Role of Vif in HIV-1 Replication/AIDS

Vif 在 HIV-1 复制/艾滋病中的作用

• 批准号: 6742442
• 负责人: David Kabat
• 金额: $ 26.37万
• 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 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742442
• 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基因对于由非腐败细胞（NP）产生感染性病毒至关重要，其中包括CD4阳性的T淋巴细胞和巨噬细胞以及一些白血病T细胞系，但与其他称为允许性（P）的细胞系无关。 NP细胞可以被P细胞衍生的VIF骨骼骨骼HIV-1感染，但感染的NP细胞仅释放非感染性病毒体。这些非传染性病毒体无法通过靶细胞中的VIF表达来挽救，并且据信它们具有降低逆转录效率的缺陷。我们和另一个实验室最近显示了NP表型在PXNP杂核中的主导性，这表明NP细胞包含一种可能使HIV-1失活的因素，并且该因素被VIF抵消。使用人类淋巴细胞cDNA文库在带有VIF作为诱饵的酵母两杂化筛选中，我们现在已经确定了干扰素诱导的蛋白SP140是该NP因子的出色候选者。简而言之，SP140发生在所有测试的NP细胞中，但在P细胞中不发生。 SP140发生在核体（NB）中，其中包含通过添加泛素相关蛋白SUMO-1的几种共价修饰的几种蛋白质。 SP140在HeLa-CD4细胞中的表达诱导至少一种蛋白质的sumoylation，而VIF的共表达阻塞了这种诱导。 HIV-1感染诱导SP140移民到细胞质及其与VIF的部分共定位。我们提出：（i）通过遗传，免疫学和生化方法验证VIF与SP140以及与其他潜在伴侣的相互作用。 （ii）分析SP140同工型和其他潜在的VIF伴侣，以使其失活VIF HIV-1的能力。 （iii）确定活跃位点与SP140以及其他潜在伙伴的相互作用。 （iv）通过二维电泳，鉴定由SP140在P细胞中诱导的Sumoypated和非肿瘤化蛋白，并确定VIF如何影响这些诱导。同样，确定NP细胞中的VIF表达是否改变了蛋白质Sumoylation，以及在P或NP细胞中制造的VIF骨中的HIV-1是否含有任何sumoyy的蛋白质。 （iv）有趣的是，单纯疱疹病毒1型ICPO蛋白靶向NB，并特别导致消除某些sumoymet的NB蛋白，以及由CMV，EBV，EBV，腺病毒和体育症病毒编码的蛋白质似乎具有相似的活性。确定这些其他病毒蛋白的表达是否会将NP细胞转化为P。这项工作可能会揭示出艾滋病中药物开发的新靶标。