Role of VPU in Retroviral Particle Assembly

VPU 在逆转录病毒颗粒组装中的作用

• 批准号: 6840086
• 负责人: PAUL W. SPEARMAN
• 金额: $ 32.7万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-20 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6840086
• 关键词: Retroviridae; cell line; clinical research; complementary DNA; expression cloning; fluorescence recovery after photobleaching; fluorescence resonance energy transfer; gag protein; heterokaryon; human immunodeficiency virus; immunoelectron microscopy; molecular assembly /self assembly; particle; protein protein interaction; protein structure function; protein transport; species difference; subtraction hybridization; virus assembly; virus genetics; virus protein

DESCRIPTION (provided by applicant): Vpu is an HIV accessory gene that induces degradation of CD4 in the endoplasmic reticulum and enhances viral particle release. This project seeks to define the mechanism by which Vpu enhances viral particle release. Preliminary studies in our laboratory show that human cells are restricted for particle assembly and require Vpu for efficient particle production (restrictive/Vpu-responsive). Other cells, notably simian cells, are permissive for efficient assembly and do not respond to Vpu (permissive/Vpu-unresponsive). In heterokaryons, the restrictive phenotype dominates, suggesting strongly that a cellular inhibitor of assembly is present in restrictive cell types. Experiments in Aim 1 of this proposal will further characterize this cellular inhibitor. The Vpu-responsive cellular factor will be measured in cell lines and in human primary T cells and macrophages, and a survey of simian cells will determine if indeed this is a species-specific factor. In Aim 2, the site of the assembly block in restrictive cells will be determined. Biochemical fractionation of pulse-labeled cells will define the kinetics of Gag trafficking in the presence and absence of Vpu. Quantitative live cell imaging techniques will be employed to identify the subcellular location where Gag accumulates in restrictive cells, and to define the subcellular site of action of Vpu. A novel Gag-Gag interaction assay based upon fluorescence resonance energy transfer (FRET) will allow the identification of intracellular Gag multimers in living cells; the influence of Vpu on these assembly intermediates will be assessed. Experiments in Aim 3 will identify the cellular inhibitor of assembly that is overcome by Vpu. An expression cloning strategy will be utilized to identify cellular factor(s) in restrictive cells that inhibit HIV assembly and are overcome by Vpu. In parallel, a subtractive hybridization approach utilizing restrictive and permissive Jurkat cell clones will identify candidate cellular cDNAs that restrict assembly. These studies will define the mechanism by which Vpu enhances HIV particle release and will characterize a novel host cell restriction to HIV replication that is present in human cells.

描述（申请人提供）：Vpu 是一种 HIV 辅助基因，可诱导内质网中 CD4 的降解并增强病毒颗粒的释放。该项目旨在明确 Vpu 增强病毒颗粒释放的机制。我们实验室的初步研究表明，人体细胞的颗粒组装受到限制，需要 Vpu 才能有效地产生颗粒（限制性/Vpu 响应型）。其他细胞，特别是猿猴细胞，允许有效组装，并且不对 Vpu 做出响应（允许/Vpu 无响应）。在异核体中，限制性表型占主导地位，强烈表明限制性细胞类型中存在细胞组装抑制剂。本提案目标 1 的实验将进一步表征这种细胞抑制剂。 Vpu 反应性细胞因子将在细胞系、人类原代 T 细胞和巨噬细胞中进行测量，并且对猿猴细胞的调查将确定这是否确实是物种特异性因子。在目标 2 中，将确定限制性细胞中装配块的位点。脉冲标记细胞的生化分级将定义在存在和不存在 Vpu 的情况下 Gag 运输的动力学。将采用定量活细胞成像技术来识别 Gag 在限制性细胞中积累的亚细胞位置，并确定 Vpu 的亚细胞作用位点。基于荧光共振能量转移 (FRET) 的新型 Gag-Gag 相互作用测定将能够识别活细胞中的细胞内 Gag 多聚体；将评估 Vpu 对这些组装中间体的影响。目标 3 中的实验将确定 Vpu 克服的细胞组装抑制剂。将利用表达克隆策略来鉴定限制性细胞中抑制 HIV 组装并被 Vpu 克服的细胞因子。与此同时，利用限制性和允许性 Jurkat 细胞克隆的消减杂交方法将鉴定限制组装的候选细胞 cDNA。这些研究将确定 Vpu 增强 HIV 颗粒释放的机制，并将描述人类细胞中存在的一种新的宿主细胞对 HIV 复制的限制。