The Molecular Virology of Hepatitis C Virus

1. The Molecular Virology of Hepatitis C Virus Stephen J. Polyak, Ph.D. Research Associate Professor Department of Laboratory Medicine
 University of Washington

2. 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

3. 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

4. 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

5. 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

6. Library Screening  Phage with portions of HCV genome Infect E.coli Phage kills E.coli and leaves plaques Screen with HCV Patient serum

7. 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

8. 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

9. 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

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

11. Happy 20th Birthday HCV! April 21, 1989

12. -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

13. 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

14. Composition of Hepatitis C Virus Lipid Envelope Capsid Protein Nucleic Acid Envelope Glycoprotein E2 Envelope Glycoprotein E1 Courtesy Michael Gale via Tenille Gale

15. HCV Life Cycle Typical virus life cycle: Binding, internalization, uncoating, protein expression/replication, assembly, release

16. 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

17. 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

18. 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

19. 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

21. -HCV replication occurs on ER membranes -many HCV proteins have membrane anchoring domains -host cell proteins required for HCV replication

24. 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

25. -HCV core protein drives assembly at the lipid droplet -LD is bound by core, then NS5A and other NS proteins

26. 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

27. 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

28. HCV Genotypes and Quasispecies

29. 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)

30. Quasispecies are kind of like siblings: highly related yet genetically distinct

31. 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

32. Drug Development is NOT Easy • one for every 1,000 drugs makes it into humans • One in 5 receive FDA approval

33. 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

34. 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!

35. 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

36. 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

37. Hepatoprotection Antiinflammatory Antiviral Antioxidant Immunomodulatory

38. Molecular Profile of Silymarin Silybum marianum seeds HPLC Fingerprint of Standardized Milk Thistle Product (MK-001)

39. 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

40. HCV RNA Synthesis HCVcc, (m.o.i. 0.01) * * * * * * Therapeutic Design

41. Infectious Virus Release Supes From 48 Hours Post-Treatment DMSO Silymarin Huh7.5.1 & Huh7

42. 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

43. 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

44. 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

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