Mechanisms Of Lipid Rafts On Cell Entry Of Hepatitis C Virus, Japanese Encephalitis Virus And Enterovirus71

Lipid rafts are specific membrane microdomains which are enriched incholesterols and sphingolipids. The microdomains have been named "rafts" becausethey can be seen as floating device within the lipid sea. Lipid rafts appear to be inmany biological events such as synthetic traffic, cell skeletal reconstruction and signaltransduction. They also provide a platform for protein-protein interactions.As the first step of infection, viral entry is of vital importance in the entire lifecycle of viruses, and therefore an effective antiviral target. Virus entry into host cellsinvolves the essential following steps:(1) attachment to host cell surface,(2)penetration into cytosol or inclusion body,(3) uncoating. Virus tends to utilize somemicrodomains or microenvironments in the cell membranes to accomplish theirtrans-membrane process from cell periphery to intracellular sites. Current studiesdemonstrate that lipid rafts are active sites where the interactions between virus andhost cell take place.There are probably four mechanisms by which lipid rafts regulate envelopedvirus entry into host cells:(1) enveloped protein anchored in lipid raft,(2) virusreceptors locating lipid raft,(3) conformational change or membrane fusion ofenveloped protein based on lipid raft,(4) lipid raft-dependent endocytosis. Humanimmunodeficiency virus (HIV) is a well-studied enveloped virus of lipid raft.Compounds targeting lipid rafts show powerful antiviral activities, indicating lipid raftas a possible antiviral target. However, different viruses hijack lipid rafts for cell entrythrough distinct mechanisms, due to diverse receptors enveloped viruses recognizeand different ways of membrane fusion, including pH-dependent and pH-independentpathways. Understanding the engagement of lipid rafts in virus entry will be helpful todesigning and developing new antiviral drugs.Compared with the thorough study of membrane fusion of enveloped viruses, theentry mechanism of non-enveloped viruses is largely unknown. Previous studies findthat some non-enveloped viruses, such as Echovirus1(EV1) and Simian Virus40(SV40), enter host cells through lipid raft-mediated endocytosis; others, likeCoxsackievirus and Rotavirus, through binding to the receptors located in lipid raft toinitiate virus entry. For this reason, it is beneficial to explore how lipid rafts mediatevirus infection in order to further elucidate the molecular mechanism of virus entry. Cell membrane is the first barrier against the virus to reach the cellularmachinery. In the present study, roles of lipid raft in cell entry of three single positiveRNA viruses were investigated, including hepatitis C virus, Japanese encephalitisvirus and enterovirus71, in order to study the molecular mechanism of theengagement of lipid rafts in interactions between virus and host cell, and discuss thedifference and relationship between different cell entry pathways through lipid raftsby diverse viruses.Part Ⅰ: Mechanism of lipid rafts on cell entry of hepatitis C virusHepatitis C virus (HCV) entry is a coordinated multistep process mediated byspecific factors, involving tetraspanin CD81, scavenger receptor class B member I(SR-BI), and the tight junction proteins claudin-1(CLDN1) and occludin. Previousstudies have suggested that the interactions between the viral envelope and host cellreceptors may depend on cholesterol-enriched membrane microdomains, termed lipidrafts, an important microdomain in cytosol membrane for many viruses'cell entry. Wefound that HCV receptors CD81, SR-BI, CLDN1and occludin all partition in lipidrafts, co-localizing with HCV envelope glycoprotein E2during viral entry. Toinvestigate whether the raft localization of CD81affects HCV cell entry, we generatedCD81partitioning mutants by substituting the CD81transmembrane and cytoplasmicdomains with those of raft proteins (SR-BI or CD9) or that of non-raft protein (CD71),while preserving the native CD81large extracellular loop (LEL) in chimeric molecule.All CD81chimeras that retain raft partitioning mediate HCV entry and CD81-inducedRho-GTPase activation which allows lateral movement of the virus and its delivery tocell-cell contact areas. Conversely, CD81LEL targeting to a non-raft membranefraction results in a CD81receptor with severely diminished capacity to mediateRho-GTPase activation and HCV entry, although this mutant binds HCV E2as well aswild type CD81. Moreover, we show that the non-raft CD81mutant fails to relocalizeHCV E2to the areas of cell-cell contact, thus disrupting the formation ofE2-CD81-CLDN1complexes which was recently described as a determinant for viralentry. Altogether, our results not only demonstrate that the association of CD81withlipid rafts plays an essential role in HCV entry, but also suggest new strategies forblocking HCV infection. Part Ⅱ: Mechanism of lipid rafts on cell entry of Japaneseencephalitis virusJapanese encephalitis virus (JEV) is an enveloped flavivirus and the mostcommon agent of viral encephalitis that enter cells through receptor-mediatedendocytosis and low pH-triggered membrane fusion. Although lipid rafts,cholesterol-enriched lipid-ordered membrane domains, have been shown to participatein JEV entry, the mechanisms of the early events of JEV infection, including thecellular receptors of JEV, remain largely unknown. In the current study, wedemonstrated that heat shock protein70(HSP70), rather than other members of theHSP70family, was required for JEV entry into a human cell line. Cell surfaceexpression of HSP70and the direct interaction between JEV envelope (E) protein andHSP70were observed. Biochemical fractionation showed that HSP70clearlymigrated into the raft fraction after viral infection and co-fractioned with E protein.The depletion of cholesterol shifted E protein and HSP70to a non-raft membrane anddecreased JEV entry without affecting viral binding to host cells. Notably, therecruitment of HSP70into lipid rafts was required for the activation of PI3K/Aktsignaling in the early stage of JEV infection. Our results indicate that lipid raftsfacilitate viral entry possibly by providing a convenient platform to concentrate JEVand its receptors on host cell membrane.Part Ⅲ: Mechanism of lipid rafts on cell entry of enterovirus71Enterovirus71(EV71) is one of the major causative agents of hand, foot, andmouth disease, a common febrile disease in children. Little is known about the earlysteps of EV71infection. Membrane cholesterol, or lipid rafts, plays an important rolein the life cycle of many viruses. In this study, we found that disruption of membranecholesterol by three pharmacological agents through different mechanisms:methyl-β-cyclodextrin (MβCD), nystatin or lovastatin, all resulted in a significantreduction of the titers of infectious EV71released into the culture supernatant, as wellas a reduction in the number of EV71genome copies in the cholesterol-depleted cells.The inhibitory effect could be reversed by the addition of exogenous cholesterol.Cholesterol depletion postinfection did not affect EV71infection, suggesting that theinhibitory effect occurs at the stage of EV71entry. We also showed that while virus bound equally to MβCD-treated cells, EV71particles failed to be internalized bycholesterol-depleted cells. Co-localization of EV71capsid protein with cholera toxinB confirmed that EV71internalization occurs in a lipid raft-dependent manner. Thesedata strongly suggest that lipid rafts are essential for the early stage of EV71infectionand highlight membrane cholesterol as potential targets to block EV71entry.SummaryThis study demonstrated the involvement of lipid rafts in HCV, JEV and EV71entry into host cells, and further illustrated the cell entry mechanisms of these threeviruses. Substituting the transmembrane domain with intracellular domain of CD81tochange its lipid raft localization provides an example for the investigation of the roleof other receptors in mediating virus entry. We first demonstrated the role of HSP70asa receptor in JEV entry into Huh7cells. Also, we first reported the engagement oflipid rafts in cell entry of the non-enveloped virus EV71.Here, we analyzed the function of lipid raft in cell entry of the three RNA viruses.It is found that the possible mechanism by which lipid rafts regulate virus-hostinteraction is that some virus receptors partition in lipid rafts, others concentrate onmembrane rafts after the attachment of viruses. Through recruiting receptor proteinsand related entry factors, lipid rafts may act as a platform for virus-cell interaction.These results serve to develop new antiviral strategies.