Overview of HCV Life Cycle with a Special Focus on Current and Possible Future Antiviral Targets

18 Pages·2017·1.19 MB·English

viruses
Review
Overview of HCV Life Cycle with a Special Focus on
Current and Possible Future Antiviral Targets
NathalieAlazard-Dany,SolèneDenolly,BertrandBoson andFrançois-LoïcCosset*
CIRI—CentreInternationaldeRechercheenInfectiologie,UnivLyon,UniversitéClaudeBernardLyon1,Inserm,
U1111,CNRS,UMR5308,ENSLyon,F-69007Lyon,France;[email protected](N.A.-D.);
[email protected](S.D.);[email protected](B.B.)
* Correspondence:fl[email protected]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:27November2018;Accepted:2January2019;Published:6January2019
Abstract: HepatitisCinfectionistheleadingcauseofliverdiseasesworldwideandamajorhealth
concernthataffectsanestimated3%oftheglobalpopulation. Noveltherapiesavailablesince2014
and 2017 are very efficient and the WHO considers HCV eradication possible by the year 2030.
Thesetreatmentsarebasedontheso-calleddirectactingantivirals(DAAs)thathavebeendeveloped
throughresearcheffortsbyacademiaandindustrysincethe1990s. AfterabriefoverviewoftheHCV
lifecycle,wedescribeherethefunctionsofthedifferenttargetsofcurrentDAAs,themodeofaction
oftheseDAAsandpotentialfutureinhibitors.
Keywords: HCV;DAA;antiviraltargets
1. Introduction
HepatitisCVirus(HCV),theleadingcauseofchronicliverdiseaseworldwide,isapositive-sense
single-strandRNAvirusclassifiedintheFlaviviridaefamilyintheHepacivirusgenus[1]. Thefight
againstthediseasestartedevenbeforethisagentwasidentifiedin1989,withtheuseofinterferon(IFN)
since1986whichwasuntilrecentlythestandardcareofthetreatmentagainstHCVinfection[2,3].
Thetherapeuticalsuccessofthistreatment,aimedatstimulatinghostantiviralresponsestoeliminate
thevirus,wasassessedbymonitoringsustainedvirologicalresponses(SVR),asdefinedbyundetectable
HCV RNA levels in the blood 12 or 24 weeks after the end of treatment. The IFN treatment was
improvedin1998withtheadditionofribavirin,anon-specificantiviralagent,andin2001,byadding
polyethylene glycol to interferon molecules (PEG-IFN) [4–7]. The main problem with IFN-based
therapies is that SVR rates remain rather modest, especially for the most common HCV genotype
worldwide,andareaccompaniedbyconsiderableadverseeffects,makinglongtreatmentduration
hardtosupport.
Inthe2010s,thehealthauthoritiesapprovedasuccessionofnewmedicinescalleddirect-acting
antivirals(DAAs). ThesemoleculesopenedanewerainthetreatmentofHCV,achievinghigherrates
ofSVRformostviralgenotypes,withshortertreatmentdurationsandfewersideeffects. Astheir
namesuggests,DAAsdirectlytargetviralproteinsthatareessentialforvirusreplication. Afteran
outlook of the mains steps of the HCV life cycle, we will review the main targets of the marketed
DAAsandthosecurrentlyunderdevelopment. Theresultsofclinicaltrialsarenotaddressedhere,
butarereviewedelsewhere[8].
ThetwomainchallengeswhenusingDAAs,asexperiencedinthefightagainstHIV,aretotreat
allgenotypesandtofighttheappearanceofresistance.ItisparticularlytrueforHCV,forwhichgenetic
variabilityisillustratedbytheexistenceofsevengenotypesandmorethan80differentconfirmed
subtypes worldwide [1]. These genotypes and subtypes show different geographical distribution,
pathogenesis and response to treatments. Whereas the first DAAs were directed against a single
Viruses2019,11,30;doi:10.3390/v11010030 www.mdpi.com/journal/viruses
Viruses2019,11,30 2of18
genotype,thenewgenerationofDAAstargetagreatervarietyofgenotypes. PangenotypicDAAswill
beparticularlyinterestinginlowandmiddle-incomecountriesastheywillallowtreatmentofHCV
patientswithoutpriorgenotypetesting. Extensionoftargetsoutsidethehepacivirusisalsoenvisioned
bysomeresearcherstryingtodevelopantiviralsactiveagainstdifferentFlaviviridae[9].
HCVshighgeneticvariabilityisalsoaproblematthelevelofindividuals. Becauseofthehigh
replicationrateandthelackofproofreadingactivityoftheHCVRNA-dependentRNA-polymerase
(vRdRp),HCVexistswithinitshostasapopulationofslightlydifferentviralvariants,formingthe
“quasispecies”[10]. Someofthemutationsinduceaminoacidchangesthatreducethesusceptibility
to one or more antiviral drugs and are therefore called resistance-associated substitutions (RASs).
VirusesharboringoneormoreRASarecalledresistance-associatedvariants(RAVs)andarefrequently
associatedwithDAAstreatmentfailureiftheirfitnessissufficient[11]. RAVscandevelopduring
treatment or may pre-exist as naturally occurring variants, albeit at low but sometimes clinically
relevantlevels,asreviewedin[12]. Inbothcases,RAVsselectedduringtreatmentandpre-existing
RAVscontributetothefailureoftreatments. Thenumberofmutationsnecessaryforavirustobecome
resistantandtheprobabilitythatthesemutationsareselectedinthepresenceofthedrugiscalled
thegeneticbarrier[13]. Inadditiontobeingpangenotypic,newantiviralsarethereforedeveloped
withtheaimofhavinghighgeneticbarrierstoresistance. Theuseofacombinationofantiviralswith
differenttargets,eachofthemwithhighpotencyandhighgeneticbarrier,nowallowsahighsuccess
ofIFN-freeoralregimensHCVtreatment.
2. OverviewoftheHCVLifeCycle
2.1. EntryofHCVParticleintoHepatocytes
HCV particles are 50–80 nm in diameter and have the particularity of being associated with
neutral lipids (cholesterol ester and triglycerides) and apolipoproteins, which confers them their
unusuallylowbuoyantdensity(Figure1a)[14,15]. HCVparticlescontainapositivesingle-strand
RNAgenomeincloseassociationwiththecoreproteins,envelopedbyalipidmembraneinwhichthe
twoviralglycoproteinsE1andE2areanchored. Associationofparticleswithlipidstendstomask
theviralglycoproteinsbutarethoughttoplayaroleinvirusentry[16],atleastintheinitialphaseof
cellattachment,duringwhichtheinteractionsofapolipoproteinEwithcellsurfaceheparansulfate
proteoglycans[16–18]orwithSR-BIhavebeenimplicated[19](Figure1b).
HCV E1 and E2 envelope proteins play a major role in virus entry and therefore in virus
tropism to hepatocytes. Overcoming the initial difficulty to obtain HCV viral particles, HCV
entry was first characterized with the use of HCV pseudo-particles (HCVpp) harboring E1 and
E2 glycoproteins [20,21]. E1 and E2 act as complexes of disulfide-bound heterodimers and E2
is clearly identified to be responsible for receptor binding [22,23], whereas E1 seems to be an
important component of an atypical fusion machinery, as reviewed recently in [24]. HCV entry
is a complex multistep process in which different cellular proteins have been demonstrated to be
involved. A cascade of interactions/co-interactions with the Scavenger Receptor class B type I
(SR-BI) [23], the human Cluster of Differentiation 81 (CD81) [22] and the tight junction proteins
claudin-1(CLDN1)[25]andOccludin(OCLN)[26]hasbeendescribed. Thesefactorsconstitutethe
minimalsetofessentialentryreceptorsthatprimetheE1-E2complexforvirusentry[27]. Thisprocess
isregulatedbymultiplehostsignalingpathways, inwhich, forexample, epidermalgrowthfactor
receptor (EGF-R) is implicated [28]. Additional accessory entry co-factors and/or regulators are
constantlydescribed,asrecentlyreviewedin[29].
Followingattachment,HCVparticlesareinternalizedviatheendocyticpathwayandmembrane
fusionisdependentonendosomalacidification(Figure1b)[30]. Intheorganism,cell-to-cellspread
couldbeanimportantmechanismthatseemstoinvolvesimilaractors[31,32].
Viruses2019,11,30 3of18
Viruses 2019, 11, x FOR PEER REVIEW 3 of 18

Figure1. HCVlipoviroparticlesandtheviruslifecycle. (a)HCVparticlescontainapositivestrand
FRigNuAreg 1e.n HomCVe (liinpogvreireonp)aarstiscolecisa atendd wthiteh vCirourse lpifreo cteyicnles,. e(an)v HelCopVe dpabryticalems ecmonbtraainn eai npowsihtiivche sEt1raanndd
REN2Agl ygceonpormoete (iinns garreeeenm) basesdodceiadteadn dwaitrhe tCigohretl ypraostseoincisa, teendvweliothpelidp ibdys aa nmdeampborlaipnoep irno tweihnisc.h( bE)1 HanCdV
Eli2f eglcyyccolpe.roTtheiends iaffreer eemntbsetdedpesdo fanHdC aVrel itfieghcytlcyl easasroeciinatdeidca wteidthi nlipbildasc ka.ndE RapEonlidpoopplraostmeiincsR. (ebti)c HulCuVm .
liMfeW cyMcleem. Tbhrae ndoiuffserWenebt .sLteDpsL oipf idHCDVro plilfeet sc.yTclhee anreeg iantdiviecastterda nidn rbelpalcikc.a tEioRn Einntdeormpleadsmiaitce Rinertiecdu.lum.
MW Membranous Web. LD Lipid Droplets. The negative strand replication intermediate in red.
2.2. HCVRNATranslationandReplication
2.2. HCV RNA Translation and Replication
TheincomingviralRNA,approximately9.6kbinlength,containsasingleopen-readingframe
flankTehde binychoimghinlyg svtriruaclt uRrNedAn, aopnp-trroaxnismlaatteedlyr e9g.6io knbs i(nN lTeRnsg)th(F, icgounrtea2inas) [a2 s].inItglise torpanensl-arteeaddiansga fsrianmglee
fplaonlykpedro bteyi nhitghhaltyis sctrou-catnudrepdo nsto-ntr-atnrasnlastliaotneadl lryegpirooncse s(sNedTRbsy) b(Foitghuvrier a2laa)n [d2]h. Iots itsp trroatnesalsaetes,da assi nad siicnagteled
pinolyFpigruorteein2 at,hraati siisn gcote- nanpdro tpeoinsts-.trTahneslaCtoiorne,alEly1 apnrdocEes2sesdtr ubcytu braolthp rvoitreainl sa,npdr ehseonstt ipnrothteeasveirsi,o anss ,
ianrdeicpartoeddu icne dFifgroumre t2hae, Nra-itseirnmg itneanl epnrodtewinhse.r eTahses eCvoerne,n Eo1n satnrudc Etu2r asltr(uNcStu)rparlo pteriontseianrse, epxrperseesnste dinf rtohme
vtihreioCn-st,e ramrei npursoodfutcheedp forloympr ottheei nN. -terminal end whereas seven nonstructural (NS) proteins are
expreTssheed NfrToRms tchoen Cta-tinermthienmusa oinf tchies- pacotliynpgroetleeimn.e nts that are essential for genome translation and
repliTchatei oNnT,Rins ccluondtianign athne inmtaeirnn aclisr-iabcotisnogm eallemenetnrtys sthitaet (aIRreE eSs)seinnttihale f5o(cid:48)r NgeTnRom(Fei gtruarnesl2aat)io[n3 3a,n34d] .
rAepnliacbatuionnd,a inntclliuvdeirnsgp aenci fiinctemrniRaNl rAib,omsoimR-a1l2 e2n,tirnyt esriatect i(nIRgEwS)i tihn tthhees e5′s NeqTuRe n(cFeigsuwrea s2ad)e m[33o,n3s4t]r. aAtend
atbournedgaunlat telivHeCr VspRecNifAic lemvieRlsNiAn,c melliRcu-1l2tu2r, ei,nstteirmacutliantgin wgittrha ntshleasteio sneaqnudengceens owmaes rdepemlicoantisotrna[te3d5, 3t6o] .
rTehguislamtei cHroCRVN RANaAct lsevinelasn inu cneclol ncuvletnutrieo, nsatilmwualyatibnygr tercarnusiltaitnigonA arngdo ngaeuntoem2et roetphlieca5t(cid:48)ioennd [3o5f,3t6h]e. Tvhiriasl
mgeicnroomRNe,Ast aacbtisl iizni nagn aunndcopnrvoetnectitoinngali twfaroym byd reegcrrauditaitnigo nAbrgyo5n(cid:48)aeuxtoen 2u tcol ethasee 5s′ [e3n7d]. oAf sthiet ivsireasls genentioamlfeo,r
sHtaCbiVlizrienpgli caantdio np,roittewcatisnigd eitn ftrifioemd dasegarpadoatetinotnia lbayn 5ti′v eirxaolntuacrgleeats.eTsh [e37v]i.r aAlsR Nit Ais iessaslesnotitahle ftoerm HpClaVte
rfeoprlivciartailorne,p itli wcaatsio indethnatitfiperdo adsu ac epsopteonstitiaivl eangteinvoirmale tsaurgseetd. Tfohrea vdirdailt iRoNnaAl pisr oatlesion thpero tdemucptiloatne afnord vviirraall
raespsleicmabtiloynt htrhoautg hpraondeugcaetsi vpeo-sseintisvee ingteenrmomedeisa tues(eFdig uforer 1abd).dNitiSo5nBali spthroetveiinra lpRrNodAu-cdteiopne nadnedn tvRiNraAl
apsoselymmbelyra tshero(vuRghd Ra pn)e,gtahteivkee-syenesnez iynmteermofedRiNatAe (Fsyignuthree s1ibs).. NWSh5eBn iss uthbeg evniroaml RicNrAep-dliecpoennsdwenetr eRNfirAst
peosltyambleisrhaesed ((vFRigduRrpe)2, bth),et hkeeym einnizmymalet roafn RslNatAio nsyanntdherseips.l iWcahtieonn smuabcgheinnoemryicw raespldiceomnos nwsterraet efdirstto
ersetqaubliirsehtehde (NFiTgRusrea n2bd),N tSh3e/ m4Ain,iNmSa4l Btr,aNnSsl5aAtioinn aadnddi trieopnlitcoatNioSn5 Bm[a3c8h]i.nAersy dwesacsr idbeemdobneslotrwat,etdh etsoe
rreeqpuliicreo ntshew NerTeRinsv aanluda bNleS3to/4oAls, fNorSe4aBr,l yNaSn5tAi- HinC Vadddriutigonsc troe eNniSn5gBa [n3d8]d. iAscso vdeersyc.ribed below, these
replicTohnes wfuenrcet iionnvaolfutahbelev tioraollsp froort eeianrslyta arngteit-eHdCbVy dDrAugA sscwreielnlibnegb arniedfl dyidsceosvcreirbye. dhereandinmore
detaTilhsei nfutnhcetifoonll oowf tihneg vsieractli ponrost.eiNnsS 2tairsgeatevdir ably pDroAtAeass ewtilhl abtec borniterfilbyu dteessctroibtehde hveirrea lapnodl yinp rmotoerien
dmetaatiulsr aitnio tnhea sfowlleolwl ainsgt osetchteioansss.e NmSb2ly iso fa vviirraall pparrotticelaesse. tNhaSt3 ciosnbtroitbhuatesv itroa lthpero vteiraasle pinolvyoplvroetdeiinn
mpraotuteroaltyiosnis aosf twheeldl oaws ntos ttrheea maspseamrtbolfyt hoef pvoirlaylp proatretiicnleasn. dNaSh3 eilsic baoseth. NaS v4iArails parcootefaacsteo irnivnotelvraecdt iinng
pwroittheoNlySs3is aonfd thaen dchoworninstgreiatmto ptahret omf etmheb praonlyep.rpot7eiwn aasndcl aas hsiefiliecdasaes. NaSv4iAro piso ar icnofaancdtohr ainstemrauclttiinpgle
with NS3 and anchoring it to the membrane. p7 was classified as a viroporin and has multiple roles
in the assembly and secretion of virions [39]. As illustrated in Figure 2c, most of the viral proteins are
Viruses2019,11,30 4of18
Viruses 2019, 11, x FOR PEER REVIEW 4 of 18
roles in the assembly and secretion of virions [39]. As illustrated in Figure 2c, most of the viral
mpreomtebirnasnae-raesmsoecmiatberda naen-ads shocaivaet edsevanerdalh afuvencsteiovnesra llifnuknecdt ioton stlhine kreedortgoatnhiezarteioonrg aonf iztahteio nceollfultahre
mceelmlublarranmees mrebqruainreesdr efoqru itrheed rfeoprlitchaetiroenp loicfa tthioen HoCftVh egHenCoVmeg eonro lmateero,r dlautreirn,gd uvririnalg avsisreamlabslsye. mTbhley .
mThasesmivea srseiavrerarenagrermanegnet mofe cnetloluflcaer lmluelamrbmraenmebs raafnteers HafCteVr iHnfCeVctiionnfe ccotinosnisctso nosf ias t“smoefmab“mraenmoubsr awneobu”s
mwaeibn”lym caoinnslytitcuotnedst ietuartelyd iena rilnyfeinctiionnfe cbtyio ndobuybldeo-mubelme-bmraenmeb vraensieclveess i(cDleMsV(D) MorVig)inoaritgininga tfirnogmf rtohme
etnhdeoepnldasompliac srmetiiccurelutimcu l(uEmR) ((EFRig)u(Freig 1ubr)e [14b0)]. [4M0o].stM oof stthoe fptrhoetepinrost eoifn sthoef rtehpelirceaptiloicna tcioomnpcolemx palreex
naerceensseacreys sfaorry tfhoirs tphrisocpersosc, ebssu,t bNutS4NBS 4aBnda nNdSN5AS5 aArea rteheth memajoarjo arcatoctrosr sini nththe e““mmeemmbbrarannoouus swweebb””
bbiiooggeenneessisis aanndd mmaaiinntteennaannccee tthhaatt ccoonnttaaiinn tthhee rreepplliiccaattiioonn ffaaccttoorriieess ooff tthhee vviirruuss [[4411]].. SSeevveerraall cceelllluullaarr
ffaaccttoorrss aarree aallssoo rreeccrruuiitteedd bbyy tthhee vviirraall pprrootteeiinnss ttoo aallllooww tthhiiss pprroocceessss..

Figure2.HCVvirusandrepliconorganizationandmembraneorganizationoftheviralproteins.(a)The
Figure 2. HCV virus and replicon organization and membrane organization of the viral proteins. (a)
HCVgenomeisapositivestrandRNAcontainingasingleopen-readingframe(fromAUGtostop)
The HCV genome is a positive strand RNA containing a single open-reading frame (from AUG to
surroundedby5(cid:48)and3(cid:48)highlystructurednon-translatedregions(NTRs).Translationofthepolyprotein
stop) surrounded by 5′ and 3′ highly structured non-translated regions (NTRs). Translation of the
isenhancedbymiR-122bindinginthe5(cid:48)NTRasindicated. Translationisinitiatedattheinternal
polyprotein is enhanced by miR-122 binding in the 5′NTR as indicated. Translation is initiated at the
ribosomalentrysite(IRES).Thepolyproteiniscleavedbycellular(whitearrowheads)orviral(black
internal ribosomal entry site (IRES). The polyprotein is cleaved by cellular (white arrowheads) or viral
arrowhead)proteases.C:Coreprotein.AllNon-Structural(NS)proteins(includingp7)areinblack.
(black arrowhead) proteases. C: Core protein. All Non-Structural (NS) proteins (including p7) are in
(b)ExampleoforganizationofaHCVsubgenomicrepliconthatwereextensivelyusedforantiviraldrug
black. (b) Example of organization of a HCV subgenomic replicon that were extensively used for
screenings,theycontainalltheproteinsnecessaryandsufficientforHCVRNAreplication.Neomycin:
antiviral drug screenings, they contain all the proteins necessary and sufficient for HCV RNA
Neomycinresistancegene. ECMV:EncephalomyocarditisVirus. (c)MembraneassociationofHCV
replication. Neomycin: Neomycin resistance gene. ECMV: Encephalomyocarditis Virus. (c)
proteins.Hatchedbarsrepresentamphipathicalphahelixortransmembranedomainsanchoringthe
Membrane association of HCV proteins. Hatched bars represent amphipathic alpha helix or
proteinstothemembrane.TwoexamplesofessentialcellularproteinsinteractingwithNS5Aareshown.
transmembrane domains anchoring the proteins to the membrane. Two examples of essential cellular
Thethreemaintypesofinhibitorsusedintheclinic(indicatedinredbytheirsuffix)areindicatednext
proteins interacting with NS5A are shown. The three main types of inhibitors used in the clinic
totheirtarget.
(indicated in red by their suffix) are indicated next to their target.
Describing the huge number of host factors that have been implicated in HCV replication is
Describing the huge number of host factors that have been implicated in HCV replication is
beyond the scope of this review but can be found elsewhere [42]. However, we will mention
beyond the scope of this review but can be found elsewhere [42]. However, we will mention a few
a few targets of particular importance in the context of antiviral research. Cyclophilins, cellular
targets of particular importance in the context of antiviral research. Cyclophilins, cellular peptidyl-
propyl cis-trans isomerase, were, for example, described as important co-factors for HCV and other
viruses replication, as shown using cyclophilin inhibitors developed against HIV [43]. Cyclophilin A
Viruses2019,11,30 5of18
peptidyl-propylcis-transisomerase,were,forexample,describedasimportantco-factorsforHCV
and other viruses replication, as shown using cyclophilin inhibitors developed against HIV [43].
Cyclophilin A interacts with NS5A and is essential for the “membranous web” formation [44,45].
Phosphatidyinositol-4-phosphate-kinase-IIIalphaisanothercentralhostfactorinteractingwithNS5A,
inducingaccumulationofPI4Pwithinthemembranousweb[46], andprovidesanexampleofthe
compleximbricationbetweentheHCVlifecycleandlipidmetabolism[17].
2.3. HCVAssembly,BuddingandSecretion
Since2005,thedevelopmentofHCVmolecularclonesthatrecapitulateallthestepsoftheviruslife
cycleallowedmuchprogressintheunderstandingofHCVassembly,buddingandsecretion[47–49].
Viralassemblyis,however,stillnotfullydescribed,asthemodelsdonotreflectalltheinteractions
withliverlipidmetabolismandgenerateparticlesthatpoorlyresemblethoseproducedininfected
individuals[16,47,50].
Viral assembly occurs near replication complexes at assembly sites associated to ER-derived
membranes,incloseproximitytolipiddropletswherecoreproteinscanaccumulate(Figure1b)[51,52].
NS5Ahasbeenidentifiedasakeyplayerinthetransitionbetweenviralreplicationandassembly,in
additiontoitsroleinreplication,andwasdemonstratedtobeinvolvedinRNAgenomedeliveryto
theCoreproteins[53,54]. NS2andp7alsoplayacentralroleinvirusassemblythroughtheirdifferent
interactionswithstructuralandnon-structuralproteins. Mostnotably,theycoordinatetherecruitment
ofCoreandtheenvelopeglycoproteinsE1E2aswellasNS3andNS5Atotheassemblysites,allowing
thegatheringofallviralactorsforassemblycorrectlyinthesameplace[55–60]. Thesmallp7viroporin
wasdemonstratedtoactinconcertwithNS2inthisprocessaswellasbeingimportantatthelaterstage
ofviralenvelopment[58,59,61]. NS4BwasalsodemonstratedtoplayaroleinHCVassembly[62].
Besidesviralproteins,severalcellularproteinshavebeendescribedasmainfactorsforHCVassembly,
suchasDGAT1[63,64]andESCRTproteins[65–68].
ViralparticlesacquiretheirenvelopebybuddingattheER.Asmentionedabove,lipidcomposition
oftheviralparticlesresemblesthatofVLDLsandHCVvirionformation,andreleasemayhighjack
theirassemblypathway[69]. Maturationofthelipoviroparticlesmayalsorequireanunconventional
passagethroughtheGolgiapparatusandatrans-endosomalsecretoryroute(reviewedin[52]),butalso
occursatapost-egressstep[16,70]. p7,ApoEandnumeroushostproteinsknowntobeinvolvedin
thesespathwaysarenecessaryforthisprocess.
TheshortoverviewofHCVlifecyclepresentedhereprovidesastrikingexampleofthefactthat
viralproteinsaremulti-purposeandexertseveralfunctionsintheviralreplicationcycle,thusreflecting
genetic economy. In an attempt to fight HCV infection, most of these proteins therefore stand as
potentialantiviraltargets,astheyareindispensableforoneormorestepsoftheviralcycle. Before
discussingthepossibilitytotargetviralentryorassembly,wewillreviewthethreemaintargetsof
DAAtreatments,whicharemainlyinvolvedinvirusreplication: NS3/4A,NS5BandNS5A.
3. CurrentAntiviralTargets
3.1. NS3/4AInhibitors
TheexperienceofantiproteasedevelopmentintheHIVfieldfacilitatedtheearlydevelopment
ofdrugsinhibitingNS3,aswellasthefactthatinvitromodelswerealreadyavailableandcomplete
replicationsystemswerenotnecessaryforpreliminaryscreenings. Theproteasedomainoftheprotein
was targeted by the first HCV antivirals that were introduced on the market and by the currently
recommendednewDAAs. Thesemoleculesaresuffixed-previrandareoftencollectivelyreferredtoas
proteasesinhibitors(PIs). NS3proteaseactivityplaysacentralroleintheviralpolyproteinmaturation,
as it cleaves four peptide junctions between the non-structural proteins, in complex with NS4A
(Figure2)[71–73]. Theproteaseactivityisalsoimplicatedinthemodulationoftheantiviralinnate
immuneresponse[74]. Thestructureoftheproteasedomainrevealedatypicalchymotrypsin-like
Viruses2019,11,30 6of18
fold,trypsin-likeproteinasewithtwoβ-barrelsandahighlyconservedcatalytictriad(His-57,Asp-81,
Ser-139),andabindingsiteforthepeptidicNS4Acofactorthatisdistantfromthecatalyticsite[75,76].
NS4AisanessentialcofactorforNS3enzymaticactivitiesthathelpstolocalizeNS3tothemembrane
andanchorstheHCVreplicationcomplexatthecellularmembrane[77–79].
Inadditiontoitstypicalserineproteasedomain,NS3containsashortN-terminalamphipathic
α helix (designated Helix α ) and a C-terminal helicase/NTPase domain, which is implicated in
0
several critical steps of the virus life cycle [75,80]. Helix α plays an important role in membrane
0
association,subcellularlocalizationandstabilityoftheNS3/NS4Acomplex,andparticipatesinHCV
virionmorphogenesis[77,81,82].Thehelicase/NTPAsedomainwasdemonstratedtobindandunwind
RNAduplexesduringviralgenomereplication,makingNS3/4Acomplexanimportantcomponentof
theviralreplicationcomplex,alsoimplicatedinvirusparticleassembly[78,82,83]. Drugstargeting
functionsotherthantheprotease,forexamplehelicaseactivity,havealreadybeenenvisionedand
couldbedevelopedinthefuture[84]. AsNS3andNS4A,butalsothedifferentNS3domains,donot
actindependently,theinhibitorscanaffectseveralfunctionsofthecomplex,allowinginhibitionof
multiplestepsbytargetingasinglemolecule[85–87].
PioneerworksoninhibitionofNS3/4aproteaseactivityidentifiedcleavagesubstrateproductsas
potentialPIs[88,89]. BoceprevirandTelaprevir,thefirsttwoDAAsthatbenefitedfromamarketing
approvalin2011,werebothpeptidomimeticlinearketoamidesthatbindtheactivesiteoftheprotease
domainofNS3[90,91]. BothDAAswereusedincombinationwithPEG-IFNandribavirin,andwere
soon discontinued in most countries due to their low genetic barriers to resistance, restriction to
genotype 1 virus and severe adverse side effects [92]. Further development of PIs resulted in
improvedantiviralpotencyandapprovalofthemainPIsrecommendedtodateindifferentcountries,
thePIsbeing: Glecaprevir,Paritaprevir,GrazoprevirandVoxilaprevir[93,94]. Theavailablecrystal
structuresofNS3incomplexwithcandidatecompoundsfacilitatedstructure-baseddesignthathas
beenextensivelyusedtoconfronttheissuesofpotency,resistanceandpharmacokinetics,leadingto
thedevelopmentofmanynewmolecules,asreviewedin[8]. Intermsofresistance,preferentiallociof
RASswereidentifiedwiththefirstgenerationofPIsandweretakenintoaccountfortheoptimization
ofthenextDAAgeneration[95,96]. Theefficiencyofthesemolecules,especiallyincombinationwith
DAAstargetingotherviralproteins,allowedthedevelopmentandapprovaloflongawaitedIFN-free
regimentreatmentswithparticularlyhighSVRandlowsideeffects[97].
3.2. NS5BInhibitors
The function of the NS5B protein at the 3(cid:48)-end of the genome was evident as soon as the
sequence of the HCV genome became available, on the basis of sequence comparison with other
viruses[98]. ThebiochemicalactivityoftheNS5Bproteinasareplicasewasconfirmedsoonafter[99]
as well as the secondary structure of its catalytic site, which was found to be typical of a vRdRp:
aright-handlikestructure,withfingers,palmandthumbsubdomains[100–102]. TheNS5Bprotein,
however,showssomeparticularities: abeta-hairpinloopinsertioninthethumbdomainthoughtto
modulate the polymerase activity and an hydrophobic C-terminal domain, anchoring the protein
tomembranes[103]. ThevRdRpwasefficientlyproducedindifferentsystemswithoutthisanchor,
allowingfurthercharacterizationoftheproteinproperties[104,105]. Playingsuchanimportantrole
in the viral life cycle and given the wealth of information available for related proteins, the NS5B
polymerasewasidentifiedveryearlyasatargetofchoiceforantiviraldevelopmentandiscertainly
oneofthebestcharacterizedHCVenzyme,asreviewedin[106]. Twotypesofinhibitorsthattarget
thecatalyticornon-catalyticsitesofthisproteinhavebeendeveloped.
Basedonknowledgeaboutcellularandviralpolymerases,thefirstinhibitorsdevelopedagainst
NS5B were nucleo(s)tides inhibitors (NIs). These inhibitors mimic the natural substrate of the
enzyme—theyaretypicallyincorporatedintothenascentRNAchainsandterminateRNAsynthesis.
Theirdevelopmentis,however,complicatedbytheirinstabilityandtendencytobeinactivatedby
thecellularmetabolism. Sofosbuvirisanuridineanalogue,thefirstNItobeapprovedbytheFDA
Viruses2019,11,30 7of18
in2013,andstillpartofmostoftherecommendedtreatmentcombinations[107]. Therealchallenge
duringthedevelopmentofSofosbuvirwastounderstandthemetabolismofthecandidateinhibitors
byliverenzymestofindtheoptimalformulationforaprodrug[108]. Shorttreatmentdurationsof
HCVtreatmentsshouldpreventtheside-effects,duetotheknowntendencyofNIstohavecumulative
toxicity[109]. Amajorconcernduringantiviraldrugdevelopmentistheappearanceofresistance,
particularlyinviruseslikeHCV,whosevRdRpwasdemonstratedtobeerror-prone. Thecatalytic
siteofthevRdRpis,however,highlyconservedbetweengenotypes,suggestingthepangenotypic
potentialofsuchadrugandaminimalriskoftheemergenceofviralresistance.Thesehypotheseswere
confirmedbytheanalysisofclinicaldatademonstratingtheextremelyrareappearanceofresistance
mutationsagainstNIs,whichusuallyinduceanimportantreductioninviralfitness[110,111]. Withits
favorablesafetyandtolerabilityprofile,itshighgeneticbarriertoresistanceanditsactivityagainst
mostgenotypes,SofosbuvirisanantiviralofchoiceinmostcurrentHCVtreatmentcombinations.
The second class of inhibitors, referred to as non-nucleoside inhibitors (NNIs), do not bind
to the catalytic site of the enzyme [106]. They were discovered as inhibitors of the polymerase
activityinscreensusingtherepliconsystem,whenresistancemutationswerediscoveredinNS5B
outside the catalytic site. These inhibitors exert their effect by inhibiting conformational changes
requiredforpolymeraseactivity. Withthedrugstestedtodate,fivedifferentbindingsiteshavebeen
discovered—twointhepalm,twointhethumbandoneinthebeta-hairpinchainofNS5B.Dasabuvir,
theonlyNNIapprovedtodatebytheFDA,targetsthepalmofNS5Bandisrecommendedforgenotype
1treatmentonly[112]. NextgenerationNNIsarecurrentlybeingtestedandcharacterized. Themode
ofactionofTegobuvir, apromisingmemberoftheimidazopyridineclassidentifiedinphenotypic
screens,was,forexample,showntocovalentlybindtheNS5Bbeta-hairpinafteractivationbycellular
enzymes[113]. NNIstargetpoorlyconservedsequencesandthereforeshowlowerbarriertoresistance
thanNIsandpoorpangenotypicactivity,evenifthesedefaultsareattenuatedintheupcomingnext
generationNNIs. Theyare,however,alreadyusefulwhenusedincombinationwithothermorepotent
drugsinsomeclinicalpresentations,toreinforcetheefficacyofthetreatmentandcontributetoreduce
treatmentduration.
3.3. NS5AInhibitors
NS5Ahaslongbeenconsideredasapotentialantiviraltarget, sinceitwasknownearlytobe
essential for viral RNA replication [114] and later also for viral assembly. The protein comprises
threedomainsseparatedbyshortlow-complexitysequences[115]. TheN-terminaldomainIisthe
bestorganizedandcharacterized. ItwasdemonstratedtobindZinc, tobewell-structured, witha
shortamphipathichelixintheveryNterminusnecessaryformembranetargetingandtobeessential
for genome replication [76,114,115]. The two C-terminal domains are, in contrast, predicted to be
intrinsicallydisordered. NS5Aisknowntoexistindifferentlyphosphorylatedformsandtointeract
withahugenumberofviralandcellularproteins,includingitselftoformdimersandmaybeoligomers.
Incontrasttotheabove-mentionedinhibitors,NS5Ainhibitorscouldnotbedevelopedusing
biochemicalassays,astheproteinhasnoknownintrinsicenzymaticactivity. Interestingmolecules
wereidentifiedduringthescreeningoflibrariesofcompoundsbydifferentcompanies. Daclatasvir
(DCV)was,forexample,identifiedbyscreeningoveramillionofcompoundsagainstHCVreplicons,
eliminatingfromthesortingboththecompoundsactiveagainstotherviruses—astheywouldnotbe
specificenough—andthoseinhibitingtheenzymaticactivityofNS3orNS5B[116]. Thedevelopment
ofresistancetothebestcandidatewasdemonstratedtobeassociatedwithspecificmutationsinthe
domainIofNS5A,therebyidentifyingNS5Aasthetargetoftheinhibitor. Inapreliminaryclinical
trial,asingledoseofDCVresultedinarapidvirologicalresponse,demonstratingthatNS5Awasa
promisingtargetforanti-HCVtherapy[116]. DCVwasshowntobindNS5AclosetotheN-terminus
ofdomain1,atthejunctionwiththeamphipathichelixthatanchorsNS5Aatthemembranes,without
perturbing the dimerization nor the stability of the protein [117–120]. It was further shown that
DCVandDCV-relatedmoleculesinhibittheformationofnewDMVsthatcontainsthereplication
Viruses2019,11,30 8of18
complexes[45,118]. OthercompoundssuchasLedipasvirandOmbitasvirhavesimilarpropertiesand
thesamebindingsitetoNS5A[121,122]. Secondgenerationinhibitorsweretestedand/oroptimized
tobepangenotypic,activeagainstpreviouslyidentifiedNS5ARAVsandcommonNS5Apolymorphs,
andhavereducedtoxicity,asexemplifiedbyElbasvirandVelpatasvir[123,124].
Better understanding of NS5A functions is now coming from studies using the inhibitors,
asreviewedin[125,126]. Asscreeningofthecompoundshasbeenmadeonreplicons, thestudies
initially focused on their impact on virus replication and cellular partners. As mentioned earlier,
NS5Awasshowntoplayanimportantroleinthemembranouswebformationnecessaryforviral
replication[41]. Testsoncell-infectiousclonesdemonstratedthatNS5Ahasmultiplecomplementary
activitiesintheviruslifecycle,suchasviralassembly[127]andmoreparticularlyonthetransferofthe
genometotheassemblysites[54]. DirectbindingofNS5AinhibitorstoNS5Ahasbeenreported,anda
RASwasshowedtoactbydecreasingthebindingofDCV[120]andLedipasvir[121]toNS5A.DCVwas
firstdescribedasinhibitingHCVreplicationbyprobablyinhibitingtheformationofdouble-membrane
vesicles required for the replication of the viral genome [118]. In addition, some studies reported
an action on assembly [127,128] by preventing the delivery of RNA to the core protein, the main
componentoftheHCVnucleocapsid[54]. Descriptionoftheexactmechanismsoftheinhibitorsare
stillongoing,butinhibitionofNS5Aseemstoactonmultiplestepsofthevirallifecycle,augmenting
itseffects.
4. PotentialFutureTargets
4.1. TargetingOtherViralProteins
Asmentionedintheintroduction,allviralproteinsessentialforviralreplicationareconsideredas
potentialantiviraltargets. Themainstrategytocombatresistanceemergenceistocombineinhibitors
thattargetdifferentproteinsinordertolimitthepossibilitiesofappearanceofcross-resistantmutations.
Thishasledtoresearchonalternativeantiviraltargetsinadditiontotheabove-describedprotease,
polymeraseandNS5A.
Thesmallp7non-structuralproteinisperhapsthemostattractiveofthepotentialalternative
antiviral targets. In early studies using HCV replicons, p7 was found to be dispensable for viral
replication [38]. But it soon became evident that p7 was essential for other steps of the virus-life
cycle [129]. Studies of the functions of p7 became possible when the HCV cellular clones became
available[130,131]andtheproteinwasdemonstratedtoplayamajorroleintheassemblyandrelease
ofinfectiousHCVparticles[44]. Indeed, p7isamultifunctionalproteinthatregulatesthecellular
secretorypathwayaswellastheinteractionsbetweenviralproteins,andthelocalizationofNS2and
coreproteinstowardscapsidenvelopment[55,56,58–61,132].
p7isalsoabletoformhomo-oligomersthatassembletoformionchannelstructuresandwas
therefore classified as a viroporin [44]. The precise role of this function, however, remains poorly
definedbutmaybeimportantfortheregulationofthesecretionofviralparticles[131,133]. Viroporins
areconsideredasanattractiveclassofantiviraltargets,withanincreasingnumberofsuchproteins
describedandcharacterized,andthesuccessofusinghostionchannelinhibitorsinothermedical
domains[134]. Inaddition,thesameinhibitorscanactonviroporinsofdifferentvirusesandmaylead
tothefindingofabroad-spectrumantiviral. TheM2viroporinofinfluenzaAvirusisthebeststudied
exampleofthisclassofmolecule[135]. M2wasalsothetargetoftheadamantaneclassofinhibitors
thathavebeenextensivelyusedagainstInfluenzaAinfectionsincethe1960s,eventhoughtheircurrent
useisnowlimitedduetotheappearanceofwidespreadresistance. Althoughp7isquitedifferent
fromM2,itwasshowntobeinhibitedbythissameclassofinhibitors[136],eventhoughclinicaltrials
incombinationwithIFNtreatmentshowednobeneficialeffects[137]. Otherp7inhibitorshavebeen
describedordesignedlike,forexample,GSK-2,hexamethylamiloride(HMA)orBIT225[138–141].
BIT225wasofparticularinterestsinceitisabletoblockbothp7andVpufromHIV,whichmightbea
Viruses2019,11,30 9of18
waytotreatHCV/HIVco-infectedpatients. Alltheseinhibitorshavedifferentefficienciesdepending
onthegenotypeofHCV[136]andapangenotypicp7inhibitorisstillnotavailable.
The last two nonstructural proteins, NS2 and NS4B, have also been targeted in preliminary
studies[142–144]. Despitetheirpotentialutilityandcomplementaritytothecurrenttreatments,these
moleculesmaybenotdevelopedfurtherforthemomentasthecurrentlyexistingtreatmentsarejudged
bymanyassufficient.
4.2. Host-TargetingAgentsagainstViralReplicationandEntry
The appearance of resistance to antivirals in HCV, a highly mutable genome, could limit the
futuresuccessofalltheabove-mentionedDAAs. Increasingthenumberofviraltargetstofightthe
appearanceofresistancesisthemainoptiontodate,buttargetingofsomehostfactorsthathavebeen
describedascrucialforviralreplicationorentryhaslongbeenenvisioned. Themainadvantagesof
theseso-calledhost-targetingantivirals(HTA)overDAAsareahighergeneticbarriertoresistance
andtheirpangenotypicantiviralactivity. Theimprovedknowledgeofvirus–hostinteractionhasledto
theidentificationofpotentialtargetssuchascyclophilinandmiR-122inhibitors[145,146].
Interferon,thefirsthistoricaltreatmentagainstHCV,wasinfactamemberofaspecialclassof
HTAsometimesreferredtoasimmunomodulatingproteins. CyclophilinAinhibitors,derivedornot
fromcyclosporins,immunosuppressivemoleculesthatwereisolatedfromfungus,weredemonstrated
earlytohavepotentanti-HCVactivities[43]. Oncedevoidoftheirimmunosuppressiveproperties,
theseinhibitorswerethefirstHTAstogototheclinicandweredemonstratedtohavesomepotential.
Cyclophilinsareagroupofmultifunctionalcellularproteinsinvolvedinproteinfolding,trafficking
andcellsignaling.ThemodeofactionofcyclophilinAinhibitorsistodisruptitsinteractionwithNS5A
andpreventMVBbiogenesis[44,45]. BecausesomeCyclophilinAinhibitorswerealsodemonstrated
to restrict HIV replication, they could potentially be of interest to treat patients with HIV/HCV
co-infection[147]. Hostproteinsarenottheonlypossibletargets—clinicalproof-of-conceptstudies
havedemonstratedthatmiR-122inhibitorsefficientlyreduceviralloadinchronicallyinfectedHCV
patients[148,149]. However,theseinhibitors,whichrequireparenteraladministration,areunlikely
to be developed further in the context of efficient oral regimen. Another problem could be that
targetingsuchanimportantmoleculeinliverfunctionmayhavesomeimpactonliverphysiologyand
deleterioussideeffects.
HTAsarelessspecificthanhighlyefficientDAAsthatwerefasterintotheclinic,butthisisalsoone
oftheiradvantages. InadditiontotheirpangenotypicpotentialintheHCVfield,theycouldalsolead
tothediscoveryofbroad-spectrumantivirals,sincethehosttargetedproteincanbeimplicatedinthe
replicationofotherviruses. Targetinghostlipidsynthesisandmetabolismtoinhibitviralreplication
(andlimitliverdiseaseprogressioninthecaseofHCV)is,forexample,envisionedagainstbothHCV
andDenguevirus[150]. Someofthesedrugsemergefromrepurposingstrategiesandthereforealso
havetheadvantageoverDAAstobeobtainedatlowcost. Oneimportantlimitation,however,isthe
riskthattheyalterimportantfunctions,andtheirsideeffectsneedthereforetobecarefullyaddressed,
evenifshorttreatmentdurationintheHCVfieldcouldlimittheseeffects.
Nomatterhoweffectivemoleculesinhibitingviralreplicationbytargetingviralorhostfactors
are,theydonotprotectnon-infectedhepatocytesfrominfection. Preventingvirusentryis,however,
an important goal, especially in the context of graft reinfection after liver transplant, but also in
preventive treatments or to fight persistent infection [151]. The understanding of the complex
mechanisms of HCV entry in cells has increased a lot in the past few years, with the description
ofanumberofreceptororco-factorswhichmakesallofthempotentiallysuitabletargets[152]. Theuse
ofneutralizingantibodiesagainstviralorhostproteinsdemonstratedthefeasibilitytofightHCVin
chronicallyinfectedanimalmodelsbytargetingvirusentry[153,154]. ITX5061,anantagonistofSR-BI,
waseventestedinaphaseItrialtopreventlivergraftreinfection,withencouragingeffects[155].Given
thenumerousessentialcellularpartnersidentifiedtodate,alltheentrysteps(attachment,binding,
fusion)couldinprinciplebetargetedbyantiviralstargetingvirusentry[151].
Viruses2019,11,30 10of18
5. ConcludingRemarks
Considerable research efforts on HCV biology by academics and industrials has led to the
discoveryofhighlyefficientDAAslessthan25yearsaftertheidentificationofthevirus. Thisgreat
achievementmayleadtoaprogressivelossofinterestinthedevelopmentoffurtherresearchinthe
field. Manypotentiallyinterestingmoleculeshavealreadybeenhaltedatearlystagesofdevelopment
andwillcertainlynotbetestedfurther. Sustainedeffortsareneeded,however,ifwearetoreachthe
2030eradicationobjectivesetbytheWHO.
Inaneradicationstrategycontext,DAAscannotbetheonlyarmoftheplan. Theyarestillhigh
costandthereforepoorlyavailableinlowincomecountrieswhereHCVincidenceishigh. Thisisnot
theironlylimitation. TreatmentwithDAAsmayleadtotheemergenceofresistantstrains,doesnot
protectfromreinfectionand,asinfectionisasymptomatic,peopleunawareoftheirstatuscontinueto
transmitthevirus. Inaddition,thehighefficiencyratesstillleavesomepatientswithouttherapeutic
options. Thesearenowaminorityofpatientsandoftenreferredtoas“hard-to-treatpatients”;they
are those who are co-infected with HBV, those who present liver or renal dysfunction and those
whounderwentlivertransplantationorprevioustreatmentfailure[97,156]. Potentiallydeleterious
drug–druginteractionswithothertreatments,againstHIVforexample,isalsoamajorconcernwith
thedevelopmentanduseofthesenewtreatments[157]. Lastbutnotleast,theinhibitorsonthemarket
havenotbeenusedforalongtimeandtheappearanceofresistantstrainsisnotunexpected,possibly
leadingtoincreasingthenumberorpatientswithouttreatmentoptionsinthefuture.
HistoryandmodelingsuggestthatHCVeradicationwillnecessitateavaccine. Progressinthe
understanding of the immune response to HCV has led to different vaccinal strategies, including
DNA,peptidesorrecombinantproteins,vector-basedvaccines,andvirus-likeparticlesordendritic
cell-basedvaccinationstrategies;theseareatvariouslevelsofdevelopment(recentlyreviewedin[158]).
ThechallengeistodevelopavaccineelicitingbothabroadT-cellresponseandneutralizingantibodies
againstahighlyvariablevirushiddeninlipoviroparticles. Theobjectiveseemsachievableinthenext
fewyears,ifresearchinthisdirectionremainsstillsupportedagainstthisnow“curable”disease.
Funding: This work was supported by the French “Agence Nationale de la Recherche sur le SIDA et les
HépatitesVirales”(ANRS),theEuropeanResearchCouncil(ERC-2008-AdG-233130-HEPCENT)andtheLabEx
Ecofect (ANR-11-LABX-0048) of the “Université de Lyon”, within the program “Investissements d’Avenir”
(ANR-11-IDEX-0007)operatedbytheFrenchNationalResearchAgency(ANR).
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
References
1. InternationalCommitteeonTaxonomyofViruses(ICTV).Availableonline:https://talk.ictvonline.org/ictv_
wikis/flaviviridae/w/sg_flavi/56/hcv-classification(accessedon22July2018).
2. Choo,Q.L.;Kuo,G.;Weiner,A.J.;Overby,L.R.;Bradley,D.W.;Houghton,M.IsolationofacDNAclone
derivedfromablood-bornenon-A,non-Bviralhepatitisgenome. Science1989,244,359–362. [CrossRef]
[PubMed]
3. Hoofnagle, J.H.; Mullen, K.D.; Jones, D.B.; Rustgi, V.; Di Bisceglie, A.; Peters, M.; Waggoner, J.G.;
Park,Y.;Jones,E.A.Treatmentofchronicnon-A,non-Bhepatitiswithrecombinanthumanalphainterferon.
Apreliminaryreport.N.Engl.J.Med.1986,315,1575–1578.[CrossRef][PubMed]
4. Poynard,T.;Marcellin,P.;Lee,S.S.;Niederau,C.;Minuk,G.S.;Ideo,G.;Bain,V.;Heathcote,J.;Zeuzem,S.;
Trepo,C.;etal.Randomisedtrialofinterferonalpha2bplusribavirinfor48weeksorfor24weeksversus
interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus.
InternationalHepatitisInterventionalTherapyGroup(IHIT).Lancet1998,352,1426–1432.[CrossRef]
5. Manns,M.P.;McHutchison,J.G.;Gordon,S.C.;Rustgi,V.K.;Shiffman,M.;Reindollar,R.;Goodman,Z.D.;
Koury,K.;Ling,M.;Albrecht,J.K.Peginterferonalfa-2bplusribavirincomparedwithinterferonalfa-2bplus
ribavirinforinitialtreatmentofchronichepatitisC:Arandomisedtrial.Lancet2001,358,958–965.[CrossRef]