Screening Of Chinese Old World Monkey TRIMCyp Gene And Construction Of Pseudotyped Human Immuno Defieiency Virus-l

Lacking proper animal models is still a major obstacle for AIDS research. Although non-human primates (NHP), such as macaques, are widely used as animal models for studies on HIV-1 infection and host immune responses, there are multiple species-specific restriction factors existing in these animals, which set up inhibitions to the cross-species transmission of HIV-1 by different ways. One key restriction factors, TRIM5α, which inhibit the proliferation of HIV-1 were identified recently in NHP. Simian TRIM5αbinds HIV-1 capsid protein (Cardarelli et al., ) and therefore restricts the replication of the human AIDS virus, but has no inhibitions on simian AIDS virus (SIV). Based on recent publications, TRIMCyp which is encoded by the TRIM5 gene inserted with a CypA peudogene by retrotransposition, the fusion protein of TRIM5αand CypA, lost its ability to restrict the replication of some retroviruses, such as human immunodeficiency virus-1 except HIV-2. Therefore, monkeys expressing TRIMCyp are predicted more susceptible to HIV-1 infection than those bearing TRIM5αand are desired candidates for cross-species transmission study of HIV-1 infection.There are two objectives in this study, which includes screening of TRIMCyp genotype macaque species and constructions of pseudotyped HIV-1 infectious clones. Results for these objectives are summarized as follows:I. In this study, the existence of TRIMCyp, the TRIM5 gene with a retrotransposon CypA cDNA, in three major Chinese Old World monkeys 655(Rhesus Macaque, Crab-eating Macaque and Thibetana Macaque) was investigated by PCR with the primers designed by the study. For the first time, the TRIMCyp chimera gene was found existing in a small population of Chinese Rhesus Macaque. In addition, TRIMCyp homozygous Crab-eating macaques were screened Several Chinese macaque species were investigated for individuals of the TRIMCyp genotype.,which provide fruitful resource for HIV-1-susceptible NHP models. The existence of TRIMCyp gene of Chinese Crab-eating macaques was statisticed. Further more, a RT-PCR protocol was developed to detect the encoding sequence of TRIMCyp gene in Rhesus Macaque and Crab-eating Macaque.Meanwhile, vectors which express kinds of TRIMCyp fusion protein and TRIM5αwere contructed in this study. And these would be used to research their retriction to retroviruses. In addition,proportion of three TRIM5-CypA splice isoforms was different in the three kinds of Chinese old world monkey ,which might represent differet retriction to HIV-2.II.HIV-1 commonly infect cells that express CD4 molecule and co-receptor CCR5 or CXCR4. It is dangerous in the experiment to use the intact HIV-1 .Therefor,in the study a single cycle replication pseudotyped HIV-1 virus vector packed by vesicular stomatitisvirus glyeoprotein (VSV-G) as its membrane protein was contructed. VSV-G could react with lecithoid component of cell membrane, which facillitated virus entry more cells.HIV-1 lacking partial env gene cotranfected 293T cells with pVSV-G could produce pseudotyped HIV-1virus. HIV-1 pNL4- 3 infectious clone was used to construct the lentivirus vector by replacing the HIV-1 env gene with a reporter gene EGFP,and this recombinant genome was packed by HIV gag protein and secreted with vesicular stomatitis virus glycoprotein (VSV-G) as its membrane protein. In the study,EGFP substituted HIV partial env gene as a reporter gene.Its expressing level reflected the biology character of pseudotyped HIV-1virus.In addition,this pseudotyped virus system could be used for the studies on cross-species transmission of retroviruses as well as on the screening of anti-HIV drugs.