Molecular Mechanism Of The Antiviral Function Of Host Restriction Factors (APOBEC3、BST-2) And The Corresponding Antagonism Against Host Defense System Mediated By Immunodeifciency Viruses

HIV-1, in common with various viruses, requires the concerted contributions ofnumerous positively acting cellular factors and pathways to achieve efficient replication.Conversely, mammalian cells also express a number of diverse, dominantly actingproteins that are widely expressed and function in a cell-autonomous manner tosuppress viral replication. These have been termed as restriction factors and/or intrinsicresistance factors, and they provide an initial line of defense against infection as acomponent of, or even preceding, innate antiviral responses. This work discusses themost extensively described examples of such factors, focusing on their impact on HIV-1.These are the APOBEC3family of proteins (in particular, APOBEC3G), BST2/tetherin/CD317, TRIM5α, MOV10and SAMHD1.Domestic cats could be infected by feline immunodeficiency virus (FIV), is apotential small animal model for the study of the immunodeficiency virus, the antiviralfunction of feline host restriction factor and the corresponding viral antagonisticmechanism has not been clearly defined yet. The feline host restriction factors whcihhave been found so far are APOBEC3family proteins, TRIM5α protein and BST-2protein. There are five feline A3(fA3) proteins, the fA3Z2(a-c) proteins targetBet-deficient feline foamy virus (FFV), while the infectivity of Vif-deficient felineimmunodeficiency virus (FIV) and feline leukemia virus (FeLV) is reduced by fA3Z3and fA3Z2-Z3. The antiretroviral activities of the fA3Z2s are inhibited by the foamyvirus accessory Bet protein via co-localizes and reduce packaging into the virion, butthe precise mechanism is not clear. It is believed that FIV Vif makes a fundamentalcontribution to overcoming the restrictions imposed by fA3Z3and fA3Z2-Z3. FIV Vifco-localizes with the feline A3proteins, targets them for proteasome degradation and rescues the infectivity of the virus. The specific interactions between FIV Vif and thefA3proteins are still unknown. The mechanism of FeLV against fA3s is not been found.The feline mRNA of TRIM5contains a premature stop codon expressing a RBCCprotein without the B30.2domain, the truncated feline TRIM5appears to be withoutany antiretroviral activity. However, a synthetic fusion of the feline TRIM5to the felineCypA generated a potent inhibitor of FIV and HIV-1. Feline BST-2has a very shortN-terminal cytoplasmic region compared with those of other mammalian andnon-mammalian homologues. Surprisingly, it remains significant antiviral activity. Anartificial feline BST-2mutant containing a longer N-terminal cytoplasmic homolog onlyexhibits limited antiviral capability. This provides clues for the functional study of theN-terminus of BST-2. Studies has shown that BCA2interacts with CT region of BST-2and improve the antiviral activity of BST-2. Whether the short CT region of felineBST-2retain this binding capability remains unclear. SAMHD1is a newly discoveredantiviral factor, which express conservatively in multiple species. The feline SAMHD1protein has not been defined, whether it is an antiviral protein, and whether HIV-2\SIVVpx antagonize feline SAMHD1would to be demonstrated.This study focus on several feline host restriction factors, discuss the precisemechanism by which the FIV Vif induces the degradation of feline A3proteins; theinteraction between feline BST-2and feline BCA2; the basic property and function offeline SAMHD1. Moreover, accordance with the conclusion, extend and discuss theimpact of N-terminal deletion of human BST-2on its biological function.In the study of degradation mechanism, our results presented here define thedetailed mechanism by which the FIV Vif induces the degradation of feline A3proteins.FIV Vif uses a conserved viral BC-box to interact with ElonginC and to recruit Cul5and ElonginB. Although lacking a Cul5box and an HCCH motif, FIV Vif specificallyselects Cul5, suggesting that it may utilize an alternative, possibly zinc-independentinterface, for Cul5selection. Future studies to define the interactions between FIV Vifand feline A3s would shed light on the novel functional domain in this lentiviral Vifprotein as well as the potential for cross-species transmission of FIV. In the feline BST-2function study, we first identified and cloned the feline BCA2gene, analysis of the sequence alignments demonstrated that feline BCA2was highlyhomologous with other species counterparts. We obtain the feline BST-2polyclonalantibody by immunizing rabbits, this provides antibody material for further research inthe future. By the virion release and co-immunoprecipitation experiments, we confirmedalthough the feline BST-2has a short CT region and could not bind with BCA2, itsantiviral function has not been affected. However, the artificial protein which caninteract with BCA2has weak antiviral activity. These implied the antiviral function offeline BST-2is unrelated with BCA2. We hypothesized that the long N-terminalcytoplasmic region of the human BST-2protein may confer an unnecessary role to itsantiviral function. These provide evidence for subsequent studies.As an extension of the above results, we explored the impact of N-terminaldeletion of human BST-2on its function. Our results provided evidence that the BST-2protein lacking nearly the entire N-terminal CT still retained antiviral activity againstVpu-defective HIV-1and could even inhibit wild-type HIV-1viral particle release.However, the fusion of different peptide labels to the N-terminus of the CT truncatedBST-2protein impaired its antiviral activity to varying degrees. The excessiveproportion of hydrophobic amino acids in the N-terminal cytoplasmic peptide may haveaffected the association of the recombinant BST-2with perinuclear lipid membranessuch as ER retention. Such impairment in intracellular trafficking influenced theN-glycosylation and reduced localization of the N-terminally tagged truncated BST-2tothe cell surface, eventually weakening its antiviral function. Importantly, theseobservations demonstrate that the CT truncated BST-2would be a more powerfulantiviral protein than the native factor. Additionally, the alteration of BST-2CT orinternal tag affects its capability to activate NF-κB. These results provide additionaldetails and implications for the structure-function model of BST-2.In the basic property and function study of SAMHD1protein from differentspecies, we constructed and expressed bovine, canis, mus, human and rhesus SAMHD1.We first identified and cloned the feline SAMHD1gene, demonstrated that feline SAMHD1was highly homologous with other species counterparts. Analysis of thesequence alignments predicted the potential function domain (HD domain) and thecapability of interacting with Vpx. These SAMHD1proteins may have anti-retroviralcapability, while the bovine SAMHD1may not be degraded by Vpx.Immunofluorescence and confocal microscopy revealed that these SAMHD1proteinsco-localized with the nucleus. Through analysis of these SAMHD1, we predicted theirfunction and basic property, which laid a certain foundation for the follow-up todetermine the antiviral function of these proteins and HIV-2/SIV Vpx proteininteraction studies.Feline host restriction factor research has a wide range of significance: primarily,FIV and HIV have similar genomic structure, virion particle morphology, infectionmechanism, life cycle, cell tropism and toxicity; the genes of domestic cat have highlyhomology with human. Therefore feline become an important animal model for study ofhuman AIDS, which is also the smallest natural animal model infected by lentivirus. Tocreate a better animal model for HIV-1research, the study of interaction between FIVand host restriction factors has a certain theoretical and practical significance.Additionally, the study of feline host restriction factors would inspire the functionresearch of human restriction factors; meanwhile, the difference of homologous proteinin different species could provide new ideas for the research of the evolution. Finally, alarge number of zoonotic disease outbreak nowadays, also highlight the practicalsignificance for host restriction factors researches of other species.