Study On Antagonistic Effect Of Tetherin Gene CSNP And HIV - 1 On Antagonistic Effect Of Tetherin On Rhesus Monkey

Background In nature, there is a relationship of co-evolution between virus and host. On the one hand, the host would develop new proteins to inhibit the invasion and replication of virus. On the other hand, the virusesare against the host by their own unique set of mechanisms to evade suppression from their host. Co-evolution not only promotes the adaptation between two interacting species, but also increased biodiversity as well as obstructed the spread of the virus across different species. The studies of human immunodeficiency virus (HIV) have found some innate immune factors in the host cells, which limited the virus’invasion, replication and budding. These factors were called host restriction factors, which participated in innate immune responses to interfere the viral different stages of life cycle. At present, there were four categories restriction factors, which were APOBEC3G (apolipoprotein B mRNAediting enzyme 3G), Trim5a (tripartite motif protein 5a), Tetherin (also known as CD317, or HM1.24) and SAMHD1 (sterile a motif domain and HD domain-containing protein 1). These restriction factors play an important role in host resistance to viral infection that effectively restrict retroviral infection. Tetherin is a transmembrane protein having a lipid raft associated special topology, which could connect viral and host membranes bounding the newly budding virus to the cell surface, thereby limiting release of the virus.In the research of AIDS animal models (simian immunodeficiency virus (SIV)/ rhesus macaque), the Tetherin protein was also found to inhibit SIV proliferation in the rhesus macaque. But rhesus Tetherin gene polymorphism and its effect on protein structure and function has not been reported. On the other hand, Tetherin as a natural anti-viral proteins, their protein structures are different in different species, This protein is species-specific. To make HIV across Tetherin restrictions of rhesus is an important step in reasch of AIDS animal model.Methods In this study, gene sequencing, sequence alignment and protein structure prediction techniques were used to explore the effect of rhesus Tetherin gene cSNP sites on its protein functions. Then different genotypes Tetherin were inserted into the eukaryotic expression vector pcDNA3.1 (-) to construct Tetherin expression vectors of different genotypes. The expression vectors of different genotypes and SIVmac239 were co-transfected into 293T cells.after 48 hours,viral load ofthe supernatant, and rhesusmacaqueswere classified by different genotypes;comparing the replication level of SIV virus in different genotypes infection-rhesus. On the other hand, the TZM-BL cell lines expressing rhesus Tetherin protein were constructed. The HIV-1NL4-3 was cultured in the mixed TZM-BL cell lines expressing rhesus Tetherin protein, and eventually HIV-1NL4-3 could across the rhesus Tetherin constraints adapting in TZM-BL cell lines by this way.Results There were eight non-synonymous mutations were found by sequence alignment. G41A, T128C, C129 and A333C cSNP sites may affect the Tetherin protein secondary structure by prediction of Psipred software. In the assay of co-transfecting 293T by different genotypes Tetherin expression vectors and SIVmac239, The SIV viral load were no significant difference between different genotype groups. But the level of Tetherin expression is different between different genotype groups. Tetherin expression of GLQ and GPH types were significantly higher than that of DLQ and DPH types. In vivio, on the plateau phase of SIVmac239 infection, reference genotype rhesus (DLQ) viral replication level was higher than that of GLQ type monkey (p<0.05). There were no significant differences between the other genotypes rhesus.After the construction of TZM-BL expressing rhesus Tetherin protein, HIV-1NL4-3 was cultured in the TZM-BL cell lines expressing rhesus Tetherin protein and TZM-BL cell line. P24 concentration of supernatant were no statistically difference between different groups.Conclusion There were eight cSNP in Chinese rhesus Tetherin gene were found. By forecastion of Psipred software, G41A, T128C, C129 and A333C cSNP sites may affect the Tetherin protein secondary structure. The results in vitro and in vivo experiments of rhesus showed that rhesus Tetherin cSNP genes could not affect their antiviral activity, but they can affect the antagonism of SIV. In the culture of HIV-1NL4-3 adaptability of TZM-BL expressing rhesus Tetherin, we did not find rhesus Tetherin inhibit replication of HIV-1NL4-3, but that does not mean that rhesus Tetherin has no inhibition of HIV-1NL4-3-Although Tetherin protein can tie the new virus at the cell surface, but we can not rule out the virus spread through the interaction between cells.