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; 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理解人类免疫缺陷病毒I型（HIV-1）编码的VIF（病毒感染因子）的功能。 VIF对于在体内或培养物中对于非抗药性细胞（例如外周血淋巴样细胞，巨噬细胞和H9 T细胞）都是必不可少的。 VIF基因的缺陷会影响病毒成分的病毒形态，而不会影响病毒成分的表达。 我们实验室和其他人的数据表明，VIF会影响逆转录，这可能是由于特定靶细胞中逆转录（RT）复合物的不稳定性。 由于VIF蛋白不是专门包装到病毒体中的，因此据信VIF在产生病毒细胞中起作用并影响病毒组装。 最近，我们证明了VIF是一种RNA结合蛋白，并与产生病毒细胞的细胞质中的HIV-1基因组RNA结合，形成了40 S的Messenger核糖核蛋白（MRNP）复合物。 VIF对病毒RNA的涂层可以保护病毒RNA免受细胞质中各种内源性抑制剂的影响。 此外，在形成萌芽粒子的过程中，MRNP复合物中的VIF可能会介导HIV-1插入前体的病毒RNA接合。 因此，可以通过VIF基因缺陷来改变适当的核蛋白酶复合物的形成。 长期目标是了解这种独特的慢病毒蛋白的分子机制，我们将执行以下一系列实验，以研究VIF的功能：（1）剖析VIF病毒RT复合物的结构和成分。 特别是在搜索异常的RNA折叠和冷凝的证据； （2）。进一步对VIF-RNA和VIF-GAG相互作用进行生化分析。 特别是，定义VIF蛋白是否是RNA伴侣； （3）。进一步定义了VIF在细胞内病毒RNA运输和包装过程中的作用。 因此，这些聚焦和相互关联的实验将允许对RT机械组装中VIF蛋白的分子机制进行精确分析，并确定VIF基因对体内HIV-1发病机理的影响。