QQ客服 官方微博 反馈留言

题名	TRIM5-CypA融合基因与北平顶猴易感HIV-1的机制研究
作者	况轶群
学位类别	博士
答辩日期	2009-06
授予单位	中国科学院研究生院
授予地点	北京
导师	郑永唐
关键词	HIV-1 北平顶猴 限制因子 TRIM5α 亲环蛋白 TRIMCyp
其他题名	A novel TRIM5-Cyclophilin A fusion gene associated with the susceptibility to infection by HIV-1 in M. leonina
中文摘要	天然免疫分子TRIM5α（tripartite motif protein 5α）是近年来发现的一种重要的宿主细胞内逆转录病毒限制因子。在灵长类动物细胞中，TRIM5α蛋白可以在病毒进入细胞后、逆转录前的阶段抑制HIV-1、N-MLV和EIAV等逆转录病毒的复制。由于TRIM5α分子的作用，绝大部分旧大陆猴（Old World monkey）都不能感染HIV-1。而在新大陆猴（New World monkey）中，鹰猴是唯一不感染HIV-1的灵长类动物。研究证明，鹰猴细胞中存在的TRIM5-CypA融合蛋白（owl monkey TRIM5-CypA，omTRIMCyp）介导了抗HIV-1的作用，从而使鹰猴不能感染HIV-1。研究证明，平顶猴是旧大陆猴中唯一报道可以感染HIV-1的灵长类动物，但是其感染HIV-1的机制并不清楚。根据现行的灵长类动物分类学，原属平顶猴群体（M. nemestrina group）的三个亚种分为猕猴属的三个不同种：巽他平顶猴（Sunda pig-tailed macaque，M. nemestrina），北平顶猴（Northern pig-tailed macaque，M. leonina）和明打威猴（Mentawai macaque，M. pagensis）。本论文对中国云南境内北平顶猴TRIM5基因座和感染HIV-1的相关性进行了研究。通过PCR和测序对北平顶猴基因组TRIM5基因座进行分析，发现一个CypA假基因的cDNA通过逆转座机制插入至TRIM5基因座的3’-UTR区域，形成了一个不同于鹰猴TRIM5-CypA的新型融合基因npmTRIMCyp（northern pig-tailed macaque TRIM5-CypA）。通过RT-PCR对npmTRIMCyp融合基因的转录本进行分析，我们鉴定出npmTRIMCyp共有3种不同的选择性剪接产物，分别为npmTRIMCypV1-V3。进一步克隆和测序这3种不同选择性剪接体，通过丰度和序列分析证实：npmTRIMCypV2是优势剪接体，可能在该融合基因产物的功能中发挥作用。研究发现北平顶猴npmTRIMCyp融合基因主要转录本中外显子7和8均被剪切掉。外显子7剪接丢失机制源于TRIM5第6内含子内 3’剪接位点的G/T突变。我们克隆了npmTRIMCyp融合基因cDNA的蛋白编码区ORF，并构建了重组表达npmTRIMCyp的载体，转染HeLa和HeLa-T4细胞并获得稳定表达的细胞株。通过感染HIV-1证实，npmTRIMCyp融合蛋白不能够限制HIV-1的感染和复制，这可能是北平顶猴作为旧大陆猴中唯一对HIV-1易感的灵长类动物的重要分子机制之一。通过HIV-1感染灵长类动物PBMCs实验证实，北平顶猴可以感染HIV-1。npmTRIMCyp可以有效地限制HIV-2ROD的复制，但对SIVmac239只有十分微弱的限制活性。通过构建鹰猴omTRIMCyp和北平顶猴npmTRIMCyp的置换剪接体（SWAP-1和SWAP-2），转染融合基因及其置换剪接体的CRFK细胞激光共聚焦实验证明，npmTRIMCyp、SWAP1和SWAP2在细胞内主要存在于胞浆中。稳定表达融合蛋白和置换剪接体的CRFK细胞感染HIV-1-GFP-VSVG分析表明，含omTRIMCyp外显子7的SWAP-1和SWAP-2均具有限制HIV-1活性，但SWAP-1的活性更强一些，这表明TRIM5结构域的外显子7可能在介导对HIV-1的限制活性中发挥了协同辅助作用。免疫共沉淀研究表明，npmTRIMCyp不能识别和结合HIV-1的衣壳蛋白。对北平顶猴中介导识别逆转录病毒区域的基因组部分进行了测序，共鉴定出46个多态性位点，表明在北平顶猴识别逆转录病毒衣壳区域存在较高的多态性。
英文摘要	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.
语种	中文
公开日期	2010-10-22
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6305]
专题	昆明动物研究所_分子免疫药理学
推荐引用方式 GB/T 7714	况轶群. TRIM5-CypA融合基因与北平顶猴易感HIV-1的机制研究[D]. 北京. 中国科学院研究生院. 2009.

个性服务

查看访问统计

相关权益政策

暂无数据

收藏/分享

除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。