Role of Vif in HIV-1 Replication/AIDS

Vif 在 HIV-1 复制/艾滋病中的作用

• 批准号: 6891314
• 负责人: David Kabat
• 金额: $ 26.43万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891314
• 关键词: AIDS; human immunodeficiency virus 1; laboratory rabbit; posttranslational modifications; protein isoforms; protein protein interaction; protein structure function; virus protein; virus replication; yeast two hybrid system

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, 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 that reduces efficiency of reverse transcription. We and another laboratory recently 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. HIV-1 infections induce Sp140 emigration to the cytosol and its partial colocalization with Vif. We propose: (i) Verify Vif interaction with Sp140 and with other potential partners by genetic, immunological, and biochemical methods. (ii) Analyze Sp140 isoforms and other potential Vif partners for their abilities to inactivate vif-deleted HIV-1. (iii) Identify active sites for interaction of Vif with Sp140 and with other potential partners. (iv) By two-dimensional electrophoresis, identify sumoylated and non-sumoylated proteins induced in P cells by Sp140, and determine how Vif influences these inductions. Similarly, determine whether Vif expression 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 work may unveil a novel target for drug development in AIDS.

HIV-1 vif基因对非受纳细胞（NP）产生感染性病毒至关重要，非受纳细胞包括cd4阳性t淋巴细胞和巨噬细胞以及一些白血病t细胞系，但与其他被称为受纳细胞(P)的细胞系无关。NP细胞可以被来自P细胞的vif缺失的HIV-1感染，但被感染的NP细胞只释放非感染性病毒粒子。这些非感染性病毒粒子不能通过靶细胞中的vif表达来拯救，并且它们被认为具有降低逆转录效率的缺陷。我们和另一个实验室最近发现NP表型在PxNP异核细胞中占主导地位，这表明NP细胞含有一种可以灭活HIV-1的因子，而该因子被Vif抵消。利用酵母双杂交筛选的人淋巴细胞cDNA文库，以Vif为诱饵，我们现在已经确定干扰素诱导蛋白Sp140是该NP因子的优秀候选者。简单地说，Sp140出现在所有的NP细胞中，而不出现在P细胞中。Sp140发生在含有几种被泛素相关蛋白Sumo-1共价修饰的蛋白的核小体（NBs）中。在Hela-CD4细胞中表达Sp140可诱导至少一种蛋白的sumo化，而Vif的共表达可阻断这种诱导。HIV-1感染诱导Sp140向细胞质迁移并与Vif部分共定位。我们建议：(i)通过遗传学、免疫学和生化方法验证Vif与Sp140和其他潜在伴侣的相互作用。（ii）分析Sp140亚型和其他潜在的Vif伙伴灭活Vif缺失的HIV-1的能力。（iii）确定Vif与Sp140及其他潜在合作伙伴相互作用的活性位点。（iv）通过双向电泳，鉴定Sp140在P细胞中诱导的sumoylated和非sumoylated蛋白，并确定Vif如何影响这些诱导。同样，确定NP细胞中Vif的表达是否会改变蛋白质的聚合化，以及P细胞或NP细胞中Vif缺失的HIV-1是否含有任何聚合化的蛋白质。（iv）有趣的是，单纯疱疹病毒1型ICPO蛋白以NB为靶点，并特异性地导致一些聚合的NB蛋白的消除，而CMV、EBV、腺病毒和沙粒病毒编码的蛋白似乎具有类似的活性。确定这些其他病毒蛋白的表达是否将NP细胞转化为p细胞。这项工作可能为艾滋病药物开发揭示一个新的靶点。