Role of VPU in Retroviral Particle Assembly

描述（由申请人提供）：Vpu是一种HIV附属基因，可诱导内质网CD4的降解并促进病毒颗粒的释放。该项目旨在确定Vpu增强病毒颗粒释放的机制。我们实验室的初步研究表明，人类细胞的颗粒组装受到限制，并且需要Vpu才能有效地产生颗粒（限制性/Vpu响应性）。其他细胞，特别是类人猿细胞，对有效组装是允许的，对Vpu没有反应（允许/不响应）。在异核细胞中，限制性表型占主导地位，这强烈表明在限制性细胞类型中存在细胞组装抑制剂。本提案Aim 1中的实验将进一步表征这种细胞抑制剂。vpu反应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，对猿类细胞的调查将确定这是否确实是一种物种特异性因子。在Aim 2中，将确定限制性细胞中组装块的位置。脉冲标记细胞的生化分离将确定在Vpu存在和不存在的情况下Gag运输的动力学。定量活细胞成像技术将用于确定Gag在限制性细胞中积聚的亚细胞位置，并确定Vpu的亚细胞作用位点。一种基于荧光共振能量转移（FRET）的新型Gag-Gag相互作用测定将允许在活细胞中鉴定细胞内Gag多聚体；将评估Vpu对这些组装中间体的影响。Aim 3的实验将确定被Vpu克服的细胞组装抑制剂。表达克隆策略将用于鉴定限制性细胞中抑制HIV组装并被Vpu克服的细胞因子。同时，利用限制性和允许性Jurkat细胞克隆的减法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，并将描述存在于人类细胞中的一种新的宿主细胞对HIV复制的限制。

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.