Studies On Mechanism Of BIV And MVV Vif-mediated Degradation Of Host APOBEC3Z2-Z3and Construction Of Sthiv-1

Most viruses including HIV-1derived a series of accessory protein to achieve efficientreplication in the long process of evolution. In the course of HIV-1infection， the host willproduce numerous cellular factors that play multiple antiviral functions through differentpathways. The roles of the host restriction factor and virus accessory protein have been a hot issuein the research field of AIDS.These positive cellular factors have been termed as host restrictionfactor. On the other hand, the virus also derived a series of accessory proteins to antagonize theantiviral effects of host restriction factors during the long-term evolution, to achieve efficientreplication. At present stage, the mechanisms between virus accessory protein and host restrictionfactor elucidated include:1. HIV-1virus infectivity factor Vif and APOBEC3G;2. HIV-1Capsidand TRIM5α;3.HIV-1Vpu and Tetherin;4.HIV-2Vpx and SAMHD-1. However, the existenceof species specificity between host restriction factor and virus accessory protein result in a seriesof problems. It mainly displays in two aspects, the cross species activity of host restriction factorand the broad-spectrum antiviral activity of virus accessory protein. First，these viruses and theirhosts have co-evolved various mutually antagonistic proteins, which over the long evolutionaryprocess have facilitated viral entry into new hosts, such as the recent outbreak of zoonosis. Thus,the study of host restriction factor of other species may shed light on the course of cross-speciestransmission. On the other side, the infection of non-human primate of HIV-1was seriouslyimpaired by the species specificity of host restriction factor, cause gorilla or monkeys whosepharmacologyand physiologyare verysimilar to human can t be used as AIDS research models.Thus the study of transformation of HIV-1was derived for the long-term replication in monkeys.In this paper, we discuss on the two aspects above based on the mechanism between virusaccessory proteins and host restriction factors.The first part of this study focused on one of the host restriction factors APOBEC3s, anddiscuss the precise mechanism between APOBEC3s of other species and the correspondinglentivirus Vif. Lentivirus (lente-, Latin for "slow") is a subfamily of viruses ofthe Retroviridae family, It was named from the long incubation period of the disease in the hostinduced by the virus. Human immunodeficiency virus type1(HIV-1), simian immunodeficiencyvirus (SIV), caprine arthritis-encephalitis virus (CAEV), feline immunodeficiency virus (FIV),bovine immunodeficiency virus (BIV), maedi-visna virus (MVV) and equine infectious anemia virus (EIAV) are lentiviruses that infect humans, monkeys, goats, cats, cattle, sheep and horses,respectively. Except for EIAV, all lentiviruses require the accessory protein viral infectivityfactor (Vif) to establish persistent infection and pathogenesis in vivo. The mechanism by whichVif of HIV, SIV and FIV suppresses the corresponding host A3s has been studied extensively,but the roles of BIV and MVV were not eclucidated yet. HIV-1Vif antagonizes the antiviralactivity of the cellular protein A3G by recruiting the transcription cofactor CBF-β and ElonginB(EloB)-ElonginC (EloC) to the Cullin5(Cul5)-Rbx complex to degrade A3G. In this part, wedetermined that BIV recruits ElonginB, ElonginC and Cullin2, MVV Vif recruits ElonginB,ElnginC and Cullin5to degrade Bos taurus (bt)A3Z2-Z3and Ovis aries (oa)A3Z2-Z3,respectively, via a proteasome-dependent but a CBF-β-independent pathway. While themembrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation ofbtA3Z2-Z3,indicating that Zn is important for the activity of BIV Vif. Furthermore, we identifieda previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC boxwhich is critical for its degradation activity. Zinc Affinity chromatographyexperiment indicatedthat the interaction between zinc and BIV Vif was depended on C-x1-C-x1-H-x19-C domain.Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC andoaEloC and dominant-negative mutants of btCul2and oaCul5could disrupt the activity of BIVand MVV Vif, respectively. TPEN had minimal effects on oaA3Z2-Z3degradation induced byMVV Vif, indicating that Zn is not important for the activity of MVV Vif.In the second part of the paper，we based on the species specificity of host restriction factor,and try to carry out the transformation of HIV-1framework to achieve the cross-speciestransmission. Since AIDS was discovered in1981，almost thirty-four million people have beeninfected by HIV. However, lack of suitable animal model is one of the key factors that hinder thedevelopment of AIDS drugs and vaccine. At present stage，two major animal models have beendeveloped for the research of AIDS in vivo. One is scid-hu-mice (severe combinedimmunodeficiency-human-mice), but the effectiveness and disease characteristics severely limitsits application. The other one is SIVs or genetically engineered SHIVs infect monkeys，butgenetic distance between HIV-1and SIVs make it can not simulate all of the primary features ofthe human infection by HIV-1. So it can not be used for direct evaluation of antiretroviral drugsor vaccines, especially evaluation of vaccine. Establishment of a valid animal model forpreclinical investigations of antiretroviral drugs or vaccines will help promote the global AIDS.Because the small non-human primate have common with human in physiologyanatomyand disease characteristics, they are still the most ideal animal model for AIDS. Monkeys were divided into two categories, one is the new world monkey，such as squirrelmonkeys and common marmosets， the other is the old world monkey，including African greenmonkey (agm) and Rhesus Macacus (mac). HIV-1infection of the new world was blocked in theinvasion stage. Because virus envelope protein gp120and the cell surface receptor proteins CD4and CCR5can not interact efficiently. Although HIV-1can effectively invasive the Old Worldmonkey, the later stage of host invasion was restricted by host restriction factors, leading to thevirus can not replicate efficiently. Three host restriction factors explained the intrusion blocking:Tripartite motif-containing protein5alpha (TRIM5α), apolipoprotein B mRNA-editing enzymecatalytic polypeptide-like3G(A3G), bone marrow stromal antigen2(Tetherin).In the second part of the paper, we try to replace several accessory proteins of HIV-1toantigonize the antiviral activities of Rhesus Macacus cells. TRIM5α blocked viral capsiduncoating, thereby inhibiting the release of virus RNA. However, the identification ofretroviruses capsid by TRIM5α was highly species-specific. It blocked the uncoating of HIV-1capsid, but did not block the uncoating of SIV capsid, so we replace HIV-1CA with SIVmac239CA; APOBEC3G can be packaged into virus particles and induce tremendous mutations on viralgenome, thereby blocking viral replication. The virus Vif protein could induce the degradation ofA3G through the ubiquitin pathway. Likewise, the degradation of A3s induced by vif wasspecies-specific. HIV-1Vif could degrade the human A3G, but not degrade the Old Worldmonkey A3G. However, SIV Vif could degrade the Old World monkey A3G, so we replaceHIV-1Vif with SIVmac239Vif; Tetherin could inhibit the release of HIV-1, and viral Vpuprotein could rescue the virus release through down-regulation the cell surface level of Tetherin.HIV Vpu could antagonize the antiviral activity of human Tetherin, but not the Old Worldmonkey Tetherin. It has been reported that HIV rare subtype DH12Vpu could effectivelyantagonize the Old World monkey Tetherin, so we replace HIV-1Vpu TM with HIV-1DH12VpuTM. We carry out the transformation of HIV-1framework, to replace HIV-1Vif, CA and VpuTM motif with SIVmac239Vif, CA and HIV-1DH12Vpu TM domain. First we detect thestructural protein of stHIV-1in293T cells, and the virus packaging. On this basis, we detectedthe infection ability of virus particle packaging of human MAGI-CCR5cells, found thatthe chimeric virus can infect human MAGI cells. In addition, we detectde the replacement partof the protein function, the results showed that stHIV-1Vif can effectively degrade RhesusMacacus A3G proteins, and HIV-1DH12Vpu could antagonize the toxic inhibition on virusinduced by Rh BST-2. In the functional test of SIVmac239CA of stHIV-1, we couldnot successfully constructed the stable cell lines of CEM×174that expressed RhTRIM5α, so wewill detect it in the next infection of RhPBMC by stHIV-1experiment indirectly. We utilized the p24protein quantitative method and TZM-bl essay to determin the virus titer of stHIV-1. Inaddition, we also successfully detected the long-term replication of stHIV-1in CEM×174cells.In the next step, we need to complete the detection the long-term reproduction of stHIV-1inRhPBMC, then we will attack Rhesus Macacus with passage virus from RhPBMC. We hope thatthe stHIV-1can replicate in Rhesus Macacus and can eventually lead to the AIDS disease ofRhesus Macacus.This study supplements our knowledge of the mechanism of degradation of host antiviralproteins induced by BIV Vif and MVV Vif. Our work may shed light on the course of disease incows and sheep, as well as the potential for cross-species transmission of BIV or MVV. Inaddition, the construction of stHIV-1is expected to become the ideal AIDS animal model, topromote the development of AIDS in the treatment of drug and vaccine.