Zakim and Boyer's Hepatology || Replication and Pathogenesis of Hepatitis C Virus

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INTRODUCTIONHepatitis C virus (HCV) infection is a major cause of chronic hep-atitis, liver cirrhosis, and hepatocellular carcinoma (HCC) world-wide.1 A protective vaccine is not available and therapeutic optionsare limited. Current standard therapy, pegylated interferon-a (PEG-IFN-a) combined with ribavirin, results in a sustained virologicresponse in 20–80% of patients, depending on the HCV genotypeand other factors.2–5 However, in clinical practice many patients donot qualify for or do not tolerate IFN-based therapy.6 As a conse-quence, the number of patients presenting with long-term sequelaeof chronic hepatitis C, including HCC, is expected to furtherincrease over the next 20–30 years.7 Thus, there is an urgent needto develop more effective and better-tolerated therapies for chronichepatitis C. A detailed understanding of the molecular virology ofhepatitis C underpins these efforts.

HCV was identified in 1989 as the most common etiologic agentof post-transfusion and sporadic non-A, non-B hepatitis by the useof recombinant DNA technology.8 Investigation of the viral life cyclehas been limited by the low viral titers found in the sera and liversof infected individuals and the lack of efficient cell culture systemsor small animal models permissive for HCV. Nevertheless, consider-able progress has been made using heterologous expressionsystems,9,10 functional cDNA clones,11 the replicon system,12,13 func-tional HCV pseudoparticles,14,15 and most recently, recombinantinfections HCV produced in vitro181–183 (see 16–19 for recent reviews).These and other milestones in HCV research are listed in Table 8-1.

TAXONOMYHCV has been classified in the Hepacivirus genus within the familyFlaviviridae, which includes the classic flaviviruses, such as yellowfever (YFV) and dengue viruses, the animal pestiviruses, such asbovine viral diarrhea virus (BVDV), and the as yet unassigned GBviruses A (GBV-A), GBV-B and GBV-C20 (Figure 8-1). GBV-C wasalso designated hepatitis G virus (HGV). However, it was subse-quently found that GBV-C/HGV is not a common agent of viralhepatitis and its pathogenic relevance, if any, remains unknown.

An important feature of HCV is its high genetic variability.21 HCV isolates fall into three major categories, depending on the degree ofsequence divergence: genotypes, subtypes, and isolates. There are sixmajor genotypes (also called ‘clades’) that differ in their nucleotidesequence by 30–35%. Within an HCV genotype, several subtypes (designated a, b, c etc.) can be defined that differ in their nucleotidesequence by 20–25%. The term quasispecies refers to the genetic het-erogeneity of the population of HCV genomes coexisting in an infectedindividual. The genetic variability of HCV may have important implica-tions for the pathogenesis, natural course and prevention of hepatitis C.

The E1 and E2 glycoprotein regions are particularly variable,whereas the core and some of the non-structural protein sequencesare more conserved. The highest degree of sequence conservation isfound in the 5¢ and 3¢ non-coding regions (NCR).

In the United States and western Europe, genotypes 1a and 1bare the most frequent, followed by genotypes 2 and 3. In Europe,genotype 3 is distributed widely among injection drug users. In

Section I: Pathophysiology of the Liver

8REPLICATION ANDPATHOGENESIS OF HEPATITIS CVIRUSDarius Moradpour and Charles M. Rice

AbbreviationsARF alternative reading frame

ARFP alternative reading frame protein

BVDV bovine viral diarrhea virus

cDNA complementary DNA

CRE cis-acting replication element

CsCl cesium chloride

DC-SIGN dendritic cell-specific intercellular

adhesion molecule-3-grabbing integrin

ER endoplasmic reticulum

F frameshift protein

GBV GB virus

HCC hepatocellular carcinoma

HCV hepatitis C virus

HCVDB Hepatitis C Virus DataBase

HGV hepatitis G virus

hVAP-A human vesicle-associated membrane

protein-associated protein A

HVR hypervariable region

IRES internal ribosome entry site

ISDR interferon sensitivity-determining region

LDL low-density lipoprotein

LDLR low-density lipoprotein receptor

L-SIGN liver/lymph node-specific intercellular

adhesion molecule-3-grabbing integrin

NCR non-coding region

NK natural killer

NS non-structural

PEG-IFN-a pegylated interferon-aRdRp RNA-dependent RNA polymerase

SR-BI scavenger receptor class B type I

TBE tickborne encephalitis

VLDL very low-density lipoprotein

VSV vesicular stomatitis virus

YFV yellow fever virus

Section I. Pathophysiology of the Liver

southern and eastern Europe, genotype 1b is most frequent. InJapan, China, and Taiwan genotypes 1b and 2 are predominant.Genotype 4 is found primarily in Egypt, North and Central Africa,and the Middle East. It is typically associated with past medicaltreatment, e.g. parenteral treatment for schistosomiasis. Genotype5 is found commonly only in South Africa and genotype 6 is foundamong intravenous drug users in Hong Kong, Vietnam and, morerecently, Australia.

Patients infected with genotype 1 have a poorer response to IFN-a therapy than those infected with genotype 2 or 3. However, theclinical significance of HCV genotypes with respect to the naturalhistory of hepatitis C is controversial.

Close to 20 000 HCV sequences, including nearly 200 full-lengthgenomes, have so far been deposited in generic databanks such asGenBank, the EMBL Nucleotide Sequence Database or the DNAData Bank of Japan (DDBJ). A number of sequence databases are dedicated specifically to HCV, including the Hepatitis C VirusDataBase (HCVDB) of the French Réseau National Hépatites(http://hepatitis.ibcp.fr), the Los Alamos Hepatitis C Virus Databases (http://hcv.lanl.gov), and the Japanese Hepatitis VirusDatabase (http://s2as02.genes.nig.ac.jp). These offer a number ofspecialized features as well as useful links for HCV sequence analysis,structure predictions, CD4+ and CD8+ T-cell epitope compilations etc.

GENETIC ORGANIZATIONHCV contains a 9.6 kb positive-strand RNA genome composed ofa 5¢ NCR, a long open reading frame encoding a polyprotein pre-cursor of about 3000 amino acids, and a 3¢ NCR (Figure 8-2).

It took 8 years from the discovery of HCV to establish the firstinfectious cDNA clone,11 because in the absence of a robust tissueculture system the only read-out for infectivity was the direct inoculation of in vitro transcribed, synthetic RNA into the liver ofa chimpanzee. In addition, owing to the variation present in thequasispecies and errors introduced by PCR (polymerase chain reac-tion) amplification, construction of infectious cDNA clones requiredthe preparation of a consensus sequence from a number of clones.Functional cDNA clones now exist for genotypes 1a,11,22–24 1b,25 and2a.26 Genetic studies using infectious clones have shown the essen-tial nature of the HCV enzymes, the conserved elements of the 3¢NCR and the difficult-to-study proteins such as p7.27,28

THE 5¢ AND 3¢ NON-CODING REGIONSThe 5¢ NCR is highly conserved among different HCV isolates andcontains an internal ribosome entry site (IRES) essential for cap-independent translation of the viral RNA.29,30 Because the vastmajority of cellular mRNAs are translated by a cap-dependentmechanism, the HCV IRES represents an attractive antiviral target.

The 5¢ NCR contains four highly ordered domains, numberedI–IV. Domain I is not required for IRES activity, but is essential forHCV RNA replication.31 Domains II and III include two large stem-loops. Subdomain IIIf forms a pseudoknot with domain IV, whichcontains the translation initiation codon. Domains II, III and IV,together with the first 24–40 nucleotides of the core coding region,constitute the IRES. The key element is domain III, which permitsdirect binding of the 40 S ribosomal subunit to subdomains IIIa,IIIc, IIId and IIIe, as well as of eukaryotic translation initiation factor3 (eIF3) to subdomain IIIb. The three-dimensional structure of theHCV IRES bound to the 40 S ribosomal subunit was resolved at 20 Å resolution by cryoelectron microscopy.32 Strikingly, it wasfound that IRES binding induces a significant conformational changein the 40 S subunit, indicating that the HCV IRES dynamically mod-ulates the host translational machinery. In addition, high-resolutionstructural information is now available for critical elements of theIRES, including stem loops II, IIIb, IIId and IIIe, as well as the IIIabcfour-way junction, facilitating the design of small molecule inhibitorsof HCV translation initiation.33–36

A current model of HCV translation initiation includes the for-mation of a binary complex between the IRES and the 40 S ribo-somal subunit, followed by assembly of a 48 S-like complex at the

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CSFVBVDV

Pestivirus

Flavivirus

YFV

JEV DENV

HCV

Hepacivirus

GBV-B

GBV-C

GBV-A

3a

1b

1a2b

2a

Figure 8-1. Simplified phylogenetic tree of the Flaviviridae family. The Flaviviridae comprise thegenera Flavivirus, Pestivirus and Hepacivirus as wellas the as yet unassigned GB viruses A (GBV-A), GBV-B and GBV-C. Only few examples of flavi- and pestiviruses, as well as few HCV genotypes and subtypes, are shown. YFV, yellow fever virus; JEV,Japanese encephalitis virus; DENV, Dengue virus;BVDV, bovine viral diarrhea virus; CSFV, classicalswine fever virus.

Table 8-1. Milestones in HCV Research

1989 Identification of HCV1993 Polyprotein processing1996 Three-dimensional structure of the NS3 serine protease1997 Infectious clone of HCV1999 Replicon system2003 HCV pseudoparticles2003 Proof-of-concept clinical studies of an HCV protease inhibitor2005 Recombinant infectious HCV

Chapter 8

REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS

AUG initiation codon upon association of eIF3 and ternary complex(eIF2•Met-tRNAi

Met•GTP) and, in a rate limiting step, GTP-dependent association of the 60 S subunit to form the 80 Scomplex.37

The 3¢ NCR is composed of a short variable region, a poly(U/UC)tract with an average length of 80 nucleotides, and an almost invari-ant 98 nucleotide RNA element, designated the X-tail.27,38–43

CIS-ACTING REPLICATION ELEMENTSPromoter elements regulating the replication of positive-strand viralRNAs, called cis-acting replication elements (CRE), are often foundat or near the 5¢ and 3¢ termini of genome RNA (or its complement).Although only the 5¢-terminal 125 bases including domain I areabsolutely required, downstream sequences that include the entire5¢ NCR dramatically enhance the efficiency of HCV RNA replica-tion.31,44 The conserved elements in the 3¢ NCR, including a minimalpoly(U) tract of about 25 bases, are also essential for replicationboth in cell culture41–43 and in vivo.27,40

Besides the 5¢ and 3¢ NCRs, conserved RNA structures have beenpredicted within the HCV open reading frame.45,46 A new CRE wasrecently confirmed in the sequence encoding the C-terminal domainof non-structural protein 5B (NS5B).47 An essential stem-loop, designated 5B-SL3.2, was identified within a larger cruciform RNA

element designated 5B-SL3 (Figure 8-2). More recently, it wasshown that the upper loop of 5B-SL3.2 is engaged in a kissing inter-action with a stem-loop in the X-tail, suggesting that a pseudoknotstructure is formed at the 3¢ end of the HCV genome that is essential for RNA replication.48

POLYPROTEIN PROCESSINGIRES-mediated translation of the HCV open reading frame yields apolyprotein precursor that is co- and post-translationally processedby cellular and viral proteases into the mature structural and non-structural proteins (Figure 8-2). The structural proteins include thecore protein and the envelope glycoproteins E1 and E2. These arereleased from the polyprotein precursor by the endoplasmic reticu-lum (ER) signal peptidase. The structural proteins are separatedfrom the non-structural proteins by the p7 polypeptide. The non-structural proteins include the NS2-3 protease and the NS3 serineprotease, an RNA helicase/NTPase located in the C-terminal two-thirds of NS3, the NS4A polypeptide, the NS4B and NS5A pro-teins, and the NS5B RNA-dependent RNA polymerase (RdRp). TheNS2-3 protease cleaves at the NS2/NS3 site, whereas the NS3serine protease is responsible for processing of the downstream non-structural proteins (Figure 8-2). These are cleaved in a preferential

127

C E1 NS2 NS4 BE2 NS3 AA BNS5

IRES-mediated translation

C E1 E2 NS2 NS3 NS4BA NS5A NS5B

Core Envelopeglycoproteins

* * ** ** * *Protease Serine

proteaseHelicase Serine

proteasecofactor

RNA-dependentRNA polymerase

Membr.web

Polyprotein processing

9.6 kb

5B-SL3

5B-SL3.2

5´ NCRIII

a bc

de

f

II

I

IV

3´ NCR

p7

1 192 747384 810 1027 1658 1712 1973 2421 3011

?

(U)n

Figure 8-2. Genetic organization and polyprotein processing of HCV. The 9.6 kb positive-strand RNA genome is schematically depicted at the top. SimplifiedRNA secondary structures in the 5¢ and 3¢ non-coding regions (NCRs) as well as in the NS5B stem-loop 3 cis-acting replication element (5B-SL3) are shown. Inter-nal ribosome entry site (IRES)-mediated translation yields a polyprotein precursor of about 3000 amino acids that is processed into the mature structural andnon-structural proteins. Amino acid positions are shown above each protein (HCV H strain; genotype 1a; GenBank accession number AF009606). Solid diamondsdenote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. The open diamond indicates further C-terminal processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS2-3 and NS3 proteases. Asterisks in the E1 and E2 regionindicate glycosylation of the envelope proteins. Note that polyprotein processing, illustrated here as a separate step for simplicity, occurs both co- and post-translationally.


order, as shown in heterologous expression systems and in HuH-7cells harboring HCV replicons (see below).10,49 The first cleavageoccurs co-translationally and liberates NS3 from the remainder ofthe polyprotein. Subsequent processing events can be mediated intrans, with rapid processing at the NS5A/NS5B site. The resultingNS4A-5A precursor is cleaved first between NS4A and NS4B,resulting in a relatively stable NS4B-5A intermediate, and subse-quently between NS4B and NS5A.

STRUCTURAL PROTEINSCOREThe first structural protein encoded by the HCV open reading frameis the core protein, which presumably forms the viral nucleocapsid.During translation of the HCV polyprotein, the nascent polypep-tide is targeted to the ER membrane for translocation of the E1ectodomain into the ER lumen, a process mediated by an internalsignal sequence located between the core and E1 sequences. Cleav-age of the signal sequence by signal peptidase yields an immature191-amino-acid core protein, which contains the E1 signal peptideat its C terminus. This signal peptide is further processed by signalpeptide peptidase (SPP), yielding the mature 21 kDa core proteinof approximately 179 amino acids.50

The N-terminal hydrophilic domain of core contains a high pro-portion of basic amino acid residues and has been implicated bothin RNA binding and homo-oligomerization. When expressed inmammalian cells, core is found on membranes of the ER, in seem-ingly ER-derived membranous webs (see below), and on the surfaceof lipid droplets.51–54 It is at present unclear whether the associationwith lipid droplets, which is mediated by the central, relativelyhydrophobic domain of core and was detected in different heterol-ogous expression systems, in transgenic mice and in liver specimensfrom HCV-infected chimpanzees, plays a role during viral replica-tion or virion morphogenesis. It has been speculated that the inter-action of core with lipid droplets may affect lipid metabolism, whichin turn may contribute to the development of liver steatosis. Theobservation that certain HCV core-transgenic mice develop steato-sis and HCC has lent further support to this hypothesis.55,56 A smallproportion of the core protein may also be found in the nucleus.

Little is known about the assembly of core into nucleocapsids. Invitro studies with recombinant HCV core proteins demonstratedthat the N-terminal 124 amino acid residues are sufficient for theassembly of nucleocapsid-like structures, and that the presence ofstructured RNA is required for this process.57 However, under theseexperimental conditions RNA encapsidation is not specific, and thesignals and processes that mediate RNA packaging and nucleocap-sid assembly during HCV replication are unknown. More recently,assembly of nucleocapsid-like particles has been observed in cell-free translation systems.58 These capsids sediment at 100S and havea buoyant density of 1.28 g/ml on CsCl gradients.

Intriguingly, the core protein has been reported to interact witha variety of cellular proteins and to influence numerous host cellfunctions, including apoptosis, cell cycle control, gene expressionand many others.59,60 However, the relevance of these observations,derived mainly from heterologous overexpression experiments, forthe natural course and pathogenesis of hepatitis C is currentlyunknown.

ARFP/F PROTEINAn alternative reading frame (ARF) was recently identified in theHCV core region which, as a result of a -2/+1 ribosomal frameshift,has the potential to encode a protein of up to 160 amino acids, des-ignated ARFP (alternative reading frame protein) or F (frameshift)protein46,61,62 (Figure 8-3). Expression of the ARFP/F protein ofHCV genotype 1a in vitro or in mammalian cells yields a 17 kDaprotein. Amino acid sequencing indicated that the frameshift prob-ably occurs at or near codon 11 of the core protein sequence.61

However, multiple frameshifting events have recently been reportedin this region, and a 1.5 kDa protein could also be produced by -1/+2 frameshifting.63 In addition, the frameshift position seems tobe genotype dependent, as a +1 frameshift at codon 42 was recentlyreported for genotype 1b.64 Detection of antibodies65 and T cells66

specific for the ARFP/F protein in patients with hepatitis C suggeststhat this protein is expressed during HCV infection. However, giventhat the ARF is not present in subgenomic HCV replicons, theARFP/F protein is not required for HCV RNA replication in vitro.Recent studies incorporating multiple stop codons into the ARF haveshown that ARFP/F protein expression is not absolutely required forreplication in vivo.67 Rather, these results suggest that the ARF mayharbor additional conserved RNA elements that are required fortranslation and/or replication of full-length HCV genome RNAs.Thus, the functions, if any, of the ARFP/F protein in the life cycleand pathogenesis of HCV remain to be elucidated.68

ENVELOPE GLYCOPROTEINSThe envelope proteins E1 and E2 are extensively glycosylated andhave an apparent molecular weight of 30–35 and 70–72 kDa, respec-tively. They form a non-covalent complex, which is believed to rep-resent the building block for the viral envelope.69 E2 is believed tomake contact with the cellular receptor(s) for HCV, whereas E1 hasbeen predicted to possess fusion activity.

128

C

ARF

ARFP/F

E1

5´ NCR

Figure 8-3. The alternative reading frame (ARF) in the HCV core coding regionand the ARF protein (ARFP)/F protein. The 5¢ non-coding region (5¢ NCR) of theHCV genome contains extensive secondary structures forming an internal ribo-some entry site. The main open reading frame of the HCV genome with thecore protein (C) and the N-terminal portion of envelope glycoprotein 1 (E1) aredepicted in gray. The ARF is illustrated in red. The putative ARFP/F protein isshown at the bottom. The frameshift from the core reading frame into the ARFoccurs at or near codon 11 of the core coding sequence.

Chapter 8


E1 and E2 are type I transmembrane glycoproteins. Interestingly,the transmembrane domains, located at their C termini, are involvedin heterodimerization and have ER retention properties. Each ofthese transmembrane domains is composed of two stretches ofhydrophobic amino acids separated by a short polar segment. Thesecond hydrophobic stretch acts as an internal signal peptide for thedownstream protein. Before signal sequence cleavage, the E1 andE2 transmembrane domains have been proposed to adopt a hairpinstructure at the translocon. After cleavage, the signal sequence isreoriented towards the cytosol, resulting in a single transmembranepassage.70

The ectodomains of E1 and E2 contain numerous highly con-served cysteine residues that may form four and nine intramolecu-lar disulfide bonds, respectively. In addition, E1 and E2 contain upto five and 11 glycosylation sites, respectively. Thus, HCV glyco-protein maturation and folding is a complex process that involvesthe ER chaperone machinery and depends on disulfide bond for-mation as well as glycosylation.

A model for E2 based on the structure of the envelope proteinfrom tick-borne encephalitis virus (TBE; a member of the flavivirusgenus)71 was proposed.72 According to this model, E2 forms an elon-gated and flat head-to-tail homodimer. The fact that the envelopeprotein of Semliki Forest virus,73 a more distantly related alphavirus,has a similar structure to the envelope proteins of TBE71 and denguevirus,74 suggests that HCV may have a similar surface architecture.However, virtually nothing is known about the actual structure ofthe HCV E1–E2 complex, and the processes that mediate viralattachment, entry, and fusion have only recently become amenableto systematic study (see below).

As discussed above, the genes encoding the envelope glycopro-teins E1 and E2 are particularly variable. A hypervariable region(HVR) of approximately 28 amino acids in the N-terminal domainof E2 has been termed HVR1.75,76 The HVR1 amino acid sequencediffers by up to 80% among HCV isolates. Interestingly, despite highvariability at the sequence level, the structure of this domain wasfound to be quite conserved.77 HVR1 appears to contain a neutral-ization epitope78 and variability, therefore, may be driven by anti-body selection. Of note, the HVR1 could be deleted from infectiousHCV cDNA clones without abrogating infectivity, although themutant virus replicated poorly and compensating changes in E1 andE2 were selected upon passaging.79 These observations suggest afunctional role of this domain, probably in virus entry into the host cell.

A second hypervariable region, HVR2, has been described atamino acid positions 91–97 of genotype 1 E2 protein.

p7p7 is a 63-amino-acid polypeptide that is often incompletely cleavedfrom E2. It has two transmembrane domains connected by a shorthydrophilic segment which forms a cytoplasmic loop, and the N andC termini are oriented toward the ER lumen.80 Both transmembranepassages have been predicted to form a-helices, and the C-terminaltransmembrane segment has been shown to function as an internalsignal peptide.

p7 of the related pestivirus BVDV is essential for the productionof infectious progeny, but not for RNA replication.81 Similarly, HCVp7 is not required for HCV RNA replication because it is not present

in subgenomic replicons. However, it is essential for virus infectiv-ity in vivo, as shown by genetic studies using infectious HCV cDNAclones.28 p7 has recently been reported to form hexamers and topossess ion channel activity.82,83 These properties suggest that p7belongs to the viroporin family, could have an important role in viralparticle maturation and release, and may represent an attractivetarget for antiviral intervention.

VIRION STRUCTUREAlthough exciting progress has recently been made with respect torelated flavi-84–86 and alphaviral virion structures,73 HCV has not sofar been conclusively visualized and its structure remains unknown.By analogy to these related viruses, it can be assumed that the coreprotein and the envelope glycoproteins E1 and E2 are the principalstructural components of the virion. E1 and E2 are presumablyanchored to a host cell-derived double-layer lipid envelope that surrounds a nucleocapsid composed of multiple copies of the coreprotein and encapsidating the genomic RNA.

The basic biophysical properties of the HCV particle wererevealed early on by experiments performed in chimpanzees. Infec-tivity was abolished by treatment with lipid solvents, indicating thatthe viral particle is enveloped.87 A rough estimate of virion size wasobtained by filtration studies demonstrating that the particle is ableto pass through 50 nm pore filters.88 Based on subsequent electronmicroscopy studies, HCV particles are believed to have a diameterof 40–70 nm.89,90 HCV circulates in various forms in the infectedhost, including virions bound to low-density (LDL) and very low-density lipoproteins (VLDL),91 which appear to represent the infec-tious fraction, virions bound to immunoglobulins, and free virions.In addition, non-enveloped nucleocapsids harboring HCV RNA havebeen described.92

NON-STRUCTURAL PROTEINSNS2-3 PROTEASECleavage of the polyprotein precursor at the NS2/NS3 junction isaccomplished by a protease encoded by NS2 and the N-terminalone-third of NS3.93,94 NS2 is dispensable for replication of sub-genomic replicons in vitro (see below) and is thus not essential forthe formation of a functional replication complex. However, theNS2-3 protease activity is essential for the replication of full-lengthHCV genomes in vivo. Site-directed mutagenesis has shown thatamino acids His 143 (i.e. His 952 of the HCV polyprotein), Glu163 (i.e. Glu 972) and Cys 184 (i.e. Cys 993) are essential for catalytic activity.93,94 The importance of these amino acid residues is consistent with a thiol protease catalytic mechanism, but NS2-3activity is stimulated by zinc or certain other divalent metal ions.Interestingly, the cellular chaperone Hsp90 was found to be essen-tial for the activation of the NS2-3 protease.95

The membrane topology of NS2 has been controversial. It hasbeen proposed that NS2 is a transmembrane protein with at leastone and up to four transmembrane segments in the N-terminaldomain.96,97 There are also data which suggest that the C terminusof NS2, after autocatalysis in the cytosol, may relocate to the ERlumen.97 Recombinant proteins lacking the N-terminal membranedomain of NS2 have been found to retain cleavage activity,

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allowing further characterization of this unique enzymatic activ-ity.98,99 Indeed, recent efforts have led to the determination of ahigh-resolution structure for the NS2 protease domain, whichreveals a dimeric structure with a thiol protease-like active site.100

NS3-4A COMPLEXA distinct serine protease located in the N-terminal one-third ofNS3 is responsible for the downstream cleavage events in the non-structural region9,101,102 (reviewed in 103). In addition, an RNA helicase/NTPase domain is found in the C-terminal two-thirds ofNS3.104 The NS4A polypeptide functions as a cofactor for the NS3serine protease and is incorporated as an integral component intothe enzyme core. Complex formation occurs via a tight interactionof the 22 N-terminal residues of NS3 with 12 amino acid residuesin the center of NS4A. NS3 by itself has no membrane anchor. TheN-terminal domain of NS4A is strongly predicted to form a trans-membrane a-helix responsible for membrane anchorage of the NS3-4A complex.105

The crystal structures of the serine protease106–108 and RNA heli-case domains of NS3109,110 as well as the entire NS3 protein111

(Figure 8-4) have been elucidated. These enzymes are essential forviral replication and have emerged as prime targets for the design ofspecific inhibitors as antiviral agents112,113 (see below).

The catalytic triad of the NS3 serine protease is formed by His57 (i.e. His 1083 of the HCV polyprotein), Asp 81 (i.e. Asp 1107)

and Ser 139 (i.e. Ser 1165). Crucial determinants of substrate speci-ficity include an acidic amino acid residue at the P6 position, a P1cysteine (trans cleavage sites) or threonine (cis cleavage sitebetween NS3 and NS4A), and an amino acid residue with a smallside chain, i.e. alanine or serine, at the P1¢ position. A consensuscleavage sequence, therefore, would read D/E-X-X-X-X-C/T | S/A-X-X-X. The three-dimensional structure of full-length NS3 revealedthat a C-terminal b strand of the helicase domain lies within theactive site of the serine protease domain, where it is expected to belocated during the cis cleavage that separates NS3 from NS4A. Thisresults in autoinhibition that is released upon trans substratebinding.111

Helicases catalyze the unwinding of doubled-stranded RNA orDNA into single-stranded nucleic acids. The energy required for thisprocess is generated by hydrolysis of NTPs by an associated NTPaseactivity. Thus, the NS3 helicase couples unwinding of RNA regionswith extensive secondary structures with NTP hydrolysis. The NS3helicase is a member of the so-called helicase superfamily 2. Theseare also called DEXH/D helicases, according to a characteristic signature sequence in one of the essential enzyme motifs. It wasrecently shown that NS3 unwinds RNA through a highly coordi-nated cycle of fast ripping and local pausing that occurs with regularspacing along the duplex substrate, suggesting that nucleic acidmotors can function in a manner analogous to cytoskeletal motorproteins.114

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Figure 8-4. Three-dimensional structure of the HCVNS3-4A complex. The structure was determined usingan engineered single-chain molecule consisting of NS3and 14 NS4A residues known to activate the serine protease linked to the N terminus of NS3. The two b-barrels in the serine protease domain are shown inmagenta and red, the helicase subdomains are shownin green, light blue and dark blue, and the central NS4Adomain interacting with NS3 is shown in olive. Residuesof the serine protease catalytic triad (His 57, Asp 81 andSer 139) are shown in ball-and-stick representation,and the protease structural zinc ion is shown as a whitesphere. The red spheres represent a phosphate mole-cule located at the NTP-binding site of the helicase.Note the interaction between the C terminus and theprotease active site region. (Reproduced from111, with permission.)

Chapter 8


NS4BNS4B, a 27 kDa integral membrane protein, is the least character-ized HCV protein.115 It is predicted to be a polytopic membraneprotein with a cytoplasmic N-terminal region followed, dependingon the prediction, by four or six transmembrane segments and a C-terminal region in the cytosol.115,116 It has been shown experimen-tally that the bulk of the protein is cytosolically oriented.115

Introduction of glycosylation acceptor sites at various positions ofNS4B recently confirmed the presence of a predicted ER luminalloop around amino acid position 161.117 Surprisingly, the N termi-nus of NS4B was found to be translocated into the ER lumen atleast partially, presumably by a post-translational mechanism.117

The NS4B proteins of HCV, pesti- and flaviviruses are similar insize, amino acid composition, and hydrophobic properties. No func-tion, however, has yet been ascribed to NS4B in any of these relatedviruses. More recently, it was found by electron microscopy thatexpression of HCV NS4B induces the formation of a seemingly ER-derived specific cellular membrane alteration, designated the mem-branous web, that harbors the viral replication complex53,118 (seebelow). Thus, a function of NS4B may be to induce the specificmembrane alteration that serves as a scaffold for the HCV replica-tion complex.

NS5ANS5A is a phosphoprotein of unknown structure and function. It isfound in a basally phosphorylated form of 56 kDa and in a hyper-phosphorylated form of 58 kDa. NS5A of HCV and BVDV, as well as NS5 of YFV, are phosphorylated by as yet unidentifiedserine/threonine kinases, suggesting that these proteins share acommon function related to their phosphorylation state.119 Basalphosphorylation requires domains in the center and C terminus of

NS5A. The centrally located serine residues 225, 229 and 232 (i.e.Ser 2197, Ser 2201 and Ser 2204 of the HCV polyprotein) areimportant for NS5A hyperphosphorylation (Figure 8-5). However,it is unknown whether these serine residues are actually phospho-rylated or whether they affect phosphorylation indirectly. The onlyphosphoacceptor sites that have been mapped experimentally areserine residues 222 (i.e. Ser 2194 of the polyprotein)120 and 349(i.e. Ser 2321) (genotype 1a HCV H strain).121 The cellularkinase(s) responsible for NS5A phosphorylation appear to belong to the so-called CMCG group of serine/threonine kinases to whichcasein kinase II, cyclin-dependent kinases and mitogen-activatedprotein kinases belong. However, the cellular kinase(s) responsiblefor NS5A phosphorylation remains elusive.

Interestingly, adaptive mutations have been found to cluster in thecentral region of NS5A in the context of selectable subgenomicHCV replicons, suggesting that NS5A is involved – either directlyor by interaction with cellular proteins and pathways – in the viralreplication process. This observation, together with the modulationof NS5A hyperphosphorylation by nonstructural proteins 3, 4A and4B,122,123 strongly supports the notion of NS5A being an essentialcomponent of the HCV replication complex.

An N-terminal amphipathic a-helix mediates membrane associa-tion of NS5A.124–126 This helix exhibits a hydrophobic, tryptophan-rich side embedded in the cytosolic leaflet of the membrane bilayer,while the polar, charged side is exposed to the cytosol. Thus, NS5Ais a monotopic protein with an in-plane amphipathic a-helix asmembrane anchor. Structure–function analyses demonstrated thatthis helix displays fully conserved polar residues at the membranesurface, which define a unique platform probably involved in specific protein–protein interactions essential for the formation of afunctional HCV replication complex.126 Comparative sequenceanalyses and limited proteolysis of recombinant NS5A protein have

131

Domain I

Membraneanchor domain 237

237

PKR interaction domain

302V3

276

ISDR 384-407

354-362

C39 Adaptive changesPutative

NLSC80C57 C59

Domain II Domain III1 213 250 342 356 447

Δ 399-441Δ 235-282222

349(1a)

384 418

GFPinsertions

Figure 8-5. Overview of the HCV NS5A protein. NS5A is drawn to scale as a box. Amino acid positions relate to the HCV Con1 sequence (genotype 1b; GenBankaccession number AJ238799; add 1972 amino acids to obtain positions relative to the HCV polyprotein). The N-terminal amphipathic a-helix that mediates mem-brane association of NS5A, the region where cell culture-adaptive changes have been found to cluster in the replicon system, the so-called interferon sensitivitydetermining region (ISDR), the double-strand RNA-activated protein kinase (PKR) interaction domain, the putative nuclear localization signal (NLS), and variableregion 3 (V3) are highlighted. The domain organization recently proposed by Tellinghuisen et al.127 is also shown. Cysteine residues 39, 57, 59 and 80, denotedby blue lines, coordinate one zinc atom per NS5A protein. Deletions identified in the replicon system are shown in light green.13,178,163 GFP insertion sites toler-ated in the replicon system are highlighted by green lines.180 Mapped phosphoacceptor sites for genotype 1b (amino acid position 222)120 and the genotype 1aHCV H isolate (amino acid position 349)121 are highlighted in red. Dashed red lines denote serine residues that affect NS5A phosphorylation.


recently led to a proposed domain organization of NS5A (Figure 8-5).127 The relatively highly conserved domain I immediately follow-ing the membrane-anchoring a-helix has been shown to contain fourabsolutely conserved cysteine residues that coordinate one zinc atomper NS5A protein.127 Mutation of these residues abolishes HCVRNA replication, indicating that the zinc is essential for NS5A structure and/or function. Thus, NS5A is a zinc metalloprotein. Thestructure of the NS5A domain I has recently been solved, revealinga completely novel protein fold, a new zinc coordination motif, anda rare cytoplasmic disulfide bond. The structure also defines surfaceproperties that may be involved in NS5A dimer formation andNS5A interaction with viral and cellular proteins, membranes andRNA128 (Figure 8-6).

HCV NS5A has attracted considerable interest because of itspotential role in modulating the IFN response. Studies performedin Japan first described a correlation between mutations within adiscrete region of NS5A, termed interferon sensitivity-determiningregion (ISDR) (Figure 8-5), and a favorable response to IFN-atherapy.129 These studies demonstrated that strains closely match-ing the prototype HCV genotype 1b (HCV-J) ISDR sequence cor-related with IFN resistance. These findings were largely confirmedin Japan, but not in Europe and North America. The reasons for thisdiscrepancy are not understood, but may involve differences in bothdoses and regimens of IFN treatment and the low prevalence of‘mutant type’ HCV genotype 1b isolates in western countries. Evenif a meta-analysis of published data seems to confirm an associationof specific ISDR sequences with the IFN response,130 this remainsa controversial issue that has thus far not translated into clinically

applicable predictors. The same is true for other regions of NS5Athat have been associated with the response to IFN therapy, such asa variable region in the C-terminal domain of NS5A termed V3(Figure 8-5). Interestingly, however, an interaction with and repres-sion of the catalytic activity of PKR by NS5A has been found bybiochemical, transfection, and yeast functional analyses.131 Muta-tions within the ISDR that were observed in clinically IFN-sensitivegenotype 1b strains disrupted the ability of NS5A to interact withand repress PKR activity, supporting the notion that NS5A mediates HCV resistance to IFN through down-regulation ofPKR.132 However, these findings are controversial, and numerousadditional potential functions have recently been attributed toNS5A (reviewed in 60,133,134). However, similar to the core protein,only very few of these postulated interactions and functional prop-erties have been validated in a meaningful context involving activeHCV RNA replication or HCV infection in vivo.

NS5BHCV replication proceeds via synthesis of a complementary nega-tive-strand RNA using the genome as a template and the subsequentsynthesis of genomic positive-strand RNA from this template. Thekey enzyme responsible for both of these steps is the NS5B RdRp.This essential viral enzyme has been extensively characterized at thebiochemical135–138 and the structural level139–142 and has emerged asa major target for antiviral intervention. The HCV NS5B proteincontains motifs shared by all RdRps, including the hallmark GDDsequence within motif C, and, based on the similarity of the enzymestructure with the shape of a right hand, possesses the classic fingers,

132

Cytoplasm

Membraneanchor

Membraneanchor

ER lumen

III

II

III

II

Figure 8-6. Structure of HCV NS5A domain I. The dimeric form of NS5A domain I (amino acids 36–198) modeled in relation to the membrane at the site of RNAreplication.128 The cytoplasmic and luminal leaflets of the membrane are indicated. The two NS5A domain I monomers are colored in blue and green. Also shownin blue and green are the N-terminal amphipathic membrane anchors of NS5A lying flat in the plane of the membrane.126 Zinc atoms coordinated by domain Iare shown as red spheres. The hypothetical locations of domains II and III of NS5A are indicated by schematic spheres. This orientation of domain I places thelargely basic surface towards the phospholipid head groups of the membrane and positions the large ‘claw’ or groove of the NS5A dimer away from the mem-brane, where it may interact with RNA. This figure was reproduced from an illustration kindly provided by Dr Timothy L. Tellinghuisen, Rockefeller University, NewYork, USA.

Chapter 8


palm and thumb subdomains. A special feature of the HCV RdRpis that extensive interactions between the fingers and thumb sub-domains result in a completely encircled active site139–142 (Figure 8-7). This feature is shared by other RdRps, including those of thebacteriophage F6 and of BVDV.143,144

As with poliovirus RdRp,145–147 oligomerization of HCV NS5B hasrecently been reported to be important for cooperative RNA syn-thesis activity.148,149

Membrane association of the HCV RdRp is mediated by the C-terminal 21 aa residues, which are dispensable for polymerase activity in vitro. Membrane targeting occurs via a post-translationalmechanism and results in integral membrane association of NS5B.150

These features, namely post-translational membrane targeting via ahydrophobic C-terminal insertion sequence; integral membraneassociation; and cytosolic orientation of the functional proteindomain, define the HCV RdRp as a member of the so-called tail-anchored proteins. The HCV RdRp insertion sequence crosses themembrane bilayer as a transmembrane segment,151 is essential forHCV RNA polymerase in cells, and is likely to possess additionalfunctions apart from its membrane anchor function.152

MODEL SYSTEMSGiven the lack of a robust cell culture system allowing natural infection, replication, and release of viral progeny, various in vitroand in vivo models have been used to study HCV (Table 8-2)(reviewed in 153).

IN VITRO MODELSInfection of primary hepatocytes and established cell lines in vitroyielded only low-level replication and often poorly reproducible

results. In their present format some of these systems may be usefulfor neutralization assays, but not for a systematic investigation of theviral life cycle.154,155 An alternative approach involves the generationof cell lines constitutively or inducibly expressing viral sequencesfrom chromosomally integrated cDNA.156 Moreover, viablechimeras of certain positive-strand RNA viruses, such as polio andSindbis virus, with HCV genetic elements, such as the IRES157 orNS3,158 have been constructed and may facilitate the screening ofselected antiviral compounds.

The Replicon SystemThe replicon system has revolutionized the investigation of HCVRNA replication.12 The prototype subgenomic replicon was a

133

UTP

tP

N-ter

C-ter

Figure 8-7. Crystal structure of the catalyticdomain of the HCV RNA-dependent RNA poly-merase (140; PDB accession code 1GX6). Ribbondiagram of the NS5A-D55 protein complexed withUTP and Mn2+. a-Helices are colored blue, b-strandsred, and connecting loops silver. The boundnucleotide and the side chains of the catalyticaspartic acids (Asp 220 and Asp 318) in the centerof the structure are represented as ball-and-stickcolored black and cyan, respectively. Mn2+ ions areshown as green spheres. Also labeled is the triphos-phate (tP) moiety of a nucleotide bound to the‘priming’ site. This figure was reproduced from anillustration kindly provided by Dr Felix A. Rey, Laboratoire de Virologie Moléculaire et Structurale,UMR 2472 CNRS/UMR 1157 INRA, Gif-sur-Yvette,France.

Table 8-2. In Vitro and in Vivo Models to Study HCV

In vitro modelsIn vitro transcription–translationTransient cellular expression systemsStably transfected cell lines (constitutive/inducible expression)Infection of primary hepatocytes and established cell linesRetroviral pseudoparticles displaying functional HCV glycoproteinsReplicons (subgenomic/full-length; selectable/transient)Chimeric viruses (e.g. poliovirus—HCV)Related viruses (e.g. BVDV)

In vivo modelsTransgenic miceImmunodeficient mice/hepatocellular reconstitution modelsChimpanzee (Pan troglodytes)Tree shrew (Tupaia belangeri chinensis)?Related viruses (e.g. GBV-B in tamarins)


bicistronic RNA where the structural region of HCV was replacedby the neomycin phosphotransferase gene and translation of thenon-structural proteins 3–5B was driven by a second, heterologousIRES from encephalomyocarditis virus (Figure 8-8). Using thisapproach it became possible, for the first time, to study efficient andgenuine HCV RNA replication in HuH-7 human hepatoma cells invitro. Interestingly, certain amino acid substitutions, i.e. cell culture-adaptive changes, can increase the efficiency of replication initiationby more than 105-fold.13,159 Adaptive changes cluster in certainregions, such as the center of NS5A (Figure 8-4), the C-terminalpart of the NS3 serine protease and the N-terminal part of the NS3RNA helicase domains, as well as two positions in NS4B.160 In NS5Athese changes often affect serine residues required for hyperphos-phorylation, suggesting that hyperphosphorylation of NS5A reducesHCV RNA replication.161–163 According to one model, hyperphos-phorylation of NS5A reduces interaction with the human vesicle-associated membrane protein-associated protein A (hVAP-A).161

However, adaptive changes are likely to increase HCV RNA repli-cation by additional, as yet unidentified mechanisms. In this context,it is interesting to note that there is an inverse correlation betweenmutations that permit efficient replication of HCV RNA in HuH-7cells in vitro and productive replication in chimpanzees in vivo afterintrahepatic inoculation.164

The replicon system has allowed genetic dissection of HCV RNAelements and proteins, provided material for biochemical and ultra-structural characterization of the viral replication complex, and facil-itated drug discovery efforts (Table 8-3). Moreover, the repliconsystem has been exploited for analysis of the effect of cytokines on

HCV RNA replication165,166 and the study of other aspects of theinteraction between HCV and the host cell.167–169a

Since the original reports of functional genotype 1b replicons,replicons for genotype 1a170 and 2a171, as well as derivatives express-ing easily quantifiable marker enzymes in a separate cistron, havebeen made to facilitate genetic studies as well as drug screening and evaluation.172–174 In addition, full-length replicons and HCVgenomes efficiently replicating in tissue culture have been devel-oped,175–177 and the spectrum of permissive host cells has beenexpanded.178,179 Finally, replicons have been established that allowtracking of functional HCV replication complexes in living cells.180

One puzzling (and disappointing) observation was that full-lengthgenome RNAs with adaptive mutations were incapable of produc-ing infectious virus. This led investigators to favor the idea that

134

3¢ NCR

(U)n

3¢ NCR

(U)n

5¢ NCR

In vitro transcription

Transfection

HuH-7 cells

Selection

Clones carryingHCV replicons

BNeoR NS3 NS4 NS5A A B

EMCVIRES

5¢ NCR

BNeoR NS3 NS4 NS5A A B

EMCVIRES

Figure 8-8. Prototype subgenomic HCV replicon.12

RNA is transcribed in vitro from a plasmid contain-ing the HCV IRES followed by a neomycin resistancecassette, a second heterologous IRES fromencephalomyocarditis virus (EMCV IRES), the HCVnon-structural region (NS3 to NS5B), and the HCV3’ NCR. RNA is subsequently transfected into HuH-7 human hepatoma cells, followed by selectionwith G418 of clones harboring autonomously repli-cating subgenomic HCV RNA.

Table 8-3. Lessons from the Replicon System

Adaptive changesCell-cycle dependence of HCV RNA replicationInverse correlation between replicon activity in vitro and HCV

replication/particle formation in vivoEvaluation of new antiviral agents and antiviral resistanceEffect of cytokines on HCV RNA replicationIdentification and characterization of the HCV replication complexHost factorsIdentification of essential RNA elementsRequirements for infectious HCV particle formation

Chapter 8


HuH-7 and other HCV permissive cell lines lacked some factor(s)necessary for particle formation and release. However, this does not appear to be the case, given recent results with a genotype 2aisolate from Japan, JFH-1. JFH-1 was isolated from a patient withacute fulminant hepatitis C, and JFH-1 subgenomic replicons canreplicate efficiently in HuH-7 cells without adaptive mutations.171

Remarkably, full-length JFH-1 produces infectious virus.181,182

Chimeras consisting of the 5¢ and 3¢ NTRs and replicase region ofJFH-1 (NS3-5B) and the C-NS2 region of other isolates will alsosometimes produce cell culture infectious particles.183 One possi-bility, which seems to be growing in popularity, is that cell culture-adaptive mutations that promote efficient RNA replication may bedeleterious in vivo because they compromise particle assembly andrelease. With these advances, the late (assembly and egress) andearly (entry) events in HCV infection can now be studied. Thesesteps can also be explored as possible new therapeutic targets.

IN VIVO MODELSThe restricted host range of HCV has hampered the developmentof a suitable small animal model of viral replication and patho-genesis. Apart from a single report on the transmission of HCV totree shrews (Tupaia belangeri chinensis),184 the chimpanzee (Pantroglodytes) is the only animal known to be susceptible to HCVinfection.185 Indeed, the chimpanzee was essential in the early char-acterization of the agent of non-A, non-B hepatitis, and has allowedthe determination of important aspects of HCV replication, patho-genesis and prevention. In this context, it would not have been pos-sible to demonstrate the functionality of infectious clones of HCVwithout chimpanzees.11 In addition, recent studies in chimpanzeeshave provided new insight into the host immune response againsthepatitis C,186–188 and the chimpanzee remains the only faithfulmodel to test the immunogenicity and efficacy of vaccine candi-dates.189–191 However, ethical and financial restrictions limit the useof primates to highly selected experimental questions.

Expression of HCV proteins in transgenic mice provided someinsights into the pathogenesis of HCV-induced liver disease.55,56

However, expression of HCV proteins from chromosomally inte-grated cDNA does not appropriately reflect the viral life cycle, andstudies on viral entry and replication are hardly conceivable in thissystem.

GBV-B, the closest relative of HCV within the family Flaviviri-dae can be transmitted to tamarins (Saguinus sp.) and may repre-sent a valuable surrogate model for HCV. Remarkably, GBV-B canbe cultured in tamarin hepatocytes in vitro.192 In addition, infectiouscDNA clones193 and replicons194 have been established for GBV-B.However, in tamarins GBV-B typically leads to self-limited infec-tion without viral persistence unless animals are immunosuppressedor molecular clones are used.195 GBV-B can also be propagated incommon marmosets (Callithrix jacchus), which are easier to breedin captivity, are smaller, and are already regularly used for drugmetabolism, pharmacokinetic and toxicology studies.196

Progress in the development of a small animal model of HCVreplication was achieved with the successful HCV infection ofimmunodeficient mice reconstituted with human hepatocytes.197

The properties of two different mouse strains, the Alb-uPA-trans-genic and the immunodeficient SCID mouse, were combined todevelop a model system that allows orthotopic engraftment of

human hepatocytes (Figure 8-9). Expression of the murine uro-kinase-type plasminogen activator under the transcriptional controlof the albumin promoter (Alb-uPA) programs murine hepatocytedeath, providing a suitable microenvironment for the engraftmentand expansion of transplanted human hepatocytes. In homozygousanimals, reconstitution with human hepatocytes was reported toreach >50% of the liver cell mass. In consequence, Alb-uPAhomozygous animals were characterized by persistent high humanalbumin production. Inoculation with serum from patients with hepatitis C resulted in persistent HCV viremia in about 75% of micewith high-level human hepatocyte engraftment. HCV RNA couldbe detected by PCR for up to 35 weeks, with titers ranging from 3 ¥ 104 to 3 ¥ 106 copies/ml. These viral titers are similar to thosefound in infected humans. Moreover, an approximately 3-log rise inviral titers after inoculation, detection of viral negative-strand RNAin the liver, and the ability to serially passage the virus throughseveral generations of animals provided convincing evidence foractive replication and production of infectious viral progeny in thissystem. However, the handling of these fragile animals affected bymajor bleeding disorders and severe immunodeficiency (approxi-mately 35% mortality in newborns) presents a non-trivial challengeand requires special expertise. Moreover, access to fresh humanhepatocytes is limited for many investigators.

REPLICATION CYCLEThe life cycle of HCV includes 1) binding to an as yet unidentifiedcell surface receptor and internalization into the host cell; 2) cyto-plasmic release and uncoating of the viral RNA genome; 3) IRES-mediated translation and polyprotein processing by cellular and viralproteases; 4) RNA replication; 5) packaging and assembly; and 6)virion maturation and release from the host cell (Figure 8-10).

RECEPTOR CANDIDATESCD81, a tetraspanin molecule found on the surface of many celltypes, including hepatocytes,198 the low-density lipoprotein recep-tor (LDLR)199 and scavenger receptor class B type I (SR-BI)200 have,among others, been proposed as HCV receptors or components ofa receptor complex.

Both CD81 and SR-BI bind E2 and are currently viewed as nec-essary, but not sufficient, for HCV entry.14,15,201–203 Expression ofCD81 in CD81-negative liver-derived cell lines confers susceptibil-ity to HCV pseudoparticles (see below), and blocking antibodiesagainst CD81 or SR-BI, recombinant CD81, or siRNA-mediateddown-regulation of CD81 expression reduces infectivity. However,additional, as yet unidentified hepatocyte-specific factors arerequired for HCV entry.

The LDLR has been attractive as a candidate receptor becauseinfectious HCV has been reported to be associated with LDL orVLDL (see above). At present, however, it is unclear whether inter-action of HCV with the LDLR can lead to productive infection. Celllines without LDLR are still infectable with HCV pseudoparticles.

HCV E2 also binds to DC-SIGN (dendritic cell-specific inter-cellular adhesion molecule-3-grabbing non-integrin) and L-SIGN(liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin). The latter is a calcium-dependent lectin expressed

135


136

Human hepatocytes

SCID Alb-uPA

Immunodeficient Alb-uPA transgenic mouse

Newborns with liver cell destruction

Partial repopulation withhuman hepatocytes

Mouse with ‘chimeric’ liver

Figure 8-9. A small animal model of HCV replication.197

1

2

35

5

(+) RNA

(-) RNA

MW

ER

E1

C

E2

p72

4A

3

4B

5A

5B

3

3

6

5

4

5

(+) RNA

3

Figure 8-10. Life cycle of HCV. 1, Virus binding and inter-nalization; 2, cytoplasmic release and uncoating; 3, IRES-mediated translation and polyprotein processing; 4, RNAreplication; 5, packaging and assembly; 6, virion matura-tion and release. The topology of HCV structural and non-structural proteins at the endoplasmic reticulum (ER)membrane is shown schematically. HCV RNA replicationoccurs in a specific membrane alteration, the membra-nous web (MW). Note that IRES-mediated translation andpolyprotein processing, as well as membranous web for-mation and RNA replication, illustrated here as separatesteps for simplicity, may occur in a tightly coupledfashion.

Chapter 8


on liver sinusoidal endothelial cells that may facilitate the infectionprocess by trapping the virus for subsequent interaction with thereceptor.204–207

Identification and validation of HCV receptor candidates has beenlimited by the paucity of systems for analysis of the early steps ofthe viral life cycle. Given the lack of native HCV particles and effi-cient cell culture systems, various alternatives have been exploredto study the early steps of HCV infection. Soluble C-terminal trun-cated versions of HCV envelope glycoprotein E2,198,200,205,208,209 lipo-somes reconstituted with HCV E1 and E2,210 and virus-like particlesexpressed in insect cells211,212 have been used to study HCV glyco-protein interactions with the cell surface. The production of virus-like particles has also been described in mammalian cells.213

However, it is unclear how virus-like particles produced in inset ormammalian cells will compare to authentic HCV virions. Pseudo-typed vesicular stomatitis virus (VSV) or influenza virus particleshave been reported incorporating chimeric E1 and/or E2 glycopro-teins whose C-terminal transmembrane domains were modified toallow transport to the cell surface.208,214–216 However, such modifi-cations may interfere with the multiple and complex roles of the E1and E2 transmembrane domains69 and may perturb the conforma-tion and functions of E1–E2 complexes. Therefore, the use of suchpseudotypes as a tool to study HCV assembly and entry remainscontroversial.216

Against this background, the recent establishment of infectiousretroviral pseudotypes displaying functional HCV glycoproteins asa robust model system for the study of viral entry represents a majorbreakthrough14,15 (Figure 8-11). HCV pseudoparticle infectivity isrestricted primarily to human hepatocytes and hepatocyte-derivedcell lines, and entry is pH dependent. Thus, HCV entry likelyinvolves transit through an endosomal low-pH compartment andfusion with the endosomal membrane.

The structural basis for low pH-induced membrane fusion hasrecently been elucidated for the dengue, TBE and Semliki Forestviruses.217–219 The envelope proteins of these related flavi- andalphaviruses possess an internal fusion peptide that is exposedduring low pH-mediated domain rearrangement and trimerizationof the protein. The scaffolds of these so-called class II fusion pro-teins are remarkably similar, suggesting that all members of the Flaviviridae, including HCV, could behave similarly.

REPLICATION COMPLEXThe formation of a membrane-associated replication complex, com-posed of viral proteins, replicating RNA, and altered cellular mem-branes, is a hallmark of all positive-strand RNA viruses investigatedthus far (see 220,221 for reviews). Depending on the virus, replicationmay occur on altered membranes derived from the ER,222–226 Golgiapparatus,227–229 mitochondria230 or even lysosomes.231 The role ofmembranes in viral RNA synthesis is not well understood. It mayinclude (i) the physical support and organization of the RNA repli-cation complex;147 (ii) the compartmentalization and local concen-tration of viral products;232 (iii) tethering of the viral RNA duringunwinding;220 (iv) provision of lipid constituents important for repli-cation;233,234 and (v) protection of the viral RNA from double-strandRNA-mediated host defenses or RNA interference.

In the case of HCV, protein–protein interactions among HCVnon-structural proteins have been described235,236 and determinants

for membrane association of the HCV proteins have been mapped.The membrane association of HCV proteins is schematically illustrated in Figure 8-12. For a more comprehensive review on theinteractions of HCV proteins, including the structural proteins, withhost cell membranes, see references.237,238

A specific membrane alteration, designated the membranous web,was recently identified as the site of RNA replication in HuH-7 cellsharboring subgenomic HCV replicons118 (Figure 8-13). Formation ofthe membranous web could be induced by NS4B alone (see above),and it was very similar to the ‘sponge-like inclusions’ previouslyfound by electron microscopy in the liver of HCV-infected chim-panzees. The membranous web was often found closely associatedwith the rough ER. Based on this observation, together with earlierstudies demonstrating the co-localization of individually expressedHCV proteins with membranes of the ER,105,115,124,150 and data

137

293T cells

Plasmid 2

CMV MLV Gag-Pol

Plasmid 1

CMV E1 E2

Plasmid 3

CMV GFPψ

HCV pseudoparticle

CMV GFP

E2E1

ψ

Figure 8-11. Generation of infectious HCV pseudoparticles. Cotransfection of293T human embryo kidney cells with plasmids allowing expression of (1)unmodified HCV E1-E2 glycoproteins, (2) retroviral core proteins, and (3) apackaging-competent green fluorescent protein (GFP) expression constructleads to secretion into the supernatant of pseudoparticles bearing HCV enve-lope glycoproteins instead of the retroviral envelope protein on their surface.CMV, cytomegalovirus promoter; y, retroviral packaging sequence.


indicating that HCV RNA replication takes place in a compartmentthat sustains endoglycosidase H-sensitive glycosylation,151 it is cur-rently believed that the membranous web is derived from mem-branes of the ER. Ongoing studies are aimed at isolating and furthercharacterizing this complex and at defining the viral and cellularprocesses involved in the formation of the membranous web.

Recent studies demonstrate a complex interaction between HCVRNA replication and the cellular lipid metabolism, presumably viathe trafficking and association of viral and host proteins with intra-cellular membranes. In this context, it was found, for example, thatgeranylgeranylation of one or more host proteins is required forHCV RNA replication.239,240 Such observations suggest that phar-macologic manipulation of lipid metabolism may have therapeuticpotential in hepatitis C.

EVOLVING THERAPEUTICSTRATEGIES

In principle, each of the steps of the HCV life cycle illustrated in Figure 8-12 represents a target for antiviral intervention.241–243

Specific inhibitors of the biochemically and structurally well-characterized NS3 serine protease, as well as the RNA helicase/NTPase and the NS5B RdRp, are currently being developed asantiviral agents, and the first candidates are already in early-phaseclinical trials.112,113 Already at this early stage it becomes evident thatthe genetic variability of HCV represents a major challenge to theclinical development of specific enzyme inhibitors and that, similar

138

A

ER

N

M

BM

N

ER

Figure 8-13. HCV replication complex.118 (A) Low-power overview of a HuH-7 cell harboring a subgenomic HCV replicon. A distinct membrane alteration, namedmembranous web (arrows), is found in the juxtanuclear region. Note the circumscript nature of this specific membrane alteration and the otherwise unalteredcellular organelles. Bar, 1 mm. (B) Higher magnification of a membranous web (arrows) composed of small vesicles embedded in a membrane matrix. Note theclose association of the membranous web with the rough endoplasmic reticulum. Bar, 500 nm. The membranous web harbors all HCV non-structural proteinsand nascent viral RNA in HuH-7 cells harboring subgenomic replicons, and therefore represents the HCV RNA replication complex. N, nucleus; ER, endoplasmicreticulum; M, mitochondria.

Cytosol

ER lumen

p7

NS4A

CC C

CCNS2

NS3NS5A

NS5B

N N N CC

NS4B

N N

E2

E1

Figure 8-12. Membrane association of HCVproteins. Note that the topologies of NS2,NS4A and NS4B are currently under investiga-tion and are illustrated only schematically. Arecent study indicated that the C terminus ofNS2 may be localized in the ER lumen, result-ing in four transmembrane domains.97 Also, itwas recently reported that the N terminus ofNS4B can be at least partially translocated intothe ER lumen.117

Chapter 8


to HIV infection, combination therapy will be necessary for thera-peutic success.244,245

In addition to these more classic pharmacological approaches,gene therapeutic strategies aimed at inhibiting HCV replication andgene expression are currently being explored in various experimen-tal systems. These include, among others, antisense oligodeoxynu-cleotides, ribozymes, and small interfering RNAs. Moreover, basedon the concept that a quantitatively and qualitatively insufficientCD4+ and CD8+ T-cell response may contribute to viral persistence,immunotherapeutic strategies aimed at enhancing the cellularimmune response against HCV are currently being investigated.

Apart from more efficient therapeutic strategies, the developmentand implementation of preventive measures is of paramount impor-tance. The development of an effective recombinant vaccine hasbeen hampered by the high genetic variability of HCV and the lackof a suitable cell culture infection system and small animalmodel.189–191 It has been shown, however, that vaccination withrecombinant envelope proteins expressed in mammalian cells canprotect chimpanzees from primary infection with a homologousvirus isolate.246 The correlates of and requirements for a broader pro-tection and a potentially neutralizing immune response still need tobe defined, however. The potential of DNA vaccination to induce ahumoral and cellular immune response is particularly interesting inthis regard. Alternative currently pursued strategies include peptideand protein vaccines, dendritic cell-based vaccines, and virus-likeparticles. Although sterilizing immunity will probably be difficult toachieve, the aim of inducing a state of immunity that prevents thedevelopment of chronic infection appears more realistic. Alongthese lines, and in contrast to earlier more pessimistic views citinglack of protective immunity,247–249 more recent observations indicatethat chimpanzees that clear infection do exhibit protective – albeitnot sterilizing – immunity upon rechallenge,250,251 i.e. they show anattenuated course with rapid control of the rechallenge inoculum.In addition, studies in intravenous drug users have shown that thereis some protective immunity in hepatitis C.252 This study showedthat the risk of developing HCV viremia was lower for intravenousdrug users who had successfully cleared a previous HCV infectionthan for those who had no evidence of previous HCV infection. Inany case, it is likely that induction of both a humoral and a cellularimmune response will be required for an effective HCV vaccine.Such a vaccine might also be useful therapeutically.253

PATHOGENESISHCV infection is a highly dynamic process with a viral half-life ofonly a few hours and an average daily virion production and clearance of up to more than 1012.254 This high replicative activity,together with the lack of a proofreading function of the viral RdRp,provides the basis for the genetic variability of HCV. In addition,these findings are similar to the dynamics of HIV infection andprovide, as discussed above, a rationale for the development andimplementation of combination antiviral therapies.

The mechanisms responsible for liver injury in acute and chronichepatitis C are poorly understood.255–257 In acute HCV infection,liver cell damage coincides with the development of the hostimmune response and not with infection and viral replication. In

addition, persistent viral replication often occurs without evidenceof liver cell damage, suggesting that HCV is not directly cytopathic.The immune response against HCV therefore plays a central role inthe HCV pathogenesis of hepatitis C.

HCV-specific major histocompatibility complex (MHC) class II-restricted CD4+ helper T-cell258,259 and MHC class I-restricted CD8+

cytotoxic T-lymphocyte (CTL) responses260,261 have been identifiedin patients with acute and chronic HCV infection. CTL-mediatedlysis of virus-infected host cells may lead to clearance of the virusor, if incomplete, to viral persistence and eventually chronic hepa-titis. Based on these observations and parallels in other viral diseases,viral persistence and immunologically mediated liver cell injury areimportant mechanisms leading to chronic hepatitis C.255

Patients who clear HCV infection have a more vigorous CD4+258,259 and CD8+ T-cell response early on.261 The role of specific CD4+ and CD8+ T-cell responses in control of HCV infection was elegantly illustrated by in vivo depletion studies inchimpanzees.187,188

Despite the presence of an immune response, however, HCV israrely eliminated. Thus, HCV may overwhelm, not induce, or evadeantiviral immune responses. Perhaps the simplest explanation isquantitative, based on the kinetics of infection relative to the induc-tion of a CTL response during the early phase of infection. Accord-ing to this model, viral persistence would be predicted if thereplication rate of the virus exceeded the kinetics of the immuneresponse. Indeed, HCV reaches high serum titers within 1 week ofinfection, whereas adaptive cellular immune responses are delayedby at least 1 and humoral immune responses by at least 2months.262–264 The rate of increase of the viral titer slows only severalweeks after infection, and HCV RNA titers decline after approxi-mately 8–12 weeks, when the serum ALT levels peak.264 Studies performed in chimpanzees showed that the appearance of adaptivecellular immune responses and the induction of type II interferon,i.e. interferon-g, coincides with the decrease in HCV RNAtiters.186,264–265 Interferon-g may have a direct antiviral effect, as itefficiently inhibits the replication of HCV replicons.166 However, theeffector functions of HCV-specific T cells appear to be reduced.266

Most patients develop chronic infection with relatively stable viraltiters, about 2–3 logs lower than in the acute phase. Only a smallproportion of patients recover and test negative for HCV RNA usingstandard assays. Whether HCV is completely cleared after recov-ery, or whether trace amounts of virus persist, similar to hepatitis Bvirus, is debated.267 HCV-specific antibodies may disappear com-pletely 10–20 years after recovery.268

Microarray analyses of serial liver biopsy samples in experimen-tally infected chimpanzees revealed that HCV induces the intra-hepatic expression of many genes, including type I interferon, i.e.interferon-a and -b, responses.265 However, even if HCV RNA repli-cation in vitro is efficiently inhibited by type I interferons,166 HCVseems to be resistant to these responses and frequently succeeds inestablishing chronic hepatitis. As discussed above, HCV may haveevolved numerous mechanisms to counteract the innate immuneresponse, including interference with the interferon system at the induction,167–169a signaling269–271 and effector levels.131,132,272 Inaddition, HCV may interfere with natural killer (NK) cell func-tions.273,274 In this context, a recent large immunogenetic studyrevealed an association between a NK cell receptor (KIR2DL3

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allele)-HLA compound genotype and HCV clearance and clinicalrecovery, pointing toward a role of NK cells in early HCV infection.275

However, to be consistent with the repeated observation that the CTL response is less vigorous in chronically infected patientsthan it is during acute, self-limited infection, additional mechanismsmust be involved. These may include the induction of peripheraltolerance or exhaustion of the T-cell response, infection of immuno-logically privileged sites, inhibition of antigen presentation, down-regulation of viral gene expression, and viral mutations that abrogate,anergize or antagonize antigen recognition by virus-specific T cells.255

An impairment of dendritic cell function has been proposed,276 butthis is controversial.277 There is some evidence that privileged sitesmay play a role, as HCV may infect extrahepatic cells and tissues.As mentioned above, the role of viral escape mutations and the quasispecies nature of HCV as a cause of viral persistence hasattracted considerable interest. In this context, HCV escape to anti-bodies278,279 and T cells280–283 has been demonstrated both in humansand chimpanzees.

The role of the humoral immune response in the natural courseand pathogenesis of hepatitis C is not well understood. Recentstudies using HCV pseudoparticles (see above) validated earlierstudies demonstrating neutralizing of antibodies.284,285 However, thehighest antibody titers are found in patients with chronic hepatitisC, and the role of antibodies capable of neutralizing a minor frac-tion of the HCV population is unknown.

CONCLUSIONS AND PERSPECTIVESThe development of powerful model systems has allowed us to systematically dissect important steps of the HCV life cycle. Theseefforts have translated into the identification of novel antiviraltargets and the development of new therapeutic strategies, some ofwhich are already in early-phase clinical evaluation. Much workremains to be done with respect to virion structure, the early andlate steps of the HCV life cycle, the mechanism and regulation ofRNA replication, and the pathogenesis of HCV-induced liverdisease. Ultimately, a detailed understanding of the viral life cycleshould result in innovative therapeutic and preventive strategies forone of the most common causes of chronic hepatitis, liver cirrhosisand HCC worldwide.

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