hepatitis c virus: molecular biology & current therapeutic options

Introduction

HepatitisCisacomplexliverdisease.Itsmedicalimportance and the need to rapidly identify newtherapeuticapproacheshasresultedinintensivestudyofitscausativeagent,hepatitisCvirus(HCV).Humansare theonlyknownnaturalhostsofHCV.Evenaftertwodecadessinceitsdiscovery,HCVcontinuestobe

IndianJMedRes131,January2010,pp17-34

17

Review Article

HepatitisCvirus:Molecularbiology&currenttherapeuticoptions

SureshD.Sharma

Department of Biochemistry & Molecular Biology, Pennsylvania State University, Pennsylvania, USA

ReceivedJuly23,2009

Hepatitis C virus (HCV) is a small (~55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent development of a robust cell culture system for HCV propagation. HCV proteins assemble and form replication complexes on modified host membranes, called as membranous webs. Even though HCV is detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments; and the viral population is known to rapidly change during the course of a natural infection thereby escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission. The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins during HCV lifecycle is vital for developing a successful cure. This review deals with current status of efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and rapidly changing hepatitis C virus.

Key wordsHCV-hepatocellularcarcinoma(HCC)-novelHCVtherapeutics-pegylated-Interferon-ribavirin-structuralandnonstructuralproteins

amajorcauseofconcernandahugeburdenonpublichealth systems worldwide. The WHO estimates thataminimumof3percentoftheworld’spopulationischronicallyinfectedwithHCV1,2.

HCV is a prototype member of the Hepacivirusgenus (from the Greek hepar, hepatos, liver) and isfurther classified into at least seven major genotypes

that differ by about 30 per cent in their nucleotidesequence.Thesegenotypes(1,2,3,4,5,6&7)showdifferenceswithregardtotheirworldwidedistribution,transmission and disease progression3, 4. Thesegenotypes have been further classified into sub-types (a, b, c, d, etc). In fact, HCV circulates in infectedindividuals as a population of diverse but closelyrelatedvariantsreferredtoas“quasispecies”.

HCV is most commonly spread by directcontact with infected blood and blood products.Availability of injectable therapies and drugs hashad a remarkable influence on HCV epidemiology. The incubation period of HCV, though ranging upto several months, averages 6-8 wk. HCV infectionis often asymptomatic, making it a very difficult to detect it at an early stage.This is the major reasonwhy early treatment is difficult. Therefore, hepatitis C isoftenreferredtoasa“silentdisease”.Inamajorityof infected people, virus infection does not resolvenaturally. Neutralizing antibodies appear to beproducedduringthecourseofanaturalinfection,yetthevirusmutatestoescapesurveillance5.Whenliverfailstoclearthevirus,theindividualsbecomechroniccarriers. However, within this chronically infectedpopulationthediseaseoutcomesvary,itcanbemild(minimal inflammation of the liver) or severe and can leadtoscartissueformation(Fig.1).Chronicinfectioneventuallycausescirrhosis leading tohepatocellularcarcinoma (HCC) and ultimately death. Currently,thereisnovaccinetopreventhepatitisC.

Liversteatosisoccursinmorethan50percentofthepatientswithchronichepatitisC(CHC)infection.Some individuals with CHC may report non specific symptoms such as fatigue, muscle aches,nausea andpain in the right upper quadrant.Antibodies directedagainst several HCV proteins can be detected inchronic patients. A variety of autoimmune andimmunecomplex-mediated diseases have also beenassociated with chronic HCV infection, autoimmunethyroiditisbeingoneofthem6.

Molecular Biology of HCV

Formanyyearsthefailuretoreplicateandproduceauthentic infectious hepatitis C virus in cell cultureremained a major obstacle for innovative and cost-effective therapy. A breakthrough in the field came withthedevelopmentofacompletein vitrocellculturesystemforHCV(JFH1)in20057,8.JFH1viralgenome(genotype 2a), cloned from a Japanese patient withfulminant hepatitis could not only replicate efficiently

incellculturebutcouldalsogenerateviralparticles.HCVcc(forHCVgrown incellculture)hasallowedresearchers for the first time to study the complete life cycleofHCV.Yet,owingtothelimitedhostrangeofHCV, the development of a small animal model (tostudyviralreplicationandpathogenesis) isstillabigchallenge in the field.

HCV virions exhibit a wide range of densities,althoughthemostinfectiousfractionhasadensityof1.15-1.17 g/ml8,9. Present inside the outer envelope,there is a (30-35 nm) inner core which encapsulatesthe single-strand viral RNA (positive-sense), whichis approximately 9.6 kb (Fig. 2). The HCV genomedoesnotenterthecellnucleus.HCV-RNAreplicationoccursinthecytoplasmofhepatocytes.Thegenomicorganization of HCV is shown schematically inFig.3.Theviral-RNAgenomeharboursasingleORF(open reading frame) which is flanked by 5’ and 3’ nontranslatedRNAsegments(NTRs).Thecis-actingreplicationelementsorCREsarelocatedinboththe5'and3'NTRsandintheNS5Bcodingsequence10.RNA

Fig. 1. WhathappenswheninfectedwithhepatitisCvirus?Acuteliverinfectionisfollowedbyachronicinfectioninalmost80percent of the infected population152. Even among them the diseaseoutcomecanbeverydifferent(mildtosevere).Chronicinfectioneventuallycausescirrhosisleadingtohepatocellularcarcinoma36.

18 INDIANJMEDRES,JANuARY2010

structuresareknowntoparticipateandplayvitalrolesinthemultiplicationofRNAviruses.

The5'-andthe3'-NTRsofthegenomearehighlyconservedandcontaincontrolelementsfortranslationoftheviralpolyprotein and replication. The 5′ UTR (+) is~341nucleotidesinlengthandcontainsaninternalribosomalentrysite(IRES).TheHCVIRESisfoldedinto four stem-loop motifs which are called as I, II,IIIandIV.TheIRESisrequiredforcap-independenttranslationofviralRNA,whichiscarriedoutbyhostcellribosome.ThedomainIIIdoftheIRESconstitutesthekeyanchoringsiteforthe40Ssubunit11.TheIRESdomainsIII-IVhavealsobeenshowntobeanactivatorofproteinkinaseR(PKR)12.However,thisactivationdoesnotinterferewithcap-independenttranslationofHCV viral proteins. HCV core protein was reportedto interact with the 5’-NTR of plus-strand RNA13.However,recentworkwithJHF1viralRNAsuggestedthat its 5’-NTR (+) does not contain RNA packaging signals14andauthorsfurtherspeculatethatitmayresideintheRNAregionencodingthereplicase.

The3'-UTR (+) is around ~200nt and is involved in RNA replication. Three different domains can berecognized in thisuTR:(i)apoly(u/uC) tractwithanaveragelengthof80nucleotides(nt),(ii)avariableregion, and (iii) a virtually invariant 98-nt X-tailregion made up of 3 stem-loops (3’SLI, 3’SLII and3’SLIII). The 3'-uTR can robustly stimulate IRES-dependenttranslationinhumanhepatomacelllines15.

Recent studies have recognized that various stem-loop structures exist in the negative strand 3’-NTR.This region is recognizedby theviralpolymeraseastheinitiationsiteforplus-strandsynthesisoftheHCVgenome16. A recent study identified a cellular factor called Far-upstream element (FuSE) binding protein(FBP) which binds to 3'NTR by interacting with thepoly(u) tract17. The importance of long-range RNA-RNAinteractionsinthemodulationofHCVlifecyclehas been well documented.Within the 3’-end of thenon-structuralprotein5B(NS5B)codingsequence,acis-actingreplicationelement(CRE)wasdiscovered18.This CRE is called as SL9266 (or 5BSL3.2) and itsdisruption blocks RNA replication19. Mutual long-range binding with both 5' and 3' sequences issuggestedtostabilizetheCREatthecoreofacomplexpseudoknot10.

Non coding RNA molecules or microRNAs(miR)areimportantinthecontrolofgeneexpressionand regulation. MicroRNA, miR-122 is specifically expressedand is found tobe abundant in thehumanliver20.ArecentdiscoveryshowedbindingofamiRNA(miR-122) to the 5’-uTR of HCV. Sequestration ofmiR-122 in liver cell lines strongly reduced HCVtranslation,whereasitsadditionstimulatedtranslationviadirectinteractionofmiR-122withtwositesinthe5'-uTR21.ThesestudieshavegeneratedalotofinterestintheroleofmiR-122inHCVmultiplicationanditspotentialasatherapeutictarget.Aroleforproteasomealpha-subunit PSMA7 in regulating HCV IRES-mediated translation has also been demonstrated22.These host factors require further scrutiny to beconsideredascandidatesfordrugtargets.

HCV Structural Proteins

HCV encodes a single polyprotein (NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH)whichisapproximately3010aminoacids(Fig.3).Thestructuralproteins(core,E1andE2)andthep7proteinare released from the polyprotein after cleavage byhost endoplasmic reticulum (ER) signalpeptidase(s).Thenonstructuralproteins(NS2,NS3,NS4A,NS4B,NS5A,andNS5B)arecleavedbyviralproteasesNS2-3 and NS3-4A. This proteolytic processing of thepolyprotein during and after translation by host andviralproteasesyieldatleast10matureviralproteins.

Core: HCV core is a multifunctional protein whichis highly basic in nature23. It forms the structuralcomponent of the virus particle. Core has beenimplicated in the development of hepatocellular

Fig. 2. HepatitisCvirusparticlestructure: TheHCVcoreproteininteracts with viral genomic RNA to form the nucleocapsid51.Twomembrane-associatedenvelopeglycoproteins,E1andE2areembeddedinalipidenvelopewhichisderivedfromthehost8,164.

SHARMA:NEWTHERAPEuTICAPPROACHESFORHCV 19

steatosisandoncogenesis.Thecoreproteinisgeneratedfromapolyproteinencodedby theviralgenomeandisprocessedbycellularproteases in theendoplasmicreticulum (ER)24. A recent study showed that coreproteincanself-assembleinHCV-likeparticles(HCV-LPs) in ER-membranes25. A region of core protein(spanning amino acids 112 to 152) is essential forassociation,notonlywiththeERbutalsowiththeoutermitochondrial membrane26.The HCV core protein isknowntopass fromtheERintomitochondriaand isinvolvedwithCa2+regulationandapoptoticsignals27.TheHCVcoreproteinwasshowntoaffectthesteadystate levels of a subset of mitochondrial proteins,including prohibitin (functions as a chaperon ofmitochondrialproteins)28.Thisinteractionofcorewiththechaperonproteinresultedinanincreasedoxidativestress due to perturbation of normal interactionsbetweencytochromecoxidaseandprohibitin.

Core has many intriguing regulatory functionswith one of the most important being recruitment ofnon structuralproteins to the lipiddroplet-associatedmembranes29. Lipid droplets (LDs) are intracellularorganelles involved in lipid storage and also takepart in intracellular vesicular trafficking30. HCVmakesuseoflipiddropletsforreplication.IninfectedHuh-7 cells, the core protein is associated with thesurface of lipid droplets31. Besides its structural andregulatory function, core plays an imperative role in

the pathogeneses of liver steatosis. upregulation ofde novo fattyacidbiosynthesisbyHCVcoreproteinin Huh7 cells has been reported. Core protein alsointeractswithapolipoproteinAII,acomponentoflipiddroplets.HCVcoreproteinistargetedtolipiddropletsby its domain 2 (D2) and this association with lipiddroplets is required for virus production. Disruptingthe association of core protein with lipid droplets isdeleteriousforHCVccproductionandthisinteractionis thought to contribute to steatosis, via depositionof triglycerides in the liver30. These data are furthersupported by studies showing that the expression ofcoreproteincanleadtothedevelopmentofsteatosisintransgenicmice28.

Clinical studies have reported that virus-inducedsteatosisisveryseverewithHCVgenotype3thanwithother genotypes32. Interestingly, core protein derivedfromgenotype3a inducedhigher fatty acid synthaseactivity than core protein derived from genotype 1b.However, no genetic or functional differences wereobserved between genotype 3a core proteins frompatientswithandwithoutHCV-inducedsteatosis,thus,suggesting a possible role of other viral proteins inthe development of hepatocellular steatosis33. HCV-subgenomicrepliconsystems(whichallowHCV-RNAto replicate autonomously), have shown subcellularlocalization of core to be both cytoplasmic andnuclear26,34.AstudywithchronicallyHCV-infectedliveralsorevealedasimilardistribution,withcorelocalizedtobothcytoplasmandthenucleus35.Inthecytoplasmthecoreproteinismostlylocalizedtotheendoplasmicreticulum(ER).HCVcoreprotein,circulating'free'innon enveloped state has also been detected in HCV-infectedpatients36.

Transgenic mice expressing the core proteindevelop HCC, indicating a direct part played by thecore protein in this process37. A study found twomutationsinthecoregene(36G/Cand209A),whichwerelinkedwithincreasedHCCrisk38.Thecoregenesequence data may provide useful information aboutHCC risk and more studies should be performed todevelopitfurther.However,itisimportanttoconsiderthetypeofgenotypeinvolved,asitisclearthatgeneexpression profile in hepatocytes is dependent on the HCV-core-genotype sequence39. Tumour suppressorprotein promyelocytic leukaemia (PML) is knownto be involved in antiviral response. Interestingly, arecent study suggests a potential mechanism for thedevelopmentoflivercancerviatheHCV-coremediatedinactivationofthePMLtumoursuppressorpathway40.

Fig. 3.AschematicrepresentationofHCVgenome,structuralandnonstructuralproteins: TheviralRNAconsistsofa5'untranslatedregion(uTR)containingtheinternalribosomeentrysite(IRES),followed by the genomic region for structural and nonstructuralgenesandthe3'uTR.HCVis translatedasapolyproteinthat isproteolyticallyprocessedbyhostandviralproteases59.Thestructuralproteins (C, core; E1, envelope protein 1; E2, envelope protein1) are located at the amino-terminal end while the nonstructuralproteins(NS2,NS3,NS4A,NS4B,NS5A,andNS5B)arelocatedat the carboxyl-terminal end70. The p7 protein (ion channel orviroporin)islocatedatthejunctionofstructuralandnonstructuralproteins59.


ARFPs:AlternatereadingframeproteinsorARFPsareencoded by the +1 reading frame of the viral genome whichoverlapswiththecoreproteincodingsequence.Theseadditionalproteinsofunknownfunctionarealsocalled as core+1 or F1 and were recently identified41.Since thenmultiplemechanismshavebeenproposedfortheexpressionofARFPs,whichinclude(i)frameshifting(ii)formtranscriptionalslippage,or(iii)frominternal initiation in the +1 open reading frame (ORF) ofthecoreproteincodingsequence.ARFPshavebeenshowntobeassociatedwiththeERandmitochondria.Interestingly, these proteins are labile and very shortlived42, 43. Bases on the ability ofARFPs to bind theproteasome subunit alpha3, it was suggested that itmayregulateproteindegradationincells44.However,thefunctional roleofARFPs in theHCVlifecycle isnotclearyet.

Envelope glycoproteins: The HCV envelopeglycoproteins,E1andE2arestructuralcomponentsofthe virion. They constitute the outer coat of fine spike-like projections of the HCV particle (Fig. 2)9. Theyundergo post-translational modifications (N-linked additionofcarbohydratechains)whilebeingtranslatedin theendoplasmic reticulum(ER).Both,E1andE2envelopeglycoproteinsarerequiredforhost-cellentryvia receptor binding. Insight into the mechanismsby which HCV gains entry into host cells is vital tounderstand primary HCV infection and re-infectionpost-transplantation.

The E2 protein has the binding sites for humanCD81,a tetraspaninexpressedonvariouscell typesincluding hepatocytes and B lymphocytes45. Thebinding of E2 was further mapped to the majorextracellular loop of CD81. CD81 along withhumanscavengerreceptorSR-BI,andtight junctionmolecules claudin-1 (CLDN) and occludin (OCLN)are the most important receptors that mediate HCVentry46. In addition, it is thought that HCV mayutilizeglycosaminoglycansandlowdensityreceptorson hepatocytes as initial attachment factors. BothCD81 and SR-BI were identified as candidate HCV receptors based on their physical interaction with asoluble version of E247,48. The HCVpp system wassubsequently used to prove that they are requiredforviralentry49.HCVppsystem(alsocalledasHCVpseudoparticle) generates virus particles, whichdisplayE1-E2glycoproteinsofHCVontheirsurface.CellentryofHCVppisHCVglycoproteinmediated.HCVpparegeneratedbyco-transfectionof3plasmidsinto 293T cells: (i) gag-pol genes of HIV or MLV,

(ii)HCVgp,and(iii)aretrovirusgenomewithLTRs,packagingsignalsandareportergene.HCVppsystemdoes not require productive HCV replication andhenceisnotrestrictedtoHuh7orHuh7.5thatsupportrobust replication50. HCVpp closely resembles thecellentrypropertiesofgenuineHCVvirionsandwasusedtoidentifyCLDNandOCLN.ThediscoveryofOCLN provides an vital advance towards efforts todevelopsmallanimalmodelsforHCV46.

HCV-likeparticles(HCV-LPs)wereisolatedfrominsect cells, infected with recombinant baculovirusexpressingHCVstructuralproteins(Core,E1andE2)51.IsolatedHCV-LPswerecomposedofallthestructuralproteins (E1, E2 and C). Further analysis of HCV-LPs by cryoelectron microscopy (CryoEM) revealedthattheyaresphericalparticleswithsmoothsurfaces.TheywerefoundtobeconsistentwiththenativeHCVvirionsisolatedfromHCVinfectedpatients.HCV-LPsfor the first time allowed 3D structural analysis of HCV particles51,whichfurtherallowsstudyingthecomplexmechanismsofHCVassemblyandmaturation.

Ahypervariabledomainnear theaminoterminusofE2isthemostvariablepartoftheviralpolyproteinandcalledasHVR-1.Ithasbeenshowntobethetargetforneutralizingantibodies.ItisinterestingtonotethatHCVcanassociatewithLDLandVLDLfrominfectedpatientserum.Amazingly,HDLsplayaveryactiveroleinHCVentry.AcomplexinterplaybetweenHDL,SR-BIandHCVenvelopeglycoproteinsleadstoenhancedHDL-mediated HCVpp entry in cells48. In addition,HDLcaninhibitHCV-neutralizingantibodiesinserumofacuteandchronicHCV-infectedpatients52,53.E2hasalsobeenshown tobindwithCD81 receptorswhichareexpressedonthyroidcellandinduceacascadeofsignalingpathwayleadingtoIL-8release.Itisthoughtthat E2 protein may induce thyroidal inflammation, therebytriggeringthyroiditisbyabystanderactivationmechanism6. Finally, E2 protein has been shown tobindPKRandasaconsequenceperturbinnateimmunepathways.

HCV Non Structural Proteins

The non structural proteins NS2, NS3, NS4A,NS4B, NS5A and NS5B are thought to be to berequiredforreplicationoftheviralgenome.Thenonstructuralprotein,p7,canformionchannels,requiredfor the production of infectious virus particles. It isnowrecognizedthatthecross-talkbetweenstructuralandthenonstructuralproteinsofHCVisrequiredforefficient virus particle production54.


The p7 ion channel: TheHCVp7 isasmall63aminoacidprotein,positionedatthejunctionofthestructuraland non structural proteins. The p7 protein belongstoa familyofviralproteinscalledasviroporins thatform ion channels. It can oligomerize following itsinclusionintoalipidmembranecreatingionchannels.Thecleavagesmediatedbysignalpeptidasesbetweenp7andNS2occursslowlyandarepartialattheE2/p7andp7/NS2sites55.ThisresultsintheformationofanE2p7NS2precursor56.Itisthoughtthatthisprecursormay have a role in the regulation of HCV lifecycle.Many models have been proposed to explain thepotential significance of these precursor forms, the simplest being, the necessity for a timely release ofthe individual proteins at the appropriate time in thevirallifecycle.However,thepreciseroleandexistenceof theseprecursors inanaturalHCVinfection isnotknown.

The p7 protein is highly hydrophobic in nature.It is localized in the ER-membranes when encodedby a replication-competent genome57. It has twoamphipathic, transmembrane regions,TM1andTM2(spanning amino acids 19-32 and 36-58) which areembeddedintheER-membrane.TheN-andC-terminiareexposedtotheextracellularenvironment58.The3-dimensionalstructureofp7-complexwasdeterminedby using single-particle electron microscopy. Thishexameric (42 kDa) protein complex was found todepict flower-shaped architecture with protruding petals oriented toward the ER lumen59.The p7 ion channelprotein serves an essential role in the production ofinfectious virus particles during HCV lifecycle60. ItappearstobedispensableforviralRNAreplication,asreplicons lacking thep7gene replicateormakeviralRNA efficiently61,62.

Theionchannelblocker,amantadine,wasthoughtto interferewith the ionchannelactivityofp7basedon studies with artificial lipid bilayer system. The p7 proteincouldformamantadine-sensitiveionchannelsin this artificial system63. However, it became clearafterclinicaltrials,thatp7ionchannelfunctionisnotaffectedbyamantadine64.Anotherstudyalsoreportedsimilar findings, where HCV strains were found resistant to the ion channel blocker, amantadine65.Nonetheless,dose-dependentreductionsofvirustitreswere achieved with iminosugars64,66. The absolutedependence of HCV on- ion channel function of p7proteinforinfectivitymakesitanattractivecandidatetargetforantiviralintervention.

HCV NS2: The non structural protein 2 (NS2) is a23-kDa transmembrane hydrophobic protein. ThemembraneassociationofNS2 isp7-independent andoccurs co-translationally67,68. NS2 is a membrane-associated cysteine protease69,70, required for HCVccinfectivity71. The cleavage between NS2 and NS3is absolutely required for persistent viral infectionin a chimpanzee72. NS2 followed by only 2 aminoacids of NS3 produces a basal proteolytic activityin vitro.However, theN-terminal180aaofNS3arerequired besides NS2 for a robust protease activity.Interestingly,all theactivesiteresidues(H952,E972and C993), needed for the catalytic activity of theNS2/3cysteineprotease,arelocatedentirelyinNS273.ThisrequirementofNS3remainedintriguinguntiltherecentdiscoveryshowingthatthezincbindingdomainofNS3couldinfactstimulatetheproteaseactivityofNS2.The functional sub-domains in NS3 essentiallyfunctionasitsregulatorycofactor73againhighlightingtight regulation of the proteolytic processing. Thisprocessisundoubtedlyvitalforvirusmultiplication.

NS2 interacts with itself forming homo-dimers.MoreoverNS2hasalsobeenshowntointeractwithalltheotherHCVnonstructuralproteins74.untilrecentlytheonlyknownfunctionforNS2wasitsautocleavingactivity at theNS2/3 junction.ExpressionofNS2 inHuh7cells resulted inupregulationof transcriptionalfactor sterol regulatory element-binding protein 1c(SREBP-1c) and fatty acid synthase. These studiesimplicate a role of NS2 in promoting steatosis. NS2proteincanbephosphorylatedbycaseinKinaseIIonSerresidueatposition168.ThisphosphorylationeventappearstoregulatethestabilityofNS2protein(atleastfrom genotype 1a). NS2 also appears to be involvedinaparticleassembly stepwhichhappenspost core,NS5A,andNS3assembly75.Thesearchformoleculesor inhibitors targeting NS2, should without a doubt,advancethedevelopmentofnewtherapeuticsagainstHCV.

HCV NS3: The non structural protein 3 (NS3) is amemberof the superfamily2DExH/D-boxhelicasesand its crystal structure has been determined. It is a67 kDa tri-functional protein with a serine protease,anRNAhelicaseandNTPaseactivities (Fig.4).TheNS3enzymehasachymotrypsin-likeserineproteaseactivity76.AlongwithitscofactorNS4Aisresponsiblefor cleavage at the NS3/4A, NS4A/4B, NS4B/5AandNS5A/5Bjunctions.NS3/4Aare localizedinERcisternaesurroundingmitochondria77.Howeverwhenco-expressed with p53 (tumour suppressor), it is


localizedalongwithp53inthenucleus.NS3unwindsboth RNA and DNA substrates, although there is noDNA intermediate involved in HCV lifecycle. Itcouples unwinding of RNA strands (with extensivesecondary structures) to ATP hydrolysis. A directinteraction between NS3 and NS5B occurs throughtheproteasedomainofNS3.RNAunwindingactivityofNS3helicaseismodulatedbythisinteractionwithNS5Bpolymerase78.Inadditiontoitsroleinviralpoly-protein processing and HCV multiplication, anotherimportantfunctionofNS3involvesantagonizinghostinnate-immunepathways.

TheinductionoftypeIinterferon(IFN)genes(TypeI IFNs include several IFN-α subtypes and a single IFN-β subtype) is regulated at the step of transcription andisbestunderstoodfortheIFN-βpromoter.Innateimmunedefensemechanismsactivatedbyalpha/betaINFs represent an essential first line of protection against viral infections. Retinoic acid induciblegeneorRIG-I is a cytoplasmicRNAhelicase79. It isan essential pathogen recognition receptor (PRR)for HCV. RIG-I upon binding viral RNA undergoeschanges in conformation and can then interact withIFN- β promoter stimulator-1 or IPS-1(IPS-1 is also knownVISA,CardiforMAVS)80.ThisinteractionofRIG-IwithIPS-1cansignaldownstreamactivationofIRFsandNFkBtotriggeralpha/beta-INFs.

ThelocalizationofMAVStothemitochondriaiscritical for its ability to induce IFNs. This functionof MAVS is abolished if the mitochondrial-targetingdomainofMAVSisdeleted81.HCVNS3/4Aproteasehas remarkably evolved to target and cleave ISP-1(or MAVS at Cys-508), thereby halting alpha/betainterferonexpression81.Thisactivitymaybenecessarybut does not appear to be sufficient for long-term viral persistence since there is (cytotoxic) T cell-mediatedclearanceofNS3/4A-expressinghepatocytesin vivo82. Therefore, other HCV proteins are mostlikely responsible for interfering with the adaptiveimmunity.

A recent study utilizing sub-genomic replicondemonstrated that besides methylation (which

regulateshelicaseactivity),NS3undergoesotherpost-translational modifications, such as phosphorylation and N-terminal acetylation83. The multifunctionalroles playedbyNS3 in the lifecycleofHCV, makesitindispensableforvirusmultiplication.NS3thereforerepresents a very promising target for anti-HCVtherapy.

HCV NS4A: Non structural protein 4A (NS4A) isa cofactor for NS3 serine protease activity. The N-terminal portion of 4A is responsible for membraneassociation of the NS3-4A complex. NS4A alsoassociateswithotherHCVproteinsontheER-derivedmembranous webs, where viral replication complexis assembled. Interestingly, NS4A is localized notonlyon theER,butalsoonmitochondria84.Thus, inaddition to its essential role in HCV replication it isalsoinvolvedinviralpathogenesisbyaffectingcellularfunctions.Further,astudyreportedalteredintracellulardistribution of mitochondria and its damage due toNS4AexpressioninHuh7cells85.TheroleplayedbythissmallpolypeptideduringHCVreplicationisverysignificant.

HCV NS4B: TheHCVnonstructuralprotein4B(NS4B)is a 27-KDa polypeptide. It is a highly hydrophobicintegral ER-membrane protein86. It contains fourtransmembrane domains and is palmitoylated at twoC-terminal cysteine residues. Palmitoylation of 4BproteinfacilitatesoligomerizationwhichappearstobeessentialforHCVreplication87,88.IthasanamphipatichelixattheN-terminalandaC-terminaldomainwhicharebothassociatedwithmembranes89.Allelicvariationin the NS4B sequence between closely related HCVisolateswasfoundtodrasticallyimpactHCVreplicationincellculture90.AcharacteristicfeatureofPlus-strandRNA viruses is their ability to induce alterations incellularmembranesandthenutilizeittoreplicatetheirowngenomes.

Backinthe1980s,cytoplasmicchangeswereseeninhepatocytesofinfectedchimpanzeeswiththehelpofelectronmicroscope.Sponge-likeinclusioncomposedofadensematrixandirregularlyarrangedmembraneswere reported91. Recently, Egger et al92 showed thatNS4B induced a distinct membrane alteration whenexpressed inculturedhumanosteosarcoma (u-2OS)cells. Thus, a protein which had no known functionuntil then was shown to induce a tight structure,designatedmembranousweb,consistingofvesiclesinamembranousmatrix.Theimmunoelectronmicroscopyof Huh7 cells supporting replication of sub-genomic

Fig. 4. AdiagrammaticrepresentationofHCVNS3protein:Theprotease and the helicase domain organization is shown withboundaries defining these domains (amino acids 1027 to 1658 in thepolyprotein)163,164.


Proteasedomain DomainI DomainII DomainIII

(Helicasedomain)

1027 1192 1214 1353 1507 1658

replicon revealed a loss of the organization andother morphological alterations of the ER showingconvolutedcisternaeandparacrystallinestructures77.

ThereplicationofHCVisaveryintricateprocess,occurring through protein–RNA and protein–proteininteractions. HCV-replication-complex was first identified in Huh7 cells supporting sub-genomic repliconin200393.Thesemembranouswebsaremadeupofsmallvesicles(80-180nmindiameter)embeddedinamembranousmatrixandarefoundcloselyassociatedwith the rough endoplasmic reticulum. NS4B co-localizedwithinthiswebtogetherwithotherstructural

and NS proteins92. Interestingly, NS4B alone couldinduce the formation of membranous web. In vitroexpressionstudiesofNS4Bfromallmajorgenotypeshave demonstrated the importance of N terminus ofNS4B94. Disrupting the amphipathic-helix in the Nterminus of NS4B abolishes the ability of NS4B torearrangemembranes. Even before an important rolein formationofmembranousweb likestructureswasdiscoveredforthisinterestingprotein,aroleforNS4BinmalignanttransformationofNIH3T3cellinassociationwiththeHa-rasoncogenehadbeenproposed95.ItisanimportantproteinforHCVlifecycle.

HCV NS5A: The non structural protein 5A (NS5A)has RNA-binding activity96. NS5A protein can befoundinbasallyphosphorylated(56kDa)andhyper-phosphorylated(58kDa)forms incells.P58formofNS5A is hyperphosphorylated at additional sites thatremain unmodified in the p56 form by casein kinase I-alpha97,98.

The first of its kind, the sub-genomic replicon utilized a HCV genotype-1b clone, called as Con1.This clone was engineered such that HCV-structuralgenes were replaced by a neomycin resistance gene.Following transfection of in vitro transcribed RNAintoHuh-7cells,antibioticG418-resistantcellscouldbeobtainedinwhichthesub-genomicRNAreplicatedautonomously.Mutationsthatenhancethecapacityofsub-genomicHCVRNAtoreplicateincellculture(Huh-7 cells) were mapped to the NS5A-coding sequenceand were called as adaptive mutations.The adaptivemutationsalterthephosphorylationstateoftheNS5Aprotein.Lossofhyperphosphorylation(orNS5A-p58form)stimulatesRNAreplicationoftheHCVgenotype1breplcioninHuh-7cells.Humanvesicle-associatedmembraneprotein (hVAP)subtypeA isknown tobeahostfactoressentialforHCVreplicationbybindingto both NS5A and NS5B. Phosphorylation status of

NS5AisalsoknowntoaffecttheinteractionofNS5Awith VAP-A99. NS5A inhibitor (BMS-824) and theNS3proteaseinhibitorblockhyperphosphorylationofNS5A;however,themechanismofinhibitionremainsunknown. Further, the involvement of a proteaseinhibitorininhibitingp58isquiteintriguing100.

NS5Ahasanamphipathicalpha-helixatitsaminoterminuswithwhichitisanchoredtotheERmembrane.NS5Aisfurtherdividedintothree-domainswhichareseparatedby linker regions.Thecrystal structureoftheconserveddomainI(DI)wassolvedrecentlyandrevealedaunique fold.DIhasazincbindingmotifandalsoformsanucleicacid-bindingdomain.NS5Aspecifically binds to the G/U rich sequences in the 3’ endsofHCVgenomicRNA96.DomainsII(DII)andIII (DIII)aremorevariableamongHCVgenotypes.Crystal structures for both DII and DIII are notavailableyet.NMRstudieshaveshownDIIofNS5Ato be flexible and disordered. NS5A-DII contains the PKR and the HCV-NS5B binding domains. AroleforNS5A-DIIIinvirusassemblywasshownviaphosphorylationofaserineresidueatposition2433byCKII101.NS5AdomainIIIisnotrequiredforRNAreplication as sub-genomic replicon lacking DIII,replicateinHuh7cells.However,arecentstudywithJFH1 virus showed that DIII of NS5A could influence RNA replication. Genotype 2 HCV isolates havea 19 residue insertion near the C terminus of DIII.upon its deletion, authors observed a delay in both

RNA replication and particle assembly102 therebysuggestingaroleinviralmultiplication.

Full length HCV NS5A has a cytoplasmiclocalization. Cleavage by caspase 3 and 6 leads togenearationofN-terminallytruncatedNS5Afragmentswhichgetlocalizedwithinthenucleus.TheC-terminaldomain of NS5A can associate with c-Raf kinase103.This interaction appears to be essential for HCVreplicationinHuh7cells,assequestrationofc-Rafbytruncated NS5A into the nucleus, negatively impactsHCVreplication104.Oxysterolbindingprotein(OSBP)caninteractwiththeN-terminalregionofDI.OSBPisco-localizedtothegolgiwithNS5AandthisfunctionalinteractionissuggestedtoplayaroleinHCVparticlerelease105. A significant association between variations insequencesbetween2209-2248nucleotidesofNS5A-gene, and response to interferon treatment has beenproposed. This region of NS5A (amino acids 237−276) iscalledasISDRorinterferonsensitivitydeterminingregion.TheISDRwasalsofoundtoassociatewiththeantiviralmolecule,PKR.Patientswithamutant-type


NS5A-ISDRhadahigher rateof sustainedvirologicresponse(SVR)thanthosewiththewild-typeNS5A-ISDR. However, recent studies have questioned theexistenceofanISDRinNS5A.ExistenceofanISDRisanongoingdebateandcontroversial.Nevertheless,NS5A can repress the PKR pathway. NS5A also hasthe ability to inhibit IFN-gamma production106. AremarkablestudywithNS5Atransgenicmicesuggestedthat NS5A protein could impair both the innate andadaptive hepatic immune response107. Indeed, NS5Ahas attracted tremendous attention in the HCV field and represents a very promising target for anti-HCVtherapy.

HCV NS5B: The HCV non structural protein 5B(NS5B)isaRNAdependentRNApolymerase(RDRP)containingtheGDDmotifinitsactivesite108.Itbelongsto a class of integral membrane proteins termedtail-anchored proteins. NS5B initiates synthesis ofcomplementary negative-strand RNA using the HCVgenome(positivepolarity)asatemplate.Subsequentlyit generates positive-strand RNA from this negative-strand RNA template. The NS5B crystal structureshows the typical fingers, palm and thumb sub-domains. NS5B is anchored to the ER-derived “membranouswebs" via its C-terminal 21 amino-acid residues109.Studies with sub-genomic replicon revealed that themembraneassociationisindispensableforviralRNAreplication110.In vitroNS5Bcannotdistinguishbetweennatural and synthetic templates. NS5A can directlybind to NS5B and modulate its polymerase (polyAtemplate/poly u primer) activity111. NS5A (at sub-stoichometric levels) stimulates replication by NS5Bon templates derived from the 3’ end of the positivestrand112. NS5A stimulates NS5B during elongation;however the mechanism of action is not clear. Itcouldeitherbedue to the stimulationof its catalyticactivityorduetoaconformationalchangeinducedbyNS5Abinding112.Thereappears tobeadifference inpolymerase specific activity in vitro,dependingonthetypeofHCVgenotype.TheJFH1NS5Benzymeshowsa 10- fold-higher specific activity when compared to J6 NS5B113.TheterminationofRNAsynthesisin vitroisnotunderstoodwell.

NS5B lacks a “proofreading” function. Due to ahigh rate of error-prone replication, complex mutantswarms are generated. However, HCV must alsomaintain highly conserved genomic segments and abalancebetweenconservedandvariableviralelementsis above all important to avoid "error catastrophe".Over the past few years numerous nucleoside and

non nucleoside inhibitors of the polymerase havebeen discovered and demonstrated clinical efficiency and advanced to clinical trials. NS5B nucleosideinhibitors (NIs) or analogs (NM-107/NM-283, PSI-6130/R7128, IDX184, MK-0608) compete withcellularribonucleosidetriphospahtesactasfunctionalchainterminators114.Non-nucleosideinhibitors(NNIs)or analogs (ABT-333, GSK625433, VCH-759, PF-868554,GS-9190)actbyallostericmechanism.Theybind to allosteric sites on NS5B and thereby inhibitRNAsynthesis114,115.

Animportantleadwasprovidedbyastudywhichestablished that NS5B associates with cyclophilinA (CypA) via its enzymatic pocket and exploits theisomerase/chaperone activity of CypA to replicate incells116.NS5BisphosphorylatedbytheproteinkinaseC-relatedkinase2(PRK2)incellculture117.Itwasfurtherdiscovered that inhibitors of NS5B phosphorylation,HA1077 (fasudil) and Y27632 suppressed theactivation of PRK2.The treatment of liver cell lines(stably replicating HCV sub-genomic replicon) withthese inhibitors cleared viral RNA suggesting thatPRK2 inhibitors act by suppressing HCV replicationvia inhibitionofNS5Bactivity118.However, itwouldbe interesting tosee if resistantmutantsemergeovera period of time. More critical would be the normalfunctionsaffectedwhichareregulatedbyPRK2.

The continuous generation and selection ofresistant variants allows HCV to escape control bytheseinhibitors/antiviraldrugs.Astudywherepatientsinfectedwithgenotype1aweretreatedwithribavirin,ledtotheemergenceofaPhe-to-Tyr(F415Y)mutationin theviral polymerase119.There is also evidence forrecombination events leading to genetic variation inHCV120. Thus, very high rates of genetic variationensurethesurvivalofhepatitisCvirusesundereverychanginghostilecellularenvironment.

Current Therapy

The very first treatment shown to be effective against HCV involved systematic administrationof INF-α. INFs are produced naturally by host’s immune system in response to a viral infection. INFwasactuallydiscovered in1957,during studieswithinfluenzavirus121.ThroughtheyearsHCVtreatmenthasimproved with the development of modified pegylated-interferon (PEG-INF), which is a stabilized version,has longer biological half-life. A SVR is defined as negativeHCVRNA,sixmonthsaftertreatment.INFisknowntoactivateseveraldirectandindirectantiviral


mechanisms(suchasviralRNAdegradation,haltviraltranslation).However,theantiviralmechanismofIFNin vivoremainsunknown.

Ribavirin was first used in treating children with respiratorysyncytialvirusinfection122.Itwasdiscoveredmorethan30yragoandiseffectiveagainstHCV,onlywhenadministeredalongwithinterferonincombinationtherapy. Ribavirin (1-beta-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide)isapurine-analogwithbroad-spectrumantiviralactivity123.AdministrationofribavirinincombinationwithPEG-INF-alfa-2a(40kDa)toHCVinfectedpatient’sresultsinanincreasedSVRrate124,125.The mechanisms or pathways involved, leading toanti-HCV effects are not fully understood yet. Someof the proposed mechanisms include: modulationof interferon-stimulated gene expression, inhibitionof host inosine monophosphate dehydrogenase (byribavirin monophosphate), modulation of the hostimmuneresponses,directinhibitionoftheviralRNApolymerase (by ribavirin triphosphate) and lethalmutagenesis of HCV-RNA genomes caused viaincorporationofribavirintriphosphatebyNS5B126,127.Thus, ribavirincanbea substrate for theHCVRNApolymerasealthoughsurprisinglyitisnotveryeffectivein monotherapy128. Therefore, pegylated interferon(PEG-INF)andribavirincombinationhasbecomethecurrent“goldstandard”regimenortreatmentofchoiceforhepatitisC.

unfortunately, both these compounds are toxicand their administration causes side effects such asheadache,fever,severedepression,myalgia,arthralgiaandhaemolyticanaemia.Moreover,thecostoftherapyisveryhigh.Severalmonthsoftherapyisrequiredforeradication of chronic HCV infection, despite whichSVR isoftennot achieved. In summary, the existingtreatment is not very effective and causes significant side effects. Development of better and improvedtreatment is requiredurgentlyandrepresentsamajorchallengeforvirologists

In addition, responses to treatment vary widelydependingonthegenotypeofHCV.Genotypes1and4 are the most difficult-to-treat. Only 40-50 per cent of thepatientsachieveSVRwithgenotype1infections.Ageisalsoanimportantfactorasolderpatientsexhibitlower response to therapy than younger patients.Moreover,currentavailabletreatmentoptionsfornonrespondersarevery few.The factorsdetermining thetriumphorfailureoftheantiviraltherapyarenotwellunderstood.A clear understanding of the function of

genes involved in the IFN-α pathway could possibly explainsomeof these leadingquestions.This insightandfurtherresearchexploitingarelationshipbetweentherapy response and host genetic polymorphisms inthe IFN-α pathway may lead to the development of newtherapeuticstrategies129.

Current State of Antivirals

At present, there is no approved (HCV specific) antiviral therapy. However, search for “specific targetedantiviraltherapyforHCV”(STAT-C)hasbeenongoingforalongtime.Advancesinthemodelsystemssuch as: (i) sub-genomic and genomic replicons, (ii)HCVpp, (iii) HCVcc-JHF1, (iv) HCV-LPs to studyHCV have further improved our understanding ofHCV lifecycle and potential therapeutic targets. Thelatestdevelopmentbeing,theabilitytogenerateHCVparticles that are infectious both in cell culture andin vivo with the Con1 wild type genome54. We areapproachingaveryproductiveaswellaschallengingphase in the field of HCV virology. Wide varieties of inhibitors or directly acting antiviral agents are atdifferentphasesofdevelopmentandsomearecurrentlyinclinicaltrials.TheseinhibitorstargetHCVreceptors,HCV-IRES, NS3/4A, NS5A and NS5B. In additionmolecules targetinghost factorswhichplaya role inHCVreplication,arealsobeingpursued.

HCVentersintohepatocytesbyinteractingwithcellsurfacereceptors.Theseinitialeventsinviruslifecycleofferanattractivetargetforantiviraldevelopment.Thelectincyanovirin-N(CV-N)wasoriginallydiscoveredagainstHIVandsubsequentlyalsoshowntobeapotentinhibitorofHCV.ItbindstoenvelopeglycoproteinsandinhibitstheinteractionbetweenE2andCD81130.Thissuggests that targeting the glycans of viral envelopeproteinsholdspotentialinthedevelopmentofantiviraltherapies.Anotherinterestingclassofinhibitorssuchascelgosivir,n-butyldeoxynojirimycinandN-(n-nonyl)

deoxynojirimycintargethostalpha-glucosidaseenzymeand may offer alternative treatment options131,132.Alpha-glucosidaseenzymeplaysacriticalroleinHCVmaturationbyinitiatingtheprocessingoftheN-linkedoligosaccharides of viral envelope glycoproteins133.Thus their inhibitorscanaffectHCVmorphogenesis.This was further confirmed by using HCV-LPs, in the presence of alpha-glucosidase inhibitors, viralglycoproteinsweremisfolded,retainedintheERandalsoimpairedintheirinteractionwithcalnexin134.Thus,this novel class of inhibitors can target host-directedglycosylationandmaypreventtheappearanceofdrug


resistancewhengivenincombinationwiththecurrentstandard of care132. Celgosivir (rapidly converted tocastanospermine) is currently undergoing clinicaltrials.

Astudywherephosphatidylinositol4-kinasetypeIII-alpha (PI4KIII-α) was knocked down prevented infection by HCVpp or by HCVcc (JFH-1). Inaddition, the susceptibility to HCVpp infection andreplicationincellswasdependentonthepresenceofphosphatidylinositol 4-kinase type III-beta (PI4KIII-β)135. Another study showed that PI4K-III-α is involved in events vital for membrane alterations, whichare essential for the formation of HCV replicationcomplexes136. PI4K-III-α also plays an essential role in membrane alterations leading to the formation ofHCV replication complexes. PI4KIII-α and β are very important host factors for HCV replication. Thesekinasesarepotentialtherapeutictargets.

Otherrecentstudieshavedemonstratedthatiridoids(suchasaglyconesofshanzhisidemethylester:loganin,verbenalin)andamphipathicDNApolymers(APs)arenovel antiviral molecules which inhibit HCV entry.Inhibitorsloganinandverbenalinwereshowntohaveanti-HCV entry and anti-infectivity activities137.APsinhibitHCVinternalizationowingtotheiramphipathicnature. ThusAPs target the cell entry step of HCV.A recent study showed thatCD81hasno role in thecell-to-cellspreadofHCVcc.Inaddition,thismodeoftransmissionappearstobeshieldedfromneutralizingantibodies. This study highlights the urgent need todevelop strategies aimed at disrupting direct cell-to-cell HCV transmission as targeting cell-free virusalone may not be enough to halt a chronic infectionandadualapproachisrequired138.

The significance of targeting NS3-4A protease is clearly evident from its vital function, requiredto process viral polyprotein. Targeting NS3-4A hasadditional advantage, since NS3-4A protease alsofunctions to inhibit host innate response by cleavingIFN-β promoter stimulator-1 (IPS-1) and TRIF139.Thelatter activity of NS3 affects signaling of the RIG-IandToll-like-receptor-3pathways, therebyperturbingthe interferon response. A specific inhibitor of NS3, telaprevir (VX-950) was put to clinical test recentlywith some success. The current thought in the field beingthatacombinationtherapyalongwithinhibitorswhich simultaneously inhibit multiple steps in theviruslifecycle(proteaseandreplicasefunctions)mayyieldbetterSVR. Indeed, recentclinical trialswhere

PEG-IFNandribavirinwereadministeredalongwithtelaprevir, showed significantlyimprovedSVRratesinpatientswithgenotype1,whichhappenstobethemostdifficult to treat140.However,therewerehigherratesofdiscontinuationinthetreatmentbecauseoftheadverseeffectssuchaspruritus,rash,andanaemia.

Another study which involved administration ofinterferon with telaprevir highlighted the importanceofretainingribavirinduringthetreatment.Asresponserates were lowest when ribavirin was not presentin the treatment141. There is supporting evidence ofa decreased SVR and relapse when ribavirin wasprematurely discontinued or ribavirin doses werefrequently missed. However, the major drawback ofribavirinissideeffects.Furtherresearchisrequiredtoadvance, refine and develop non-toxic versions of these inhibitors.Thereissomeprogressinthisdirectionandseveralribavirinanalogues(suchas4-iodo-1-beta-D-ribofuranosylpyrazole-3-carboxamide, 4-propynyl-1-beta-D-ribofuranosylpyrazole-3-carboxamide, and4-phenylethynyl-1-beta-D-ribofuranosylpyrazole-3-carboxamide)arebeingevaluatedandcouldofferanalternativeandbettertherapeuticoption142.

NS5A plays a vital role in HCV replication andisemergingasan important target for the therapyofHCV infection.BMS-824 is a smallmoleculewhichtargetsNS5Aandinhibitshyperphosphorylationorp58formation100. Inhibitors that disrupt phosphorylationwith a concomitant decrease in virus replication areindeedveryattractiveandshouldbedevelopedfurther.Another molecule, zinc mesoporphyrin (a synthetichemeanaloguewithacentralzincofthemesoporphyinmacrocycle)causesadramaticdownregulationoftheNS5A protein by enhancing its polyubiquitinationand proteasome-dependent degradation. Zincmesoporphyrin holds immense potential as a noveldrugtotreatHCVinfection143.

HCVpolymeraseisanattractivetargetowingtoitsessentialroleinviralRNAreplication.Itisbeyondanydoubt the most validated target for the developmentofantiviraltherapy.Valopicitabine(NM-283)wasthefirst NS5B analogue to demonstrate proof of concept for 5B nucleoside inhibitors in the clinic. CurrentlyonlyRoche-1728NIisunderactivedevelopmentandclinical trials144. IdenixNI IDX-184 is underphase Itrialinpatientsinfectedwithgenotype1.AnotherNIfrom Merck, MK-0608 is in preclinical trials. TheNIscanbeutilizedbythepolymeraseonceconvertedto the 5’-triphosphate form. NNIs on the other hand


are non-competitive inhibitors which target the viralpolymerase, preferentially in the initiation phase. ANNI, PF-00868554 (Pfizer) is currently in phase II clinical trials where it is given along with PEG-INFandribavirin.GS-9190(Gilead)isunderdevelopmentand currently a study with VCH-759 (ViroChemPharma) isongoing.SeveralNNIssuchasVCH-916(ViroChemPharma),GSK-625433(GlaxoSmithKline)andABT-333 (Abbott) have reached phase I clinicaltrials.

A further advance came with the concept ofadministering inhibitors of both, viral and hostproteins. This seems a very intelligent choice for arapidlymutatingviruslikeHCVandmightbevitaltosuccess in forthcoming therapies. Remarkable workis being done in this regard with a specific host factor cyclophilin(Cyp),whichbelongstoafamilyofcellularpeptidyl-prolyl isomerases. It is required for HCVreplication.CyclosporineAinhibitsHCVreplicationbybindingtocyclophilin.IthasnowbeenestablishedthatcyclosporineAandothernonimmunosuppressiveCypinhibitors(suchasDebio025,NIM811,andSCY-635)effectivelyblockHCVreplicationin vitro116.Arecentstudy investigated theeffectsofcombiningNIM-811(host inhibitor) with specific inhibitors of viral protein using repliconsystem145. Interestingly,acombinationof specific viral inhibitors (viral protease or polymerase inhibitors)withNIM811preventedthedevelopmentofresistanceveryeffectively.CyclosporineAderivativessuch as Debio 025 that lack the immunosuppressivefunction,arecurrentlyinclinicaltrials146.

HCVhasdevelopedcomplexmechanismstousethevarietyandintricacyofthehostlipidome.Apromisingcandidateforanti-HCVtherapyemergedwiththerecentdevelopmentofstatins,adrugthatinterfereswithlipidmetabolism. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.Statinshavebeenapprovedclinicallyforthetreatmentofhypercholesterolaemia.Thesecausea reduction inintracellular mevulonate, LDL and geranylgeraniol,thereby inhibiting cholesterol biosynthesis and alsocausing a decrease in prenylated proteins147,148. ThisdrughasalsobeenusedtofurtherenhancetheantiviraleffectsofINF148.Aninterestingstudyutilizingrepliconsystemshowedthatstatins(mevastatinorsimvastatin)incombinationwithpolymeraseorproteaseinhibitorscouldeffectivelycleartheculturesfromHCVreplicons.Inaddition,mevastatinwasfoundtopreventselectionof replicons resistant to the non nucleoside inhibitor

HCV-796149.Thesecell culture studies suggest thatacombination of specific viral inhibitors with statins maydelayorpreventthedevelopmentofresistancetoviralinhibitors.However,thisremainstobetestedinclinicaltrials.

MicroRNAs(miR)are(~22nucleotide)naturallyoccurringnoncodingRNAs.Thesebelongtoafamilyof small RNA molecules that perform and regulatecritical cellular functions. MicroRNAs serve asgene regulators controlling the cell cycle and tissuedifferentiation150.Thesearealsoimplicatedinapoptosisandoncogenesis151.ThemiRNA(miR-122)isrequiredfor HCV replication and development of antiviraltherapies targeting on specific miRNA has great potential forexploration.However,abetterunderstandingoftherole formiR-122binding to theHCVis required foreffective design of antiviral therapies. Attempts areunderway utilizing RNAi technology and one suchattemptisthedesigningofTT-033,whichcontainsthreeseparateRNAimoleculestoshutdownreplicationofallstrainsofthehepatitisCvirus.Thisstrategysoundspromisingandcouldpotentiallystopthegenerationofviralescapemutants165.

Anotherpromisingareaforexploring therapeuticoptions are targeted at processing bodies (P bodies).ThesePbodiesareimportantformanyvirusesintheirlifecycles.Recentstudieshavefurtherhighlightedtheimportance of miRNAs and P bodies in modulatinghost cell interactions with viruses152. An interestingstudyestablishedtheroleofmiR-29aandPbodiesinregulatingHIV-1productionandinfectivity152.AlinkbetweenHCVandPbodieshasalsobeenobserved153.TheRNAhelicaseDDX3isacomponentofPbodiesandisrequiredforHCVRNAreplication154.HCVcoreproteinbindsandcolocalizeswithDDX3inPbodies155.The significance of core-DDX3 helicase interaction in HCVpathogenesisisanactiveareaofresearch.

There are also reports indicating progress withtherapeutic vaccines for HCV. IC41, a syntheticpeptide vaccine, containing HCVT-cell epitopes hasbeen recently shown to be safe and is currently inclinical trial. IC41 can induce HCV-specific INF-gamma-secreting CD4+ and CD8+ T cells in healthy individuals. It was shown to induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCVnon-responderpatients156.Thereisalsoevidencethat broadly neutralizing antibodies against antigenicregions of E2 can protect Alb-uPA/SCID mouse(human liver-chimeric), against HCV challenge157.


Another interesting candidate for a peptide vaccineis the HCV-specific HLA-A2-restricted NS3 (1073) epitope158.As with any RNA virus, rapidly mutatinggenomessuchasHCVposeahugeobstacletoantiviral/vaccinedevelopmentandrepresentsamajorchallengeto the field. In addition, HCV acquires a lipid envelope, whichoriginatesfromhostmembranes.Thus,thelipidcompositionoftheHCVenveloperesemblesthatofthehostcellmembraneandthisclevermimicrymayallowHCVtoavoiddetectionbytheimmunesystem159.

Conclusions

Recent advances in our understanding of HCVstructure, genome and its lifecycle have revealednumerous target sites for potential pharmacologicalintervention.TheseshouldhelpinfurtherimprovingHCV treatment. Efforts by investigators across theglobehaveledtodetailedmolecularcharacterizationof the virus, but despite these advances in ourunderstanding, many fundamental questions stillremain unanswered. For example, we are yet to find themechanismsbywhichHCVreplicates inhumanliver?Whatarethemolecularmechanismsbywhichviral proteins initiate hepatocarcinogenesis? Whatare the underlying defense mechanisms that play arole in thenatural resolutionofHCV infection in avery small percentage of people160. The membrane-associated RNA replication complex presumablyinvolvesvarioushostproteins.However, theprecisehost components and mechanisms for replication

are not entirely understood yet. Current efforts todevelop antiviral therapeutics are haunted by theuniquenatureoftheHCV,i.e.,theerror-pronenatureof NS5B, generation of resistance mutants whichpresentsaselectivegrowthadvantage.TocircumventthismajorhurdleinHCVtherapy,thecurrentresearchefforts are aimed at combination therapy targetingmultipleproteinsbothviralandhost.NumerousnovelHCV specific inhibitors such as nucleoside and non nucleosidepolymerase inhibitors,protease inhibitors,as well as non HCV specific compounds with antiviral activity such as nitazoxanide, cyclophilin inhibitors(Debio025),silibininareunderdevelopmentinclinicaltrials. An immunotherapeutic vaccine along withthe current therapy may also be beneficial especially in immunocompromised patients. In the meantime,combination therapyutilizingPEG-INFandribavirinwillremainstandardofcare.Arecentestimateprojectsthe current treatment market for HCV to be arounduS$3billionperyear.ThismarketisexpectedtogrowrapidlyandreacharounduS$8billionby2010161.

Acknowledgment Author thanks Dr Craig E. Cameron (Pennsylvania Stateuniversity)forencouragementandsupport.

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Reprint requests:DrSureshD.Sharma,ThePennsylvaniaStateuniversity,201AlthouseLaboratoryuniversityPark,PA16802,uSA e-mail:[email protected]


hepatitis c virus: molecular biology & current therapeutic options

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