Role of Vif in HIV-1 Replication/AIDS

描述：（由申请人提供）HIV-1 Vif基因对于以下方面至关重要： 非允许细胞（NP）的感染性病毒生产，包括 CD 4阳性T淋巴细胞和巨噬细胞以及一些白血病T细胞系， 与HUT 78和MT-2一样，但在被称为 允许的（P）。NP细胞可以感染来自P 细胞，但感染的NP细胞仅释放非感染性病毒体。这些 非感染性病毒体不能通过靶细胞中的Vif表达来拯救，并且 据信它们的基因组RNA或核心蛋白存在缺陷， 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表明，NP的优势 PxNP异核体中的表型，表明NP细胞含有一种因子， 可以抑制HIV-1，并且这种因子被Vif抵消。使用人 利用酵母双杂交技术，以Vif为诱饵， 现在确定干扰素诱导蛋白Sp140是一个很好的候选者 对于这个NP因子。简言之，Sp140存在于所有测试的NP细胞中，但不存在于P 细胞Sp140存在于含有几种蛋白质的核体（NB）中 通过加入泛素相关蛋白Sumo-1进行共价修饰。 Sp140在Hela-CD 4细胞中的表达诱导至少一种蛋白的类小泛素化 蛋白，并且Vif的共表达阻断这种诱导。因此， Sp140的至少一种生化作用。我们建议：（i）分析Sp140 同种型和相关蛋白质的能力，以表达它们对Vif缺失的 H1V-1 (ii)确定Vif与Sp140和Sp140相互作用的活性位点。 通过我们的双杂交筛选确定的其他潜在合作伙伴。(iii)通过 双向电泳，鉴定所有sumoylated和non-sumoylated 蛋白诱导的P细胞的Sp140，并确定如何Vif影响这些 诱导。类似地，确定NP细胞中的Vif表达是否改变 蛋白质类小泛素化和Vif缺失的HIV-1是否在P或NP细胞中产生 含有任何sumoylated蛋白质。(iv)有趣的是，单纯疱疹病毒 1型ICPO蛋白靶向NB，并特异性地消除一些 sumoylated NB蛋白和由CMV、EBV、腺病毒和腺病毒编码的蛋白质。 沙粒病毒似乎具有类似的活性。确定是否表达 这些其他病毒蛋白质将NP细胞转化为P。该项目旨在 在HIV-1的天然细胞靶标中鉴定Sp140或另一种蛋白质， 如果不被病毒中和的话就有可能根除感染这项工作 可能会为艾滋病药物开发揭示一个新的靶点。

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.