| 其他摘要 | Recently, the innate immune factor TRIM5α (tripartite motif protein 5α) was identified as a important host cell restriction factor inhibited retroviruses. In primate cells, it potently blocks the replication of retroviruses, including HIV-1, N-MLV, EIAV, etc., post-entry but before reverse transcription. In Old World monkeys, TRIM5α confers most primates are not infectious to HIV-1. In New World monkeys, owl monkey is the only primate that is not prone to infection by HIV-1. The TRIM5-CypA fusion gene in owl monkey cells inhibits the replication of HIV-1, make it is not susceptible to infection by HIV-1. Extensive work suggest that the pig-tailed macaque (M. nemestrina) is the only primate species susceptible to HIV-1 infection in Old World monkeys. The cause of why pig-tailed macaques are prone to infection by HIV-1 is still yet not fully understood. According to current widely-accepted taxonomy of primate, the previously reported Macaca nemestrina group is divided into three species: Sunda pig-tailed macaque (M. nemestrina), Northern pig-tailed macaque (M. leonina), and Mentawai macaque (M. pagensis). We analyzed the association of the TRIM5 locus with the susceptibility to infection by HIV-1 of local M. leonina in Yunnan, China. We identified a novel TRIM5-CypA fusion gene npmTRIMCyp (northern pig-tailed macaque TRIM5-CypA) in M. leonina through PCR and sequencing assays. The npmTRIMCyp is caused by retrotransposition of CypA pesudogene cDNA into 3’-UTR of TRIM5 gene which differs from TRIM5α-CypA fusion gene in owl monkeys. The npmTRIMCyp contains three different alternative splicing isoforms, i.e. npmTRIMCypV1-V3, the npmTRIMCypV2 as the major isoform may involved in the function of the fusion gene product. More work indicated that both exons 7 and 8 were spliced out in the major splicing isoforms during the reverse transciption of the fusion gene . The G-to-T mutation in the 3’ splicing site of intron 6 should prevent the inclusion of exon 7 during splicing. We cloned the cDNA of npmTRIMCyp and omTRIMCyp, the recombinant expression vectors were constructed and transfected into HeLa or HeLa-T4 cell lines. Cells stably expressing fusion proteins were challenged with HIV-1. Our functional assay demonstrated that the fusion gene lost the ability of restricting post-entry replication of HIV-1. It provides the potential molecular mechanism of why M. leonina are prone to HIV-1 infection, the only known exception in Old World monkeys. The infection assay in PBMCs confirmed that M. leonina are susceptible to infection by HIV-1. Moreover, we demonstrated that npmTRIMCyp could inhibit the replication of HIV-2ROD, while it only showed very moderate restriction activity to SIVmac239. By constructing the swap splicing isoforms (SWAP-1 and SWAP-2) between npmTRIMCyp and omTRIMCyp, the confocal microscopy assay demonstrated that npmTRIMCyp, omTRIMCyp, SWAP-1 and SWAP-2 are all sublocalized in the cytoplasm of the host cells. Further functional experiments demonstrated that both SWAP1 and SWAP-2 restricted HIV-1-GFP-VSVG replication, and the SWAP-1 containing the omTRIMCyp exon 7 showed higher restriction activity, which indicated that the exon 7 of TRIM5 plays a critical synergic role in the restriction activity to HIV-1. Furthermore, we performed the Co-ImmunoPrecipitation to detect the interaction between the fusion proteins and incoming HIV-1 capsids, the results showed that the npmTRIMCyp could not recognize and bind to the incoming HIV-1 capsids. Finally, we amplified and sequenced a fragment empassing the genomic region may be involved in the recognition region，and 46 polymorphic sites were identified in this region, which suggested that high polyphisms exist in the npmTRIMCyp recognition domain in M. leonina. |
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