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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

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slide1

The Molecular Virology of Hepatitis C Virus

Stephen J. Polyak, Ph.D.

Research Associate Professor

Department of Laboratory Medicine


University of Washington

overview
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
HCV Research Timeline

2009

Infectious HCV cell culture system

2005

2003

Functional HCV

pseudoparticle system

Infectious cDNA clone constructed

Non-infectious replicon system

1997

1999

HCV serine protease structure (NS3-4A)

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
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 identification5
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
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
Southern Blots with Clone 81

Infected Chimp Liver

Acute Chronic

Placenta

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
Northern Blots with Clone 81

RNase A

DNaseI

Ctrl

2 4 12 g

Infected Chimp Liver

Uninfected Chimp Liver

Oligo dT

unbound

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
Western blot

Clone 5-1-1 expressed as SOD fusion protein called PS5

SOD

PS5

NANBH patient serum

Experimentally infected chimp serum

Protein from clone 5-1-1 closely associated with NANBH infections

slide10

HCV was the first virus to be isolated and characterized solely by molecular methods, ie without culture methods

slide12

-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

hcv genome
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

slide14

Composition of Hepatitis C Virus

Lipid Envelope

Capsid Protein

Nucleic Acid

Envelope Glycoprotein E2

Envelope Glycoprotein E1

Courtesy Michael Gale via Tenille Gale

slide15

HCV Life Cycle

Typical virus life cycle:

Binding, internalization, uncoating, protein expression/replication, assembly, release

tools to study hcv entry
Tools to study HCV entry
  • Pseudoparticles
  • (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 genes

HCV E1E2

HIV particle

Reporter gene

Bartosch, J Exp Med 2003

Hsu, PNAS 2003

Wakita, Nat Med 2005

Lindenbach, Science 2005

Zhong, PNAS 2005

Courtesy of Matt Evans

hcv receptors entry factors

Claudin-1

SR-BI

GAGs

CD81

(Evans, von Hahn et al., 2007)

(Scarcelli et al., 2002)

(Barth et al., 2003)

(Pileri et al., 1998)

HCV receptors/entry factors

LDL-R

(Agnello et al., 1999)

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)

Form the backbone of tight junction strands in epithelial tissues

Highest expression in hepatocytes (less elsewhere)

Low density lipoprotein receptor

Intracellular

Silencing confers resistance

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

Expression confers susceptibility

Ligands influence infection

Expression confers susceptibility

occludin 1 the newest hcv entry factor
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

slide19

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. Nature000, 1-5 (2009

10.1038/nature07684

slide21

-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

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

slide25

-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
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
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
slide29

G

A/E

G 93FIHH87

G 93SE6165

J

AE 93TH253

J 94SE7887

A

J 93SE7022

A 85UGU455

K

K 96CMMP53

A 94KEQ23

K 97CDEQTB

H

H BEVI997

F2 95CMMP2

H 90CF056

F

F1 93BR020

C 95IN2106

D 83CDNDK

D 83CDELI

C 92BR025

C 86ETH222

D

C

B 86USJRFL

B 83FRHXB2

B

10%

HIV-1 group M

8316 sites

HCV

9213 sites

4

1

2c BEBE1

4a ED43

2

1b J

1c G9

1b A

2a J6

1a H77

1a HCV1

2b J8

5

5a SA13

9a VN004

10a JK049

8a VN405

6b Th580

3a NZL1

7a VN235

11a JK046

3 (10)

6a HK2

3b Tr

10%

6 (7 8 9 11)

drugs to fight hcv infection
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

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
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
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
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
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
slide37

Hepatoprotection

Antiinflammatory

Antiviral

Antioxidant

Immunomodulatory

molecular profile of silymarin
Molecular Profile of Silymarin

Silybum marianum

seeds

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

slide39

JFH-1

M

E

1 10 20 50 100

I

M=mock

E=EtOH

I=IFN

NS5A

GAPDH

US Pharmacopoeia Milk Thistle

Silymarin Inhibits HCV Infection

HCVcc,

(m.o.i. 0.01)

Silymarin

Preps

m

- - D MK UT SB E IFN

NS3

NS5A

Actin

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

Therapeutic

Design

slide40

HCV RNA Synthesis

HCVcc,

(m.o.i. 0.01)

*

*

*

*

*

*

Therapeutic Design

slide41

Infectious Virus Release

Supes From 48 Hours Post-Treatment

DMSO

Silymarin

Huh7.5.1 & Huh7

intravenous silymarin reduces viral loads in ifn nonresponders
Intravenous Silymarin Reduces Viral Loads in IFN Nonresponders

PEG/RBV

Silybinin

Silibinin

5 mg/kg

10 mg/kg

15 mg/kg

20 mg/kg

Ferenci et al., Gastroenterology 2008

Courtesy of Peter Ferenci

summary
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
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
Acknowledgements
  • Polyak Lab:
    • Jessica Wagoner, Michael Austin, Jessica Brownell
  • NIH
    • NIDDK, NIAID, NCCAM