Role of Vif in HIV-1 Replication/AIDS

描述：(申请人提供)HIV-1 Vif基因对 由非允许细胞(NP)产生的传染性病毒，包括 CD_4阳性T淋巴细胞和巨噬细胞以及某些白血病T细胞系 如HUT78和MT-2，但在其他被称为 允许(P)。P来源的Vif缺失HIV-1可感染NP细胞 细胞，但受感染的NP细胞只释放非传染性病毒粒子。这些 非感染性病毒粒子不能通过Vif在靶细胞中的表达来拯救，并且 他们被认为在基因组RNA或核心蛋白上存在缺陷 降低了逆转录的效率。我们和另一个实验室 最近进行的互补研究显示了NP的优势 PxNP异核体的表型，表明NP细胞含有一种 可以使HIV-1失活，这一因素可以被VIF抵消。使用人类 以Vif为诱饵的酵母双杂交筛选中的淋巴细胞c DNA文库 现在确定干扰素诱导蛋白Sp140是一个很好的候选蛋白 对于这个NP因素。简而言之，Sp140在所有被测试的NP细胞中都存在，但在P 细胞。Sp140存在于含有多种蛋白质的核小体中。 通过添加泛素相关蛋白SUMO-1进行共价修饰。 Sp140在Hela-CD4细胞中的表达诱导至少一个 蛋白质，Vif的共表达阻断了这种诱导。因此，VIF会起到抵消作用 Sp140至少有一种生化作用。我们建议：(I)分析Sp140 异构体及其相关蛋白灭活Vif缺失的能力 H1V-1。(Ii)确定VIF与Sp140和与VIF相互作用的活性部位 通过我们的双混合屏幕确定的其他潜在合作伙伴。(Iii)由 双向电泳法，鉴定所有的总和和非总和 Sp140诱导P细胞中的蛋白质，并确定Vif如何影响这些蛋白质 引诱。同样，确定NP细胞中的Vif表达是否改变 蛋白质总甲基化与P细胞或NP细胞中Vif缺失的HIV-1的形成 含有任何苏木糖化蛋白。(四)有趣的是，单纯疱疹病毒 I型ICPO蛋白以NBS为靶标，并特异性地导致某些 Sumoylated Nb蛋白，以及由CMV、EBV、腺病毒和 禽流感病毒似乎也有类似的活动。确定是否表示 这些其他病毒蛋白将NP细胞转化为P细胞。这个项目试图 识别HIV-1天然细胞靶点中的Sp140或其他蛋白质 如果不被VIF中和，它可能会根除感染。这部作品 可能会揭开艾滋病药物开发的新靶点。

DESCRIPTION: (provided by applicant) The HIV-1 Vif gene is essential for infectious virus production by nonpermissive cells (NP), which include CD4-positive T-lymphocytes and macrophages and some leukemic T-cell lines such as HUT78 and MT-2, but is irrelevant in other cell lines that are termed permissive (P). NP cells can be infected with Vif-deleted HIV-1 derived from P cells, but the infected NP cells release only noninfectious virions. These noninfectious virions cannot be rescued by Vif expression in target cells, and they are believed to have a defect in their genomic RNA or core proteins that reduces the efficiency of reverse transcription. We and another laboratory recently performed complementation studies which showed the dominance of the NP phenotype in PxNP heterokaryons, suggesting that NP cells contain a factor that can inactivate HIV-1 and that this factor is counteracted by Vif. Using a human lymphocyte cDNA library in a yeast two-hybrid screen with Vif as bait, we have now identified the interferon-inducible protein Sp140 as an excellent candidate for this NP factor. Briefly, Sp140 occurs in all tested NP cells but not in P cells. Sp140 occurs in nuclear bodies (NBs) that contain several proteins covalently modified by addition of the ubiquitin-related protein Sumo-1. Expression of Sp140 in Hela-CD4 cells induces sumoylation of at least one protein, and coexpression of Vif blocks this induction. Thus, Vif counteracts at least one biochemical effect of Sp140. We propose: (i) Analyze Sp140 isoforms and related proteins for their abilities to inactivate Vif-deleted H1V-1. (ii) Identify active sites for interaction of Vif with Sp140 and with other potential partners identified by our two-hybrid screen. (iii) By two-dimensional electrophoresis, identify all sumoylated and non-sumoylated proteins induced in P cells by Sp140, and determine how Vif influences these inductions. Similarly, determine whether Vif expession in NP cells alters protein sumoylation and whether Vif-deleted HIV-1 made in P or NP cells contains any sumoylated protein. (iv) Interestingly, the herpes simplex virus type 1 ICPO protein targets NBs and specifically causes the elimination of some sumoylated NB proteins, and proteins encoded by CMV, EBV, adenoviruses and arenaviruses appear to have similar activities. Determine whether expression of these other viral proteins converts NP cells to P. This project seeks to identify Sp140 or another protein in the natural cellular targets of HIV-1 that could potentially eradicate infection were it not neutralized by Vif. This work may unveil a novel target for drug development in AIDS.