HEPATITIS C ADVANCED THERAPY FOR€¦ · Laurent Chatel-Chaix, Martin Baril and Daniel Lamarre ... Kenneth Yan and Amany Zekry 13 Altered Dosage or Durations of Current Antiviral

ADVANCED THERAPY FOR

HEPATITIS CEdited by

Geoffrey W. McCaughanJohn G. McHutchisonJean-Michel Pawlotsky

McC

aughan • M

cHutchison •

Pawlotsky

ADVANCED THERAPY FOR

HEPATITIS C

ADVAN

CED

THER

APY FOR

HEPATITIS

C

Edited by

Geoffrey W. McCaughan, University of Sydney, Sydney, AustraliaJohn G. McHutchison, Gilead Sciences, Inc., USA

Jean-Michel Pawlotsky, Université Paris Est, Créteil, France

According to the WHO, 170 million people, or 3% of the world’s population, are infected with Hepatitis C and at risk of developing liver cirrhosis and/or liver cancer. Three to four million people each year are newly diagnosed carriers of the virus.

Advanced Therapy for Hepatitis C provides you with expert guidance from the world’s leading hepatologists on the very latest treatment options for patients with the HCV virus. Focusing mainly on the efficacy and clinical use of antiviral therapies, key topics include:

• Treatment of recurrent hepatitis C following liver transplantation

• Antivirals in cirrhosis and portal hypertension

• HIV and hepatitis C co-infection

• Cytopenias: how they limit therapy and potential correction

• The problem of insulin resistance and its effect on therapy

• Antivirals in acute hepatitis C

In addition, it fully covers the foundations for understanding antiviral therapies in HCV, such as the complex pharmacology and mechanisms of antiviral drugs. Finally, a chapter on New Horizons: Interleukin 28 and direct-acting antiviral therapy for HCV, offers you a glimpse into the future possibilities for HCV therapy.

Edited by a team of outstanding international reputation, Advanced Therapy for Hepatitis C is an essential tool for all hepatologists and gastroenterologists involved in the management of patients with hepatitis C. Titles of related interest

Sherlock’s Diseases of the Liver 12th editionDooley, ISBN 9781405134897

Medical Care of the Liver Transplant Patient, 4th editionClavien, ISBN 9781444335910

Cover design: Meaden CreativeCover image: Hepatitis C virus © Science Photo Library

9 781405 187459

ISBN 978-1-4051-8745-9

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Advanced Therapy for Hepatitis C





Advanced Therapyfor Hepatitis CE D I T E D B Y

Geoffrey W. McCaughan MBBS, PhD, FRACPHead of Liver Immunobiology Program

Centenary Research Institute

A.W. Morrow Professor of Medicine

Director A.W. Morrow GE/Liver Center

Director Australian Liver Transplant Unit

Royal Prince Alfred Hospital

University of Sydney

Sydney, NSW, Australia

John G. McHutchison, MDSenior Vice President, Liver Disease Therapeutics

Gilead Sciences, Inc.

Foster City, CA, USA

Jean-Michel Pawlotsky, MD, PhDDirector, French National Reference Center for Viral Hepatitis B, C and delta

Head, Department of Virology, Bacteriology, and Hygiene

INSERM U955

Hopital Henri Mondor

Universite Paris Est

Creteil, France

A John Wiley & Sons, Ltd., Publication



This edition first published 2012 C© 2012 by Blackwell Publishing Ltd

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Library of Congress Cataloging-in-Publication DataAdvanced therapy for hepatitis C / edited by Geoffrey W. McCaughan, John G.McHutchison, Jean-Michel Pawlotsky.

p. ; cm.Includes bibliographical references and index.ISBN-13: 978-1-4051-8745-9 (hardcover : alk. paper)ISBN-10: 1-4051-8745-X (hardcover : alk. paper)ISBN-13: 978-1-4443-4631-2 (ePDF)ISBN-13: 978-1-4443-4634-3 (Wiley Online Library)[etc.]

1. Hepatitis C–Treatment. 2. Antiviral agents. I. McCaughan, Geoffrey W. II. McHutchison, J. G.III. Pawlotsky, Jean-Michel.

[DNLM: 1. Hepatitis C–therapy. 2. Antiviral Agents–therapeutic use.WC 536]

RC848.H425A38 2012616.3′62306–dc23

2011016561

A catalogue record for this book is available from the British Library.

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1 2012

http://www.wiley.com/wiley-blackwell



Contents

Contributors, viiPreface, xi

Section I Foundations for Understanding Antiviral Therapies in HCV

1 HCV Replication, 3Michael R. Beard

2 Hepatitis C Virus Genotypes, 12Scott A. Read and Mark W. Douglas

3 Immune Responses to HCV: Implications for Therapy, 17David G. Bowen

4 Mechanisms of Action of Antiviral Drugs: The Interferons, 25Edmund Tse and Michael R. Beard

5 Pharmacology and Mechanisms of Action of Antiviral Drugs: Ribavirin Analogs, 36Fred Poordad and Grace M. Chee

6 Pharmacology and Mechanisms of Action of Antiviral Drugs: PolymeraseInhibitors, 43Lotte Coelmont, Leen Delang, Mathy Froeyen, Piet Herdewijn and Johan Neyts

7 Pharmacology and Mechanisms of Action of Antiviral Drugs: Protease Inhibitors, 53Laurent Chatel-Chaix, Martin Baril and Daniel Lamarre

8 Measuring Antiviral Responses, 60Jean-Michel Pawlotsky and Stephane Chevaliez

Section II Efficacy and Clinical Use of Antiviral Therapies

9 Genotype 1: Standard Treatment, 67Rebekah G. Gross and Ira M. Jacobson

10 Individually Tailored Treatment Strategies in Treatment-naıve ChronicHepatitis C Genotype 1 Patients, 74Johannes Wiegand and Thomas Berg

v



vi Contents

11 Genotype 1 Relapsers and Non-responders, 84Salvatore Petta and Antonio Craxı

12 Standard Therapy for Genotypes 2/3, 90Kenneth Yan and Amany Zekry

13 Altered Dosage or Durations of Current Antiviral Therapy for HCVGenotypes 2 and 3, 97Alessandra Mangia, Leonardo Mottola and Angelo Andriulli

14 Genotypes 2 and 3 Relapse and Non-response, 104Stella Martınez, Jose Marıa Sanchez-Tapias and Xavier Forns

15 Hepatitis C Genotype 4 Therapy: Progress and Challenges, 113Sanaa M. Kamal

16 Antivirals in Acute Hepatitis C, 127Heiner Wedemeyer

17 Antivirals in Cirrhosis and Portal Hypertension, 132Diarmuid S. Manning and Nezam H. Afdhal

18 Treatment of Recurrent Hepatitis C Following Liver Transplantation, 140Ed Gane

19 Antiviral Treatment in Chronic Hepatitis C Virus Infection with ExtrahepaticManifestations, 150Benjamin Terrier and Patrice Cacoub

20 Cytopenias: How they Limit Therapy and Potential Correction, 160Mitchell L. Shiffman

21 The Problem of Insulin Resistance and its Effect on Therapy, 169Venessa Pattullo and Jacob George

22 HIV and Hepatitis C Co-infection, 177Gail V. Matthews and Gregory J. Dore

23 HCV and Racial Differences, 185Andrew J. Muir

24 HCV and the Pediatric Population, 190Kathleen B. Schwarz

25 New Horizons: IL28, Direct-acting Antiviral Therapy for HCV, 196Alexander J. Thompson, John G. McHutchison and Geoffrey W. McCaughan

Index, 215


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Contributors

Nezam H. Afdhal MDBeth Israel Deaconess Medical CenterBoston, MA, USA

Angelo Andriulli MDGastroenterology DepartmentIRRCS Casa Sollievo della SofferenzaSan Giovanni Rotondo, Italy

Martin Baril PhDResearch AssociateInstitut de Recherche en Immunologieet Cancerologie (IRIC)Montreal, Quebec, Canada

Michael R. Beard PhDHead, Hepatitis C Virus Research LaboratorySchool of Molecular and Biomedical ScienceThe University of Adelaide & Center for Cancer Biology,SA PathologyAdelaide, SA, Australia

Thomas Berg MDProfessorDepartment of Gastroenterology and RheumatologyDivision of HepatologyUniversity of LeipzigLeipzig, Germany

David G. Bowen MBBS, PhDSydney Medical School, University of SydneyRoyal Prince Alfred HospitalSydney, NSW, Australia

Patrice Cacoub MD, PhDProfessorDepartment of Internal MedicineGroupe Hospitalier Pitie-SalpetriereUniversite Pierre et Marie CurieParis, France

Laurent Chatel-Chaix PhDPost-doctoral FellowInstitut de Recherche en Immunologieet Cancerologie (IRIC)Montreal, Quebec, Canada

Grace M. Chee PharmDHepatology DepartmentCedars-Sinai Medical CenterLos Angeles, CA, USA

Stephane Chevaliez PharmD, PhDNational Reference Center for Viral Hepatitis B, Cand deltaDepartment of Virology & INSERM U955Hopital Henri MondorUniversite Paris-EstCreteil, France

Lotte Coelmont PhDLaboratory of Virology and ChemotherapyRega Institute for Medical ResearchUniversity of LeuvenLeuven, Belgium

Antonio Craxı, MDFull Professor of GastroenterologySezione di Gastroenterologia, Di.Bi.M.I.S. PoliclinicoPaolo GiacconeUniversity of PalermoPalermo, Italy

Leen Delang PhDLaboratory of Virology and ChemotherapyRega Institute for Medical ResearchUniversity of LeuvenLeuven, Belgium

vii



viii Contributors

Gregory J. Dore BSc, MBBS, MPH, PhD, FRACPProfessor and Head, Viral Hepatitis ClinicalResearch ProgramNational Centre in HIV Epidemiology andClinical ResearchThe University of New South Wales;Infectious Diseases PhysicianSt Vincent’s HospitalSydney, NSW, Australia

Mark W. Douglas BSc (Med)(Hons), MBBS (Hons),PhD, FRACPSenior Lecturer, Hepatology and VirologyStorr Liver Unit, Westmead Millennium InstituteSydney Emerging Infections and Biosecurity InstituteUniversity of SydneySydney, NSW, Australia

Xavier Forns MD, PhDLiver Senior SpecialistLiver Unit, Hospital ClinicIDIBAPS and Ciberehd (Centro de Investigacion en Redde Enfermedades Hepaticas y Digestivas)Barcelona, Spain

Mathy Froeyen PhDAssistant Professor of PharmacyLaboratory of Medicinal ChemistryRega Institute for Medical ResearchUniversity of LeuvenLeuven, Belgium

Ed Gane MB ChB, MD, FRACPAssociate Professor and HepatologistNew Zealand Liver Transplant UnitAuckland City HospitalAuckland, New Zealand

Jacob George MBBS (Hons), FRACP, PhDDirector of Gastroenterology and Hepatic ServicesStorr Liver Unit, Westmead Millenium InstituteUniversity of SydneySydney, NSW, Australia

Rebekah G. Gross MDAssistant Professor of MedicineDivision of Gastroenterology and HepatologyWeill Cornell Medical CollegeNew York, NY, USA

Piet Herdewijn PhDProfessor of PharmacyLaboratory of Medicinal ChemistryRega Institute for Medical ResearchUniversity of LeuvenLeuven, Belgium

Ira M. Jacobson MDVincent Astor Professor of MedicineChief, Division of Gastroenterology and HepatologyDivision of Gastroenterology and HepatologyWeill Cornell Medical CollegeNew York, NY, USA

Sanaa M. Kamal MD, PhDDepartment of Gastroenterology and Liver DiseaseAin Shams Faculty of MedicineCairo, Egypt;Department of GastroenterologyTufts School of MedicineBoston, MA, USA

Daniel Lamarre PhDFull Professor, Department of MedicineFaculty of MedicineInstitute for Research in Immunology and Cancer (IRIC)Universite de MontrealMontreal, Quebec, Canada

Alessandra Mangia MDLiver UnitIRRCS Casa Sollievo della SofferenzaSan Giovanni Rotondo, Italy

Diarmuid S. Manning MB, BChBeth Israel Deaconess Medical CenterHarvard Medical SchoolBoston, MA, USA

Stella Martınez MDLiver Unit, Hospital ClinicIDIBAPS and Ciberehd (Centro de Investigacion en Redde Enfermedades Hepaticas y Digestivas)Barcelona, Spain

Gail V. Matthews MBChB, MRCP (UK), FRACP, PhDClinical AcademicNational Centre in HIV Epidemiology and ClinicalResearchUniversity of New South WalesSydney, NSW, Australia



Contributors ix

Geoffrey W. McCaughan MBBS, PhD, FRACPHead of Liver Immunobiology ProgramCentenary Research InstituteA.W. Morrow Professor of MedicineDirector A.W. Morrow GE/Liver CenterDirector Australian Liver Transplant UnitRoyal Prince Alfred HospitalUniversity of SydneySydney, NSW, Australia

John G. McHutchison, MDSenior Vice President, Liver Disease TherapeuticsGilead Sciences, Inc.Foster City, CA, USA

Leonardo Mottola PhDLiver UnitIRRCS Casa Sollievo della SofferenzaSan Giovanni Rotondo, Italy

Andrew J. Muir MD MHSDirector, Gastroenterology/Hepatology ResearchDuke Clinical Research InstituteDuke University Medical CenterDurham, NC, USA

Johan Neyts PhDProfessor of VirologyLaboratory of Virology and ChemotherapyRega Institute for Medical ResearchUniversity of LeuvenLeuven, Belgium

Venessa Pattullo MBBS, FRACPStorr Liver Unit, Westmead Millennium InstituteUniversity of Sydney, Sydney, NSW, Australia;Division of GastroenterologyToronto Western HospitalUniversity Health Network, University of TorontoToronto, Ontario, Canada

Jean-Michel Pawlotsky MD, PhDDirector, French National Reference Center for ViralHepatitis B, C and deltaHead, Department of Virology, Bacteriology, andHygieneINSERM U955Hopital Henri MondorUniversite Paris EstCreteil, France

Salvatore Petta MD, PhDSezione di Gastroenterologia, Di.Bi.M.I.S. PoliclinicoPaolo GiacconeUniversity of PalermoPalermo, Italy

Fred Poordad MDAssociate Professor of MedicineDavid Geffen School of Medicine at UCLA;Chief, Hepatology and Liver TransplantationCedars-Sinai Medical CenterLos Angeles, CA, USA

Scott A. Read MScStorr Liver Unit, Westmead Millennium InstituteUniversity of SydneySydney, NSW, Australia

Jose Marıa Sanchez-Tapias MD, PhDSenior ConsultantLiver Unit, Hospital ClinicIDIBAPS and Ciberehd (Centro de Investigacion en Redde Enfermedades Hepaticas y Digestivas)Barcelona, Spain

Kathleen B. Schwarz MDDirector, Pediatric Liver CenterDivision of Pediatric Gastroenterology and NutritionProfessor of PediatricsJohns Hopkins University School of MedicineBaltimore, MD, USA

Mitchell L. Shiffman MDDirectorLiver Institute of VirginiaBon Secours Virginia Health SystemRichmond and Newport News, VA, USA

Benjamin Terrier MDDepartment of Internal MedicineGroupe Hospitalier Pitie-SalpetriereUniversite Paris 6 Pierre et Marie CurieParis, France

Alexander J. Thompson MD, PhDSt. Vincent’s Hospital Melbourne, University ofMelbourne, Melbourne, VIC, Australia;Victorian Infectious Diseases Reference Laboratory(VIDRL), North Melbourne, VIC, Australia;Department of Gastroenterology and Duke ClinicalResearch InstituteDuke UniversityDurham, NC, USA



x Contributors

Edmund Tse MBBS, FRACPSchool of Molecular and Biomedical ScienceThe University of Adelaide and the Center for CancerBiologySA PathologyAdelaide, SA, Australia

Heiner Wedemeyer MDProfessorDepartment of Gastroenterology, Hepatology andEndocrinologyMedizinische Hochschule HannoverHannover, Germany

Johannes Wiegand MDPrivate LecturerDepartment of Gastroenterology and RheumatologyDivision of HepatologyUniversity of LeipzigLeipzig, Germany

Kenneth Yan MBBS, Mmed (Clin Epi), FRACPConjoint Associate LecturerSt George Clinical SchoolFaculty of MedicineUniversity of New South WalesSydney, NSW, Australia

Amany Zekry MBBS, PhD, FRACPDepartment of Gastroenterology and HepatologyClinical School of MedicineSt George HospitalSydney, NSW, Australia


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Preface

Hepatitis C virus results in chronic liver disease in over170 million people worldwide. This book arrives at awatershed in the history of antiviral treatment of the hep-atitis C virus. It is the beginning of the end of non-specificantiviral approaches via interferon-based therapies. Fromnow on the field will be dominated by the arrival of HCV-specific direct antiviral agents. Initially these agents willstill require interferon and ribavirin but already clinicaltrials are under way that do not include either of theseagents.

This publication outlines the current standard of careup until this time and includes therapeutic approachesto wide patient groups. We believe that the structureof the book will remain relevant for future editions asthe new therapies are gradually rolled out across thesepatient groups, as well as across an increasing number ofcountries.

G.W.M.J.G.M.J-M.P.

xi


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I Foundations for UnderstandingAntiviral Therapies in HCV





1 HCV ReplicationMichael R. BeardSchool of Molecular and Biomedical Science, University of Adelaide, and Centre for Cancer Biology,SA Pathology, Adelaide, SA, Australia

Introduction

Hepatitis C virus (HCV) is classified in the Hepacivirusgenus within the family Flaviviridae and is the leadingcause of chronic hepatitis and liver disease related mor-bidity worldwide. With an estimated 170 million peopleinfected worldwide and the ability of the virus to estab-lish a chronic infection in approximately 70% of cases,it is not surprising that HCV represents a major cause ofglobal suffering and morbidity and a burden to many pub-lic health systems. Chronic HCV infection is often asso-ciated with development of serious liver disease, includ-ing cirrhosis, liver failure, and hepatocellular carcinoma.Accordingly, a thorough understanding of the life cycleand molecular biology of HCV and its interaction withthe host are essential in the development of treatmentand vaccine strategies. Although these studies have beenhampered by the lack of a small-animal model and, untilrecently, a lack of a tissue culture system that accuratelyreflects the life cycle of HCV, significant progress has beenmade in the understanding of HCV molecular biology andpathogenesis. In this chapter we discuss recent advancesin models to study HCV replication and the HCVlife cycle.

The HCV Genome

HCV possesses a 9.6 kb single-stranded, positive-senseRNA genome composed of a 5′ UTR (untranslatedregion), a long open-reading frame (ORF) encoding apolyprotein of approximately 3000 amino acids, and a 3′

UTR (Figure 1.1). The polyprotein can be divided into

three segments based on the functional aspects of the pro-teins: the NH2 terminal region comprises the structuralproteins (core, E1, and E2); a central region consists oftwo proteins (p7 and NS2) that are not involved in HCVreplication or are structural components of the virus, butprobably play a role in virion morphogenesis; and theCOOH-terminal proteins (NS3, NS4A, NS4B, NS5A, andNS5B) that are required for HCV replication (Figure 1.1).A detailed description and function of the HCV proteinscan be found in an excellent review from Moradpourand colleagues [1]. After release of the HCV genome intothe cytoplasm the genome is exposed to the host cellularmachinery for translation of the viral polyprotein. The5′ and 3′ UTRs are highly conserved and critical to viralgenome replication and translation of the viral polypro-tein. The 5′ UTR is approximately 341 nucleotides longand contains a highly structured RNA element known asthe internal ribosome entry site (IRES) that is recognizedby the cellular 40S ribosomal subunit to initiate transla-tion of the RNA genome in a cap-independent manner.The importance of the secondary and tertiary structureof the IRES domain for initiation of translation has beendemonstrated by mutational analysis. However, the pri-mary sequence, particularly in stem-loop IIId and IIIe, isalso critical for efficient HCV IRES activity [2,3]. Recently,the structural nature of HCV IRES interactions with the40S ribosomal subunit and the eIF3 complex has beenrevealed by cryo-electron microscopy [4,5]. Preceding theIRES at the extreme 5′ end are elements required for viralreplication that overlap partially with the IRES region(domain II), leading to speculation that this region isinvolved in regulation of a viral translation to replicationswitch [6]. Consistent with this speculation is the recentobservation that a short highly conserved RNA segmentat the 5′ end of the HCV genome binds a liver-specific

Advanced Therapy for Hepatitis C, First Edition. Edited by Geoffrey W. McCaughan, John G. McHutchison and Jean-Michel Pawlotsky.c© 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

3



4 Chapter 1

Structural proteins Non-structural proteins

C E1 E2 NS2 NS3 4a NS4b NS5a NS5bp7

9.6 kb

C E1 E2 p7 NS2 NS3 NS4b4a NS5a NS5b

Core Envelopeglycoproteins

Protease?assembly

Serineprotease

Helicase Replicationcomplex

Multifunctionalreplication

host interactions

RNA-dependentRNA polymerase

?viroporin NS3 serineprotease cofactor

IRES directed polyproteintranslation

Polyprotein processing

Host signal peptidase

NS2/3 cysteine proteinase

NS3 serine protease

5′NTR 3′NTR

IRES

Figure 1.1 Genomic organization and polyprotein processing of the HCV genome. The HCV genome consists of a positive-strandedRNA genome that is flanked by 5′ and 3′ UTRs of highly ordered secondary structure. The polyprotein is cleaved by either host- orviral-encoded proteases (depicted by triangles) to liberate the mature structural and non-structural proteins.

microRNA, miR-122, that is required for efficient HCVreplication in cultured hepatoma cells [7]. However, inthe HCV-infected liver no correlation was noted betweenmiR-122 abundance and levels of HCV RNA in the liveror serum, which suggests that the impact of miR-122 maybe less prominent in vivo than in vitro [8]. miR-122 mayimpact HCV replication indirectly through stimulationof translation of the viral polyprotein by enhancing asso-ciation of ribosomes with the IRES [9]. Although morework is required to determine its role in the HCV life cycleand pathogenesis, modulation of miR-122 expression andactivity presents as an attractive target for future therapy.

Like the 5′ UTR, the 3′ UTR of the HCV genome con-tains a high degree of secondary structure. This regionis 200–300 nucleotides in length and is comprised ofthree major elements involved in replication: (i) a vari-able region (30–50 nucleotides), which directly followsthe NS5B stop codon; (ii) a polyuridine (U/C) tract(20–200 nucleotides); and (iii) a highly conserved region(98 nucleotides), known as the 3′ X region, which formsa three stem-loop structure [10–12]. Mutational analysishas revealed that the poly-U/C tract and the 3′ X regionplay a more important role than the variable region in thesynthesis of negative-strand RNA [13].

Models to Study HCV Replication

HCV RepliconsThe development of the subgenomic HCV repliconsystem, first reported in 1999, significantly enabledthe study of HCV replication in cultured cells forthe first time [14]. Replicons represent autonomouslyreplicating HCV RNAs, and typically contain an in-frame insertion of a selectable antibiotic marker (e.g.,neomycin phosphotransferase: G418) within the aminoterminal HCV core sequence, followed downstream bya heterologous IRES from encephalomyocarditis virus(EMCV), a picornavirus, to drive internal translationof the downstream HCV open reading frame (NS2 toNS5B) (Figure 1.2). The minimal requirements for aviable HCV replicon are HCV-derived 5′ and 3′ ter-mini and the non-structural proteins (NS3 to NS5B)that form the replication complex, however, replication-competent HCV replicons encoding the complete HCVpolyprotein are viable [15,16]. Transfection of Huh-7 (hepatoma-derived) cells with synthetically derivedtranscripts followed by selection with G418 results in theestablishment of cell lines that harbor autonomous



HCV Replication 5

Neo EMCVIRES

Neo EMCVIRES

C E1 E2 NS2 NS3 4a NS4b NS5a NS5bp7

NS3 4a NS4b NS5a NS5b

(a)

(b)

5′NTR

IRES

5′NTR

IRES

3′NTR

3′NTR

Figure 1.2 Schematic diagram of the organization of the (a) genomic and (b) subgenomic HCV replicons. In both cases the HCVIRES drives expression of the neomycin phosphotransferase gene while the EMCV IRES drives expression of the HCV proteins.

replication of the virus. HCV RNA isolated from celllines under antibiotic selection often contains cell-culture adapted mutations that greatly enhance replica-tion, although the molecular basis for this increased repli-cation is unclear [15,17,18]. These adaptive mutationsoften map to the NS5A protein and may potentially influ-ence phosphorylation, resulting in a hypophosphorylatedstate and increased replication. Adaptive mutations havealso been mapped to NS3, NS4A, NS4B, and NS5B. Inter-estingly, these replication-adaptive cell-culture mutationshave been shown to reduce in vivo infectivity in chim-panzees, highlighting the adaptive nature of these virusesderived from cell culture [19]. HCV replicons are notrestricted to Huh-7 cells, and other cell lines such as HeLaand cells of murine origin have also yielded selected clonesof replicating HCV, highlighting that HCV replicationis not restricted to liver-derived cells of human origin[20,21].

The antibiotic selection process not only selects forHCV genomes with high replication capacity but alsoclones of Huh-7 cells that are highly permissive for HCVinfection. One such cell line, Huh-7.5, has been “cured”of HCV by treatment with low doses of interferon-�, is hyperpermissive for HCV replication [22], andis clearly enriched for factors that promote replicationand/or defects in innate viral sensing pathways. For exam-ple, Huh-7.5 cells have a spontaneous knockout of thedsRNA cellular sensing protein RIG-1 and do not mounta robust antiviral response to viral infection that allowsfor HCV replication. This highlights the importance ofinnate immune sensing in HCV infection and is con-

sistent with the ability of the HCV NS3/4A protein tocleave IPS-1, which is integral to the innate immune RIG-Ipathway [23].

HCV replicons have been valuable tools for study-ing numerous aspects of the HCV life cycle andinteraction with the host cell. However, their major lim-itation has been inability to produce infectious virusparticles even when the complete complement of HCVproteins is expressed, for reasons that are not entirely clear[15,16,22]. The original replicon concept has undergoneevolution and replicons are now available that containvarious markers (e.g., GFP, luciferase) that allow quanti-tative assessment of HCV replication and have been usefulin high-throughput screening of antiviral compounds.

Productive Viral Infection in Cell CultureThe recent identification in 2005 of a cloned HCV genome(genotype 2a), known as JFH-1, that is capable of initiat-ing high-level replication in cell culture and production ofinfectious virus particles represents a major breakthroughin the pursuit of a cell-culture model for HCV [24,25]. Incontrast to HCV replicon systems, transfection of Huh-7 cells with RNA synthesized in vitro from the clonedJFH-1 cDNA genome and a related genotype 2a chimera,FL-J6/JFH replicate efficiently in Huh-7 cells without theneed for cell-culture adaptive mutations. Moreover, virusparticles produced by these cells are infectious in chim-panzees and can be serially passaged in vivo [25] whilethe FL-J6/JFH virus can infect mice containing humanliver grafts [24]. Interestingly, virus produced in vivohas a lower buoyant density than virus produced in cell



6 Chapter 1

culture, suggesting association with low-density lipids[26]. This system represents a major advance in the studyof virus-host interactions and the virus life cycle, all in thecontext of replicating virus. Similarly, the highly adaptedgenotype 1a HCV isolate known as H77-S (derived fromthe H77 isolate) [27] is also capable of instigating HCVRNA replication and production of infectious virus par-ticles [28]. This represents another breakthrough in thegeneration of tools to study the HCV life cycle, particularlybecause this genotype is more prevalent worldwide and isassociated with more significant liver disease. Intrageno-typic and intergenotypic chimeras of HCV that containthe non-structural protein-encoding regions of JFH-1 andthe structural protein-encoding regions of other HCVgenomes may help define regions of structural proteinsthat influence the efficiency of virus particle synthesisand secretion [29]. This relatively new cell-culture modelsystem will be invaluable in the study of many aspects ofvirus-host interaction, including viral entry, and assemblyand release, which were previously inaccessible to manip-ulation.

The HCV Virion and Entry

The relatively low levels of HCV in plasma samples havehampered visualization of viral particles; however, virus-like particles have been identified by electron microscopy,which has indicated that infectious HCV virions areroughly spherical particles of diameter 55–65 nm withfine projections of approximately 6 nm ([25] and ref-erences therein). The major protein constituents of thehost-derived lipid bilayer envelope are the highly glyco-sylated HCV envelope glycoproteins E1 and E2 that sur-round the viral nucleocapsid, composed of many copiesof the HCV core protein and the genomic HCV RNA (Fig-ure 1.3). HCV from serum and plasma fractionates witha wide range of buoyant densities that can be attributedto association of the virus with lipoproteins, in particularapolipoprotein-B (Apo-B) and apolipoprotein-E (Apo-E), which are components of host low density lipoprotein(LDL, Apo-B) and very low density lipoprotein (VLDL,Apo-B, Apo-E) and suggest a close association with cir-culating LDL/VLDL [26]. The physiological associationof HCV with LDL/VLDL remains unexplained mecha-nistically; however, it could be involved in viral uptake(see below), or alternatively the association of Apo-Bwith HCV virions may indicate a role for the hepaticLDL/VLDL secretory pathway in release of the virus.

E1

E2

Core

RNA(+)

Figure 1.3 The HCV particle. The RNA genome isencapsidated by the icosahedral nucleocapsid consisting of thecore protein. The nucleocapsid is enveloped by a host-derivedspherical lipid bilayer that is enriched with heterodimers of theenvelope glycoproteins E1 and E2.

Hepatocytes are the main target for infection with HCV;however, identification of the cellular receptors responsi-ble for HCV entry has proven difficult due to the lack ofappropriate model systems. However, using a combina-tion of HCV pseudotyped particles (HCVpp) [30,31] andcell-culture-derived HCV (HCVcc) [25], the complementof HCV receptors now seems complete.

The 25 kDa tetraspanin molecule CD81 and the humanscavenger receptor class B type I (SR-BI) both bind HCVE2 and are necessary but not sufficient for HCV entry [32].For example, CD81 ectopic expression in hepatocyte-derived cell lines that are negative for CD81 confers sus-ceptibility to HCVpp and HCVcc; however, expression ofboth factors in non-hepatocyte-derived cell lines does notconcur infectivity [24,30]. Clearly additional hepatocytefactors are required for HCV entry. Using an interac-tive cloning and expression approach, the tight junctionprotein claudin-1 (CLDN1) was recently identified as anHCV co-receptor [33]. CLDN1 was found to be essentialfor entry into hepatic cells and rendered non-hepatic cellssusceptible to infection. However, despite the identifica-tion of CD81, SR-B1, and CLDN1 as essential HCV entryco-factors, a number of human cell lines and those of non-primate origin remained resistant to HCV infection, sug-gesting an additional entry factor. Using a cyclic lentivirus-based screen of a cDNA library derived from a highlyHCV-permissive hepatocarcinoma cell line (Huh-7.5) for



HCV Replication 7

genes that render the non-permissive CD81+, SR-BI+293T cell line infectable with HCVpp, the remaining cru-cial factor was recently identified as occludin (OCLN), alsoa tight junction protein [34]. Although expression of allfour entry factors (CD81, SR-B1, CLDN1, OCLN) rendersmouse cell lines susceptible to HCVpp infection, thesecells could not support HCVcc infection. This is not sur-prising given past reports of inefficient replication of HCVRNA in mouse cell lines, and suggests that specific hepato-cyte factors are crucial for efficient HCV replication. Theidentification of CD81 and OCLN as the minimal human-specific entry factors (HCV can bind to murine SR-B1 andCLDN1) not only significantly advances our understand-ing of the molecular mechanisms of HCV entry but alsoprovides important steps for the development of a mousemodel of HCV infection and provides an attractive targetfor the development of novel antiviral strategies.

Other molecules have been suggested to be involved inHCV entry. The association of HCV virions in serum withLDL and VLDL suggests that the LDL receptor (LDLR)may be an attractive candidate receptor. However, its pre-cise role remains to be determined [26,35]. LDLR is notsufficient itself for entry and it does not bind directlyto HCV E2 [30]. Together with the glycosaminoglycans,the LDLR in concert with other cell-surface proteins mayserve to collect HCV at the cell surface and facilitate bind-ing with receptors crucial for HCV entry. Consistent withthis, a role has been proposed for L-SIGN and DC-SIGNin HCV attachment although they do not seem to mediatecell entry of HCV and their role is unclear [36].

The precise molecular events underlying HCV bind-ing and entry are not well understood. However, HCVbinding to the cell surface is thought to occur in a step-wise process by binding to several receptors followed bytransfer to the tight junction proteins CLDN1 and OCLNthat may facilitate cellular uptake (Figure 1.4). Similar toother flaviviruses, HCV entry is thought to be mediated byclathrin-mediated endocytosis with delivery of the nucle-ocapsid from the endosome in a pH-dependent manner[37–39]. Furthermore, the E1 and E2 proteins are class IIfusion proteins that result from the production of a fusionpore in the endosome membrane that facilitates genomerelease to the cytoplasm [40].

HCV Replication

The HCV replication process is summarized in Figure 1.5.After translation of the HCV proteins from the positive-sense RNA genome by direct interaction of the host 40S

ribosomal subunit with the IRES within the 5′ UTR ofthe genome, HCV replication begins. This IRES-directedtranslation is cap-independent and enables virus trans-lation/replication to continue even after host cell cap-dependent translation has been shut down in response toviral infection.

Similar to other positive-stranded viruses, HCV isbelieved to replicate in association with intracellularmembranes in a complex called the membranous web,although the exact details of this association are not wellunderstood. It is thought that the association predom-inantly with endoplasmic reticulum (ER) membranesmay provide support for the organization of the replica-tion complex, compartmentalization of the viral products,concentration of lipid constituents important for replica-tion, and protection of the viral RNA from host-mediatedinnate immune defenses. This membranous web was firstnoticed in cultured cells harboring HCV replicons andcontains detectable concentrations of the non-structuralproteins NS3, NS4A, NS4B, NS5A, and NS5B, and is verysimilar to sponge-like inclusions noted in liver tissue fromHCV-infected chimpanzees [41–44]. Expression of NS4Balone induces the formation of the membranous web, andrecent work has shown that membrane association is facil-itated by amino acids 40 to 69 of the N-terminal region ofNS4B [45].

The phosphorylation status of NS5A appears to be adeterminant of HCV RNA replication with mutationsthat reduce hyperphosphorylation of NS5A dramaticallyenhancing HCV RNA replication [46,47]. In this manner,hyperphosphorylation of NS5A may induce a switch fromgenome replication to viral protein translation. NS5A alsointeracts with several host proteins that may be importantin HCV replication through formation of the replicationcomplex or facilitating assembly. NS5A interacts with theSNARE-like vesicle-associated membrane host proteins,VAP-A and VAP-B [48]. NS5A also interacts with ger-anylgeranylated F-box protein, FBL2, which is essentialfor replication and seems to be part of the replicationcomplex [49]. How this interaction contributes to repli-cation is unclear but it may help anchor the replicasecomplex to membranes. Its involvement in the replica-tion process highlights the close interaction between HCVreplication and the host cholesterol biosynthetic pathway[50]. Another host factor, cyclophilin B, has also beenimplicated in HCV replication through interaction withNS5B and stabilization of RNA binding, and was originallydiscovered through the ability of the powerful immuno-suppressive drug cyclosporin A (CsA) to inhibit HCVreplication [51]. However, more recent work suggests that



8 Chapter 1

CD81SR-BI

CLDN1OCLN

Lipoproteins

LDLR

GAGs

Clathrin-dependent endocytosis and

endosome acidification

Fusion and genome release Replication

Cytosol

Extracellular space

Plasma membrane

Receptor binding

Figure 1.4 Model for HCV entry. HCV particles associatedwith LDL and VLDL are thought to be tethered to thehepatocyte surface by the LDL-R and GAGs and subsequentstepwise interactions with CD81 and SR-B1. HCV is transferredto the tight junction proteins OCLN and CLDN-1 from where

virus enters the cell by endocytosis. Release of the HCV corecontaining the RNA is mediated by fusion of the E1 and E2proteins with the endosome. The relative roles and the spatialdistributions of each of the HCV receptors remain to bedetermined.

cyclophilin A plays a critical role in cleavage of NS5A/5Band assembly of the replication complex [52,53]. CsAanalogs are currently being developed as antivirals againstHCV [54].

The precise details of the HCV RNA replication processare still unclear but comparison with other flavivirusessuggests that the positive-stranded genome serves as atemplate for the synthesis of negative-strand RNA. Com-ponents of the membrane-bound replication complexassociate with the 3′ end of the positive strand of thegenome, with NS5B at the catalytic core, and initiate denovo synthesis of negative-strand RNA. These two strandsremain base-paired, which results in the formation of

a double-stranded RNA molecule that is copied multi-ple times by semiconservative replication by the RNA-dependent RNA polymerase (RdRp) NS5B to generatemultiple progeny, positive-strand viral RNA genomes.Importantly, the NS5B RdRp has no proofreading capacityand as such is error prone. This lack of proofreading abil-ity results in the generation of many different but closelyrelated genomes, often referred to as quasispecies. Thisgenetic diversity is ideally suited to escape of immunecontrol and is a significant factor in the generation ofantiviral resistance to select antiviral agents. While a pro-portion of new positive-strand genomes serve as tem-plates for viral protein translation, others associate with



HCV Replication 9

5′

3′

5′3′

3′5′

+ RNA

+ RNA

- RNA

Endoplasmicreticulum

Lipid droplets

Membranous web

Nucleus

1

2

3

4

5

6

Hepatocyte

Figure 1.5 Lifecycle of HCV. 1, Virus binding and internalization; 2, release of HCV RNA and translation of viral proteins; 3,association of HCV proteins in ER and formation of the replication complex in association with lipid droplets, replication of HCVRNA; 5, virion maturation and packaging of RNA; 6, release of virions.

the core protein and form dimers within core-protein-enriched nucleocapsids. The association of the core pro-tein with cytoplasmic lipid droplets has emerged as acritical determinant of nucleocapsid and infectious viralparticle assembly. It is thought that the core uses thisplatform to recruit replication complexes and associatednew genomes from closely associated ER-derived “lipid-droplet-associated membranes” in the assembly process[55,56]. Core particles may then become enveloped viabudding through the ER where viral glycoproteins (E1/E2heterodimers) become embedded. Little is known aboutthe process of viral particle egress except that particleschange in their biophysical properties (increased density)upon exit. Recent studies have indicated that the processesof HCV particle assembly, maturation, and secretion aredependent upon the machinery involved in the assemblyand secretion of VLDL by hepatocytes [57].

The development and use of in vitro cell-culture modelsystems described above has been and continues to befundamental in dissecting the stages of HCV replicationand identification of viral-host interactions at the molec-ular level (Figure 1.5). While these studies are importantfor our understanding of HCV biology, they also providespecific targets for the development of novel therapeuticsdesigned to completely eradicate HCV infection across allgenotypes. Current therapies for HCV focus on modula-tion of the host immune response. However, with a greaterunderstanding of HCV replication and host interactions,we are currently in a phase of developing therapeutics thatdirectly target various stages of the HCV life cycle. Drugstargeting HCV entry and fusion, viral helicase, and poly-merase or protease function are all under clinical inves-tigation, with some showing exceptional promise. Thesetargeted therapies when used in combination with the



10 Chapter 1

current therapeutic regime of Peg-IFN alfa-2 and rib-avirin will provide the foundation for systemic eradicationof HCV in infected persons. Furthermore, defining novelhost factors essential for HCV replication and a greaterunderstanding of the immunological correlates of immu-nity to HCV will provide the cornerstone for further devel-opment of novel therapeutics to combat HCV infection.

References

1. Moradpour D, Penin F, Rice CM. Replication of hepatitis Cvirus. Nat Rev Microbiol 2007;5(6):453–63.

2. Kieft JS, Zhou K, Jubin R, et al. The hepatitis C virus internalribosome entry site adopts an ion-dependent tertiary fold. JMol Biol 1999;292(3):513–29.

3. Psaridi L, Georgopoulou U, Varaklioti A, Mavromara P.Mutational analysis of a conserved tetraloop in the 5′

untranslated region of hepatitis C virus identifies a novelRNA element essential for the internal ribosome entry sitefunction. FEBS Lett 1999;453(1–2):49–53.

4. Siridechadilok B, Fraser CS, Hall RJ, et al. Structural rolesfor human translation factor eIF3 in initiation of proteinsynthesis. Science 2005;310(5753):1513–15.

5. Spahn CM, Kieft JS, Grassucci RA, et al. Hepatitis C virusIRES RNA-induced changes in the conformation of the 40sribosomal subunit. Science 2001;291(5510):1959–62.

6. Appel N, Schaller T, Penin F, Bartenschlager R. From struc-ture to function: new insights into hepatitis C virus RNAreplication. J Biol Chem 2006;281(15):9833–6.

7. Jopling CL, Yi M, Lancaster AM, et al. Modulation of hep-atitis C virus RNA abundance by a liver-specific MicroRNA.Science 2005;309(5740):1577–81.

8. Sarasin-Filipowicz M, Krol J, Markiewicz I, et al. Decreasedlevels of microRNA miR-122 in individuals with hepati-tis C responding poorly to interferon therapy. Nat Med2009;15(1):31–3.

9. Henke JI, Goergen D, Zheng J, et al. MicroRNA-122stimulates translation of hepatitis C virus RNA. EMBO J2008;27(24):3300–10.

10. Blight KJ, Rice CM. Secondary structure determinationof the conserved 98-base sequence at the 3′ terminusof hepatitis C virus genome RNA. J Virol 1997;71(10):7345–52.

11. Kolykhalov AA, Feinstone SM, Rice CM. Identification ofa highly conserved sequence element at the 3′ terminus ofhepatitis C virus genome RNA. J Virol 1996;70(6):3363–71.

12. Tanaka T, Kato N, Cho MJ, et al. Structure of the 3′ ter-minus of the hepatitis C virus genome. J Virol 1996;70(5):3307–12.

13. Yi M, Lemon SM. 3′ nontranslated RNA signals requiredfor replication of hepatitis C virus RNA. J Virol 2003;77(6):3557–68.

14. Lohmann V, Korner F, Koch J, et al. Replication of subge-nomic hepatitis C virus RNAs in a hepatoma cell line. Science1999;285(5424):110–13.

15. Ikeda M, Yi M, Li K, Lemon SM. Selectable subgenomicand genome-length dicistronic RNAs derived from an infec-tious molecular clone of the HCV-N strain of hepatitis Cvirus replicate efficiently in cultured Huh7 cells. J Virol2002;76(6):2997–3006.

16. Pietschmann T, Lohmann V, Kaul A, et al. Persistent andtransient replication of full-length hepatitis C virus genomesin cell culture. J Virol 2002;76(8):4008–21.

17. Blight KJ, Kolykhalov AA, Rice CM. Efficient initia-tion of HCV RNA replication in cell culture. Science2000;290(5498):1972–4.

18. Lohmann V, Korner F, Dobierzewska A, Bartenschlager R.Mutations in hepatitis C virus RNAs conferring cell cultureadaptation. J Virol 2001;75(3):1437–49.

19. Bukh J, Pietschmann T, Lohmann V, et al. Mutations thatpermit efficient replication of hepatitis C virus RNA inHuh-7 cells prevent productive replication in chimpanzees.Proc Natl Acad Sci U S A 2002;99(22):14416–21.

20. Kato T, Date T, Miyamoto M, et al. Nonhepatic cell linesHeLa and 293 support efficient replication of the hep-atitis C virus genotype 2a subgenomic replicon. J Virol2005;79(1):592–6.

21. Zhu Q, Guo JT, Seeger C. Replication of hepatitis C virussubgenomes in nonhepatic epithelial and mouse hepatomacells. J Virol 2003;77(17):9204–10.

22. Blight KJ, McKeating JA, Rice CM. Highly permissive celllines for subgenomic and genomic hepatitis C virus RNAreplication. J Virol 2002;76(24):13001–14.

23. Foy E, Li K, Wang C, et al. Regulation of interferon regula-tory factor-3 by the hepatitis C virus serine protease. Science2003;300(5622):1145–8.

24. Lindenbach BD, Evans MJ, Syder AJ, et al. Completereplication of hepatitis C virus in cell culture. Science2005;309(5734):623–6.

25. Wakita T, Pietschmann T, Kato T, et al. Production of infec-tious hepatitis C virus in tissue culture from a cloned viralgenome. Nat Med 2005;11(7):791–6.

26. Nielsen SU, Bassendine MF, Burt AD, et al. Associationbetween hepatitis C virus and very-low-density lipoprotein(VLDL)/LDL analyzed in iodixanol density gradients. J Virol2006;80(5):2418–28.

27. Kolykhalov AA, Agapov EV, Blight KJ, et al. Transmissionof hepatitis C by intrahepatic inoculation with transcribedRNA. Science 1997;277(5325):570–74.

28. Yi M, Villanueva RA, Thomas DL, et al. Production of infec-tious genotype 1a hepatitis C virus (Hutchinson strain) incultured human hepatoma cells. Proc Natl Acad Sci U S A2006;103(7):2310–15.

29. Pietschmann T, Kaul A, Koutsoudakis G, et al. Construc-tion and characterization of infectious intragenotypic andintergenotypic hepatitis C virus chimeras. Proc Natl Acad SciU S A 2006;103(19):7408–13.

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