteases
-core, E1 and E2 are the structural proteins which form the virus particle
-remaining proteins are nonstructural and have roles in viral replication
Composition of Hepatitis C Virus
Lipid Envelope
Capsid Protein
Nucleic Acid
Envelope Glycoprotein E2
Envelope Glycoprotein E1
Courtesy Michael Gale via
Tenille Gale
HCV Life Cycle
Typical virus life cycle:
Binding, internalization,
uncoating, protein
expression/replication,
assembly, release
Tools to study HCV entry
Pseudoparticles
(HCVpp)
Cell culture virus
(HCVcc)
HCV
E1E2
HIV particle
Reporter gene
• Gutted HIV-virion studded
with envelope proteins
from other viruses
– Only measure viral entry
– Delivers reporter gene to
infected cell
– Allows study of cells that
do not support RNA
replication
Bartosch, J Exp Med 2003
Hsu, PNAS 2003
Courtesy of Matt Evans
• Authentic HCV virion
– Generated from
infectious JFH-1
subtype 2a genome
– Capable of
productively infecting
cells in culture or
animals
– Detected by viral
antigens or with
incorporated reporter
genes
Wakita, Nat Med 2005
Lindenbach, Science 2005
Zhong, PNAS 2005
HCV receptors/entry factors
GAGs
(Barth et al., 2003)
Linear polysaccharides
on proteins of all human
cell surfaces
CD81
(Pileri et al., 1998)
Tetraspanin superfamily member
Expressed in all nucleated cells
Part of B-cell receptor complex
SR-BI
(Scarcelli et al., 2002)
Claudin-1
(Evans, von Hahn et al., 2007)
Scavenger receptor class B member I Form the backbone of tight
HDL receptor (multiple other ligands) junction strands in epithelial
tissues
Expressed in hepatocytes, adrenal
Highest expression in
cortex, gonads (less elsewhere)
hepatocytes (less elsewhere)
LDL-R
(Agnello et al., 1999)
Low density lipoprotein
receptor
Intracellular
Silencing confers resistance
Provides a way for
the virus to stick to
cells
Expression confers
susceptibility
Ligands influence
infection
Expression confers
susceptibility
viral RNA
accumulation
increased or
decreased in
parallel with LDLR
mRNA expression
and LDL entry
Occludin 1: The Newest
HCV Entry Factor
Huh-7.5 cDNA library in a retroviral vector
packaged into VSVGpp
NIH3T3 mouse cells expressing human CD81, SR-BI and CLDN1
challenged with HCVpp, encoding antibiotic
Surviving clones tested for susceptibility to GFP-HCVpp
Expression of human OCLN and CD81
determines HCV species tropism
Mouse NIH3T3 cells are only
permissive for HCVpp entry
when CD81, SR-B1, CLDN1
and OCLN expressed
Hamster CHO cells are only
permissive for HCVpp entry
when human OCLN expressed
A Ploss et al. Nature 000, 1-5 (2009
10.1038/nature07684
-HCV replication occurs on
ER membranes
-many HCV proteins have
membrane anchoring
domains
-host cell proteins required
for HCV replication
Lipid Droplets and HCV
Assembly
-immunofluorescent detection of HCV core protein,
NS5A protein and an LD associated protein, ADRP,
staining of lipid droplets with a fluorescent dye
-confocal microscopy shows how all these
interactions take place
-HCV core protein drives assembly at the lipid
droplet
-LD is bound by core, then NS5A and other NS
proteins
HCV Life Cycle: Key Features
• Multiple proteins mediate HCV entry:
– CD81, Scavenger Receptor B1, Claudin 1, Occludin, Very Low
Density Lipoprotein Receptor
• Input HCV RNA is translated, a polyprotein is formed,
and individual viral proteins are released from
polyprotein by cellular and viral proteases
• HCV proteins associate with endoplasmic reticulum
membranes, the site of HCV replication
• Virion assembly occurs at lipid droplets
• HCV leaves the cell by hitching a ride on the
apolipoprotein B secretion pathway
• HCV life cycle is intimately tied with lipid metabolism
HCV Variability
• RNA virus, RDRP lacks proof-reading
function
• Mutations arise during replication are
not corrected
– Genotypes
• genetically divergent HCV isolates that can be
grouped phylogenetically
– Quasispecies
• Highly related yet genetically distinct viruses
HCV Genotypes and
Quasispecies
Definition
Nucleotide
Similarity
Genotype
Heterogeneity
among different
viruses
66% – 69%
Subtype
Closely related
viruses within each
genotype
77% – 80%
Quasispecies
Complex of genetic
variants within
individual viruses
91% – 99%
Term
HCV
9213 sites
4
2c BEBE1
1
4a ED43
2
1b J
2a J6
1b A
1c G9
1a H77
1a HCV1
2b J8
G
5a SA13
A/E
G 93FIHH87G 93SE6165
AE 93TH253
J 94SE7887
J 93SE7022
A 85UGU455
K 96CMMP53
A 94KEQ23
K 97CDEQTB
H BEVI997
F2 95CMMP2
H 90CF056
F1 93BR020
C 95IN2106
D 83CDNDK
J
K
A
9a VN004
H
F
D 83CDELI
D
C 92BR025
C 86ETH222
B 86USJRFL B 83FRHXB2
5
10a JK049
8a VN405
C
B
10%
HIV-1 group M
8316 sites
6b Th580
3a NZL1
7a VN235
6a HK2 11a JK046
3b Tr
10%
6 (7 8 9 11)
3 (10)
Quasispecies are kind of like siblings:
highly related yet genetically distinct
Drugs to Fight HCV Infection
• Interferon Alpha based therapy has been the
standard of care for 20 years
– Recombinant protein that induces an antiviral response in most cells (M.
Gale lecture)
– Pegylated IFN plus ribavirin combination therapy is now the standard of
care (D. Gretch lecture)
• Therapy has improved over 2 decades but still about
half of all patients are not cured of their infection
• Viral Factors:
– Genotype and quasispecies impact response to therapy
– Virus employs strategies to counteract cellular antiviral defenses
• Host Factors
• Drug Factors:
– Nasty side effects: flu-like symptoms, depression, effects on blood
system
– Cost
– Some patients refuse western medicine
Drug Development is NOT Easy
-one for every 1,000 drugs makes it into humans
-One in 5 receive FDA approval
HCV Drugs in Development
(as of April 21st, 2009)
• 23 drugs against HCV targets:
–
–
–
–
•
12 targeting NS3/4a protease
8 targeting NS5B polymerase
2 targeting NS5A
1 entry inhibitor
15 general drugs:
– 6 against cellular targets: cyclophilin, miRNAs, caspases,
glucosidase, phospholipids
– 9 Immunomodulators (stimulators/inhibitors): TLR9 agonists, A3AR
agonists, anti-inflammatory, anti-fibrotic
•
6 Interferons:
– IL-29, oral IFN, albuferon, consensus IFN
• 6 vaccines
• 4 liver cancer drugs
• 42 studies cancelled
http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs.html
Targeting Host Cell Functions
Required for HCV Replication
• Debio-025, NIM811
– cyclophilin B inhibitor
– CYPB binds NS5B and enhances binding
to HCV RNA
• Nitazoxanide
– A broad spectrum antibiotic!
Complementary and
Alternative Medicine (CAM)
• Used for centuries by billions of people
– Traditional Chinese Medicine
– Kampo Medicine (Japan)
– Herbal Supplements (America)
• Improves “liver function” (hepatoprotection)
– Antiviral, Immunomodulatory, Anti-inflammatory,
Antioxidant
Silymarin
 Extract of crushed milk thistle seeds:
 Milk Thistle:
Silymarin:
Extract from seeds of Milk Thistle
a complex of at least 7 flavonolignans and 1
flavonoid that comprise 65-80% of milk thistle
extract
 Prevents liver disease in many experimental animal
models
 Used widely by HCV patients as a hepatoprotectant
 Clinical studies indicate that Silymarin is very well
tolerated and safe
Hepatoprotection
Antiviral
Antioxidant
Antiinflammatory
Immunomodulatory
Molecular Profile of Silymarin
Silybum marianum
seeds
HPLC Fingerprint of Standardized Milk Thistle Product (MK-001)
Silymarin Inhibits HCV Infection
HCVcc,
(m.o.i. 0.01)
m
-
Therapeutic
Design
-
Silymarin
Preps
D MK UT SB E IFN
NS3
NS5A
Actin
Polyak et al., Gastroenterology. 2007. 132(5):1925-36
M
E
JFH-1
1 10 20 50 100 I
M=mock
E=EtOH
I=IFN
NS5A
GAPDH
US Pharmacopoeia Milk Thistle
Therapeutic Design
m
oc
k
I
12 FN
hr
I
18 FN
hr
I
24 FN
hr
I
48 FN
hr
I
72 FN
hr
IF
N
4000.0
6h
r
U
S
12
hr P
U
S
18
hr P
U
S
24
hr P
U
S
48
hr P
U
S
72
hr P
U
SP
6h
r
JF
6h H
r
J
12 FH
hr
J
18 FH
hr
J
24 FH
hr
J
48 FH
hr
J
72 FH
hr
JF
H
0h
r
0h
r
HCV copies/10ng
HCV RNA Synthesis
HCVcc,
(m.o.i. 0.01)
7000.0
6000.0
5000.0
* *
* * * *
3000.0
2000.0
1000.0
0.0
Infectious Virus Release
Supes From 48 Hours Post-Treatment
DMSO
Silymarin
Huh7.5.1 & Huh7
Intravenous Silymarin Reduces Viral
Loads in IFN Nonresponders
Silybinin
Ferenci et al., Gastroenterology 2008
PEG/RBV
Silibinin
5 mg/kg
10 mg/kg
15 mg/kg
20 mg/kg
Courtesy of Peter Ferenci
Summary
• HCV is a positive sense RNA virus that causes chronic liver
infection in the majority of people it infects
• HCV is a global health problem
• During its life cycle, HCV interacts with numerous cell surface
proteins on liver cells, replicates in association with cellular
membranes, and assembles at lipid droplets
• HCV mutates during replication, generating genotypes and
quasispecies which influence response to therapy
• IFN therapy is the main treatment option for patients with
chronic hepatitis C
• New treatments are in development, some that target the virus,
some that target cellular factors needed for HCV replication
• Complementary and Alternative Medicines are self-prescribed
by HCV patients so it is imperative to understand their
mechanisms of action and possible interactions with westernbased treatments
The Future
• Better Drugs
– fewer side effects
• Vaccine?
• Limitations:
–
–
–
–
–
Compliance
Resistance, resistance, resistance
funding, not on global scene
expensive meds won’t work for all
need more affordable therapies that can reach
patients in less developed countries
Acknowledgements
• Polyak Lab:
– Jessica Wagoner, Michael Austin, Jessica
Brownell
• NIH
– NIDDK, NIAID, NCCAM