The Molecular Virology of Hepatitis C Virus

Stephen J. Polyak, Ph.D.Research Associate Professor

Department of Laboratory MedicineUniversity 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

1975

Description of non-A, non-B hepatitis

1989

IdentificatioIdentification of n of

hepatitis C hepatitis C virusvirus

IdentificatioIdentification of n of

hepatitis C hepatitis C virusvirus

1993Delineation of HCV genome organization and polyprotein processing

1996HCV serine protease structure (NS3-4A)

1998 IFN-alpha and ribavirin combination therapy

1997Infectious cDNA clone

constructed

2003Functional HCV pseudoparticle system

2005

Infectious HCV Infectious HCV cell culture cell culture

systemsystem

Infectious HCV Infectious HCV cell culture cell culture

systemsystem

2009

1999Non-Non-infectious infectious replicon system

Non-Non-infectious infectious replicon system

Courtesy of Charlie Rice

HCV Research Timeline

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 IdentificationHigh 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

Phage with portions of

HCV genome

Infect E.coli

Phage kills E.coli and

leaves plaques

Screen with HCV Patient

serum

Library Screening

Southern Blots with Clone 81

Placenta

Infected Chimp LiverAcute Chronic Placenta

Clone 81 probe 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

Clones Hybridize to RNA from infected animals, RNA is single stranded, size approx 5-10,000 nucleotides

Infected Chimp Liver

Uninfected Chimp Liver

Uninfected Chimp Liver

2 4 12 g DNaseI

RNase

A

Strand 1 Clone 81 probe

Strand 2 Clone 81 probe

Viral RNA ds 81

Olig

o dT

unbo

und

Ctrl

Western blotClone 5-1-1 expressed as SOD fusion protein called PS5

PS5

SO

D

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

Flavi

GBV-B

GBV-AHCV

Pesti

GBV-CHGVFlaviviridae

-enveloped -ssRNA -(+) sense

-HCV is related to flaviviruses (West Nile virus, -HCV is related to flaviviruses (West Nile virus, Dengue virus) and PestivirusesDengue virus) and Pestiviruses

-HCV occupies a unique branch on the flavivirus -HCV occupies a unique branch on the flavivirus phylogenetic tree, known as hepaciviridaephylogenetic tree, known as hepaciviridae

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:Typical virus life cycle:

Binding, internalization, Binding, internalization, uncoating, protein uncoating, protein

expression/replication, expression/replication, assembly, releaseassembly, release

Tools to study HCV entryPseudoparticles

(HCVpp)

• 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

Cell culture virus(HCVcc)

• 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 genesBartosch, J Exp Med 2003

Hsu, PNAS 2003Wakita, Nat Med 2005 Lindenbach, Science 2005Zhong, PNAS 2005

HIV particle

Reporter gene

HCV E1E2

Courtesy of Matt Evans

HCV receptors/entry factors

Intracellular

GAGs(Barth et al., 2003) (Pileri et al., 1998)

CD81(Scarcelli et al., 2002)

SR-BI

Linear polysaccharides on proteins of all human cell surfaces

Tetraspanin superfamily member

Expressed in all nucleated cells

Part of B-cell receptor complex

Scavenger receptor class B member I

HDL receptor (multiple other ligands)

Expressed in hepatocytes, adrenal cortex, gonads (less elsewhere)

(Evans, von Hahn et al., 2007)

Claudin-1

Form the backbone of tight junction strands in epithelial tissues

Highest expression in hepatocytes (less elsewhere)

Expression confers susceptibility

Silencing confers resistance

Ligands influence infection

Expression confers susceptibility

LDL-R

Low density lipoprotein receptor

(Agnello et al., 1999)

viral RNA accumulation increased or decreased in

parallel with LDLR mRNA expression

and LDL entry

Provides a way for the virus to stick to

cells

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

A Ploss et al. Nature 000, 1-5 (200910.1038/nature07684

Expression of human OCLN and CD81 determines HCV species tropism

Mouse NIH3T3 cells are only Mouse NIH3T3 cells are only permissive for HCVpp entry permissive for HCVpp entry when CD81, SR-B1, CLDN1 when CD81, SR-B1, CLDN1

and OCLN expressedand OCLN expressed

Hamster CHO cells are only Hamster CHO cells are only permissive for HCVpp entry permissive for HCVpp entry

when human OCLN expressedwhen human OCLN expressed

-HCV replication occurs on -HCV replication occurs on ER membranesER membranes

-many HCV proteins have -many HCV proteins have membrane anchoring membrane anchoring

domainsdomains

-host cell proteins required -host cell proteins required for HCV replicationfor HCV replication

Lipid Droplets and HCV Assembly

-immunofluorescent detection of HCV core protein, -immunofluorescent detection of HCV core protein, NS5A protein and an LD associated protein, ADRP, NS5A protein and an LD associated protein, ADRP,

staining of lipid droplets with a fluorescent dyestaining of lipid droplets with a fluorescent dye

-confocal microscopy shows how all these -confocal microscopy shows how all these interactions take placeinteractions take place

-HCV core protein drives assembly at the lipid -HCV core protein drives assembly at the lipid dropletdroplet

-LD is bound by core, then NS5A and other NS -LD is bound by core, then NS5A and other NS proteinsproteins

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

Term DefinitionNucleotide Similarity

GenotypeHeterogeneity among different viruses

66% – 69%

SubtypeClosely related viruses within each genotype

77% – 80%

QuasispeciesComplex of genetic variants within individual viruses

91% – 99%

10%

5a SA13

10a JK049

3a NZL1

3b Tr

7a VN23511a JK0466a HK2

6b Th580

8a VN405

9a VN004

2b J8

2a J6

2c BEBE1 4a ED43

1b J1b A 1c G9

1a H77

1a HCV1

2

41

5

6 (7 8 9 11)

3 (10)

HCV9213 sites

10%

H BEVI997

H 90CF056

C 95IN2106

C 92BR025C 86ETH222

B 83FRHXB2B 86USJRFL

D 83CDELID 83CDNDK

F1 93BR020

F2 95CMMP2

K 97CDEQTBK 96CMMP53

J 93SE7022J 94SE7887

G 93FIHH87G 93SE6165

AE 93TH253

A 85UGU455

A 94KEQ23

G

A

A/E

H

CB

D

F

K

J

HIV-1 group M8316 sites

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

Extract of crushed milk thistle seeds:Milk Thistle:

Silymarin: Extract from seeds of Milk Thistlea 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

Silymarin

Immunomodulatory

AntiinflammatoryAntiviral

Antioxidant

Hepatoprotection

Silybum marianumseeds

HPLC Fingerprint of Standardized Milk Thistle Product (MK-001)

Molecular Profile of Silymarin

NS3NS5A

m - - D MK UT SB E IFN

Actin

SilymarinPreps

Silymarin Inhibits HCV Infection

NS5A

GAPDH

M E 1 10 20 50 100 IM=mockE=EtOHI=IFN

JFH-1

US Pharmacopoeia Milk Thistle

TherapeuticDesign

Polyak et al., Gastroenterology. 2007. 132(5):1925-36

HCVcc,(m.o.i. 0.01)

HCV RNA Synthesis

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

0hr m

ock

0hr J

FH

6hr J

FH

12hr

JFH

18hr

JFH

24hr

JFH

48hr

JFH

72hr

JFH

6hr U

SP

12hr

USP

18hr

USP

24hr

USP

48hr

USP

72hr

USP

6hr I

FN

12hr

IFN

18hr

IFN

24hr

IFN

48hr

IFN

72hr

IFN

HC

V c

op

ies/1

0n

g

* * * * * *

Therapeutic Design

HCVcc,(m.o.i. 0.01)

DMSO Silymarin

Supes From 48 Hours Post-Treatment

Infectious Virus Release

Huh7.5.1 & Huh7

Intravenous Silymarin Reduces Viral Loads in IFN Nonresponders

SilibininSilibinin5 mg/kg5 mg/kg10 mg/kg10 mg/kg15 mg/kg15 mg/kg20 mg/kg20 mg/kg

PEG/RBVPEG/RBVSilybininSilybinin

Ferenci et al., Gastroenterology 2008Ferenci et al., Gastroenterology 2008 Courtesy of Peter FerenciCourtesy 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 western-based 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