" HEPATITIS CVIRUS LIFECYCLE[Year] HEPATITIS C VIRUS LIFE CYCLEThe HCV life cycle is a complex series of interactions including attachment, entry, fusion andrelease of mature virions. 1. Virion Attachment HCV life cycle begins with the virion attachment with the cell surface receptors onhepatocytes. Multiple cellular receptors and entry factors for HCV have been identified,1 2 including the Glycosaminoglycans, scavenger receptor class B type I (SRB1) and CD81as3 4 well as tight junction proteins, claudin-1 (CLDN1) and occludin(OCLN) (Table 2). Table 2- The essential factors involved in HCV entry.Receptor DescriptionActionGlycoprotein receptor , amajorSRRB1 It is a binding partner of HCV E2receptor for HDLs along with CD81Adhesion, morphology, proliferation,CD81has 4 transmembrane domains and differentiation; binds HCV throughE2interact with CD81 as a part of HCVCLDN1Tight junction proteinreceptor complexInvolved in HCV entry after SRB1 andOCLN Tight junction proteinCD81GAG Polysaccharides Capturing Virus particle and attachment HCV entry is a very crucial and complex step involving lipoprotein components, viralstructural mediators and a number of cellular receptors.? GlycosaminoglycansGlycosaminoglycans (GAG) are the large polysaccharides expressed on the surface of mostmammalian cells. Heparan sulphate proteoglycans (HSPG) is an attachment factor forbinding to cellular receptors. It was shown that HCV E2 attachment was prevented by a5, 6heparan sulphate homolog and by treatment with heparinase . Syndecan 1 identified2 through a screening study is pointed out as a major HSPG attachment molecule involved in7 HCV entry . apoE mutant deficient studies have shown that apoE mutants produced poorly8infectious viral particles . Thus GAG is mainly involved in capturing viral particles from lowand high density fractions and help in attachment to other cellular receptors. ? SRB1 and CD81- the binding receptors for E2SRBI is Scavenger receptor class B type 1, a 509 amino acid glycoprotein receptor highlyexpressed in the liver. It is a receptor for high density lipoproteins and promotes uptake ofHDL cholesterol esters into hepatocytes.SRB1 binds to the HVR 1 of E2 and mediate HCV9,10entry and cell to cell transmission . CD81 is another receptor involved in HCV entry,11. where the large extracellular loop of CD81 binds to E2 protein of HCV ? Claudin and occludinClaudin 1 and occludin are the tight junction proteins which play role in virus entry aninternalisation. Claudin-1 is composed of two extracellular domain, EL1 and EL2 anchored to12 the cell membrane through four transmembrane domains . Interactions of claudin withCD81 has shown to enhance HCV association with cell receptor complexes and promote13,14internalisation . Downregulation of claudin-1 in Huh7.5 cells was reported to inhibit HCV15infection . The role of occludin in HCV entry need further studies. But studies show thatdownregulation of occludin decrease both HCV entry and glycoprotein-mediated cell fusion16 . These suggest that the tight junction proteins act as late mediators of virus entry and areimportant in virus internalisation.? Role of Envelop proteins E1 E2 are functional proteins that require proper heterodimerisation and glycosylation. E1and E2 harbour 8 and 18 cysteines respectively that form disulfide bridges. E2 disulfidebridges are essential for CD81 binding , antibodies recognition and play an active role in17,18 budding process of HCV particle . Glycosylation play a role in heterodimerisation, viralparticle secretion, proper protein folding and viral entry.2. Postbinding Steps3 The interactions of the viral particles with the receptors, coreceptors and cofactors result inthe internalisation of the viral particle-receptor complexes through clathrin-dependent19,20 endocytosis. Association of CD81- claudin interaction is crucial for virus internalisation .Following cell entry through receptor mediated endocytosis, the HCV particle undergoes pHdependent membrane fusion within an acidic endosomal compartment. Decapsidation of theviral nucleocapsids releases the positive stand HCV genomic RNA into the cell cytoplasm.HCV genomic RNA is used for both polyprotein translation and replication in the cytoplasm.The RNA directly serves as template for translation into the HCV polyprotein. Thetranslation is initiated in a cap-independent manner via the Internal Ribosome entry site(IRES) in the 5’UTR that form a stable pre-initiation complex by directly binding to the 40Sribosomal subunit. The 40S subunit assembles with eIF3 and this complex joins the eIF2,GTP and the initiator tRNA. The eIF2 with the hydrolysis of GTP positions the tRNA in theP site and the tRNA base pair with the start codon on the mRNA. A second GTP hydrolysisalong with the eIF5B enables the 60S ribosomal subunit to associate forming a functional 80S21,22 ribosome which initiates viral protein synthesis . After its synthesis, the HCV polyprotein is cleaved by host and viral proteases to producestructural and non structural proteins (Figure 2). During the polyprotein processing the HCVproteins are associated with the membranous web which includes double-membrane vesiclescontaining HCV nonstructural proteins, HCV RNA , ER membranes and lipid droplets.NS5B RNA dependent RNA polymerase replicates the genome by the synthesis of negativestrand RNA. This negative strand RNA serves as a template for the synthesis of positivestrand RNA. This positive strand RNA can be further translated to produce new viral proteins23 or serve a template for further replication or be assembled to infectious virion . Viralassembly occurs within lipid raft like structures. HCV nucleocapsid is built from units of thecore protein with RNA, surrounded by a membrane derived from the human cell withembedded heterodimers of the envelop glycoprotein E1 E2. The virions associate with lowdensity and very low density lipoproteins forming lipoviroparticles that arepolymorphic(Merz A J Bio chem 2011).4 Figure 2- HCV polyprotein processing –cleavage at C-E1, E1-E2, E2-p7 and p7-NS2junctions requires host signal peptidase. NS2 cleaves the site between NS2 and NS3; NS3/4Acleaves the sites at NS3-NS4A,NS4A-NS4B, NS4B-NS5A and NS5A-NS5BThe very low-density lipoproteins are involved in the production of the infectious HCVparticles. HCV uses this lipoprotein biosynthetic pathway to form the mature viral particlesand export them for infecting new cells and creation of new virions.Genome encapsidationoccur in the endoplasmic reticulum and the nucleocapsids are enveloped and matured in thegolgi apparatus and the newly produced virions are released in the pericellular space byexocytosis. HCV infection causes changes in the lipid droplets (LDs) distribution andaccumulation around the perinuclear region in HCV infected cells. The assembly initiates onthe cytosolic side of the ER membrane where the core proteins associate with the LDsalongwith the other viral proteins. The viral replication complex (RC) is recruited by the lipid LDsand the replicated RNA is transferred from the RCs to associate with the core for itsencapisdation. NS3 is required for formation of core containing particles. E1 E2glycoproteins are incorporated with the help of NS2 to the immature virions. The maturevirions assisted by VLDL and apoE are released by exocytosis to infect new hepatocytes(Figure 3).5 Figure 3 : Schematic diagram of HCV life cycle. 1. Interaction of the virions with the cell surface receptors.2. Receptor-mediated endocytosis. 3. HCV glycoprotein-mediated membrane fusions releasing the virion's nucleocapsid(viral RNA) into the cytoplasm. 4. Genomic RNA is translated and processed into the 10 mature HCV proteins. Theendoplasmic reticulum is modified by viral and cellular factors to form a membranousweb, which is the major site of viral RNA amplification.5. Replication of viral RNA via synthesis of positive strands (+) from a replicativeintermediate negative RNA strand (-) template. 6. Virion assembly, budding.7. Mature virions are released from the cell to complete the life cycle.REFERENCES1. Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, et al. Thehuman scavenger receptor class B type I is a novel candidate receptor for the hepatitisC virus. EMBO J 2002;21:5017-5025.2.Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, et al. Binding ofhepatitis C virus to CD81. Science 1998;282:938-941.3.Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, et al. Claudin-1is a hepatitis C virus co-receptor required for a late step in entry. Nature2007;446:801-805.6 4.Liu S, Yang W, Shen L, Turner JR, Coyne CB, Wang T. Tight junction proteinsclaudin-1 and occludin control hepatitis C virus entry and are downregulated duringinfection to prevent superinfection. J Virol 2009;83:2011-2014. 5. Koutsoudakis G, Kaul A, Steinmann E, et al. Characterization of the early steps ofhepatitis C virus infection by using luciferase reporter viruses. J Virol. 2006;80:5308– 5320. 6. Jiang J, Cun W, Wu X, Shi Q, Tang H, Luo G. Hepatitis C virus attachment mediatedby apolipoprotein E binding to cell surface heparan sulfate. J Virol. 2012;86:7256– 7267.7. Shi Q, Jiang J, Luo G. Syndecan-1 serves as the major receptor for attachment ofhepatitis C virus to the surfaces of hepatocytes. J Virol. 2013;87:6866–6875. 8. Jiang J, Cun W, Wu X, Shi Q, Tang H, Luo G. Hepatitis C virus attachment mediatedby apolipoprotein E binding to cell surface heparan sulfate. J Virol. 2012;86:7256– 7267.9. Brimacombe CL, Grove J, Meredith LW, et al. Neutralizing antibody-resistant hepa-titis C virus cell-to-cell transmission. J Virol. 2011;85:596–605.10. Catanese MT, Loureiro J, Jones CT, Dorner M, von Hahn T, Rice CM. Differentrequirements for scavenger receptor class B type I in hepatitis C virus cell-free versuscell-to-cell transmission. J Virol. 2013;87:8282–8293.11. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A.J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, S. Binding of hepatitis C virusto CD81. Science.1998; 82, 938-94112. Gunzel D, Fromm M. Claudins and other tight junction proteins. Compr Physiol.2012;2:1819–1852.13.Harris HJ, Davis C, Mullins JG, et al. Claudin association with CD81 defineshepatitis C virus entry. J Biol Chem. 2010;285:21092–21102.14. Farquhar MJ, Hu K, Harris HJ, et al. Hepatitis C virus induces CD81 and claudin-1endocytosis. J Virol. 2012;86:4305–4316.15. Evans MJ, von Hahn T, Tscherne DM, et al. Claudin-1 is a hepatitis C virus co- receptor required for a late step in entry. Nature. 2007;446:801–805.16. Ploss, A.; Evans, M.J.; Gaysinskaya, V.A.; Panis, M.; You, H.; de Jong, Y.P.; Rice,C.M. Human occludin is a hepatitis C virus entry factor required for infection ofmouse cells. Nature 2009, 457,882–8867 "