Role of HIV-1 VIF in Assembly of the RT Complex

HIV-1 VIF 在 RT 复合物组装中的作用

• 批准号: 6648418
• 负责人: Hui Zhang
• 金额: $ 27.48万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-17 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648418
• 关键词: RNA binding protein; RNA directed DNA polymerase; RNase protection assay; SDS polyacrylamide gel electrophoresis; autoradiography; density gradient ultracentrifugation; enzyme biosynthesis; enzyme complex; gag protein; human immunodeficiency virus 1; immunoprecipitation; intracellular transport; molecular chaperones; northern blottings; nucleic acid hybridization; pathologic process; polymerase chain reaction; protein protein interaction; ribonucleoproteins; scintillation counter; virion; virulence; virus RNA; virus assembly; virus protein

This research plan will seek to further the understanding of the function of Vif (virion infectivity factor) encoded by human immunodeficiency virus type I (HIV-1). Vif is essential for viral replication either in vivo or in culture for non-permissive cells, such as peripheral blood lymphoid cells, macrophages and H9 T-cells. Defects in the vif gene will affect the virion morphology, but not the expression of viral components. Data from our laboratory and others have indicated that Vif affects reverse transcription, possibly because of the instability of reverse transcription (RT) complex in specific target cells. As Vif protein is not specifically packaged into virions, it is believed that Vif functions in the virus-producing cells and affects viral assembly. Recently, we demonstrated that Vif is an RNA binding protein and binds to HIV-1 genomic RNA in the cytoplasm of virus-producing cells to form a 40 S messenger ribonucleoprotein (mRNP) complex. The coating of Vif to viral RNA may protect the viral RNA from various endogenous inhibitors in the cytoplasm. In addition, Vif in mRNP complexes may mediate viral RNA engagement with HIV-1 Gag precursors during formation of a budding particle. As such, the formation of a proper nucleocapsid complex could be altered by vif gene defect. With a long term goal of understanding the molecular mechanism(s) underlying this unique lentiviral protein, we will perform the following series of experiments to study the function of Vif:(1) Dissect the structure and components of RT complexes of vif- viruses. Especially, search the evidence of aberrant RNA folding and condensation; (2). Further perform biochemical analysis for Vif-RNA and Vif-Gag interactions. Especially, define whether Vif protein is an RNA chaperone; (3). Further define the role of Vif in the process of intracellular viral RNA trafficking and packaging. Thus, these focused and interrelating experiments will allow precise analyses of the molecular mechanism(s) of Vif protein in the assembly of RT machinery, and determine the effects of vif gene on HIV-1 pathogenesis in vivo

该研究计划将寻求进一步了解 I 型人类免疫缺陷病毒 (HIV-1) 编码的 Vif（病毒粒子感染因子）的功能。 Vif 对于体内或培养中不允许的细胞（例如外周血淋巴细胞、巨噬细胞和 H9 T 细胞）的病毒复制至关重要。 vif基因的缺陷会影响病毒粒子的形态，但不会影响病毒成分的表达。 我们实验室和其他实验室的数据表明，Vif 会影响逆转录，可能是因为特定靶细胞中逆转录 (RT) 复合物的不稳定性。 由于 Vif 蛋白没有被特异性包装到病毒粒子中，因此人们认为 Vif 在病毒产生细胞中发挥作用并影响病毒组装。 最近，我们证明Vif是一种RNA结合蛋白，可以与病毒产生细胞的细胞质中的HIV-1基因组RNA结合，形成40S信使核糖核蛋白（mRNP）复合物。 Vif 包裹在病毒 RNA 上可以保护病毒 RNA 免受细胞质中各种内源抑制剂的影响。 此外，mRNP 复合物中的 Vif 可能在出芽颗粒形成过程中介导病毒 RNA 与 HIV-1 Gag 前体的结合。 因此，适当的核衣壳复合物的形成可以通过 vif 基因缺陷来改变。 为了了解这种独特的慢病毒蛋白的分子机制，我们将进行以下一系列实验来研究Vif的功能：（1）解析vif病毒RT复合物的结构和成分。 特别是寻找异常RNA折叠和缩合的证据； （2）。进一步对 Vif-RNA 和 Vif-Gag 相互作用进行生化分析。 特别是，确定Vif蛋白是否是RNA伴侣； （3）。进一步明确Vif在细胞内病毒RNA运输和包装过程中的作用。 因此，这些集中且相互关联的实验将允许精确分析 Vif 蛋白在 RT 机器组装中的分子机制，并确定 vif 基因对体内 HIV-1 发病机制的影响