The Cell Biology of HIV-1 Genome Trafficking

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)导致获得性免疫缺陷综合征(AIDS)。艾滋病毒-1疫苗的缺乏、多药耐药性和治疗的并发症(以及费用)强调了继续需要确定新的病毒-宿主接口，并有可能通过抗病毒战略进行靶向。我们最近发现了细胞内CRM1核输出受体的一个物种特异性属性，它可以抑制HIV-1‘S内含子的mRNAs(包括gRNAs)在小鼠细胞中的核质转运。CRM1在脊椎动物中非常保守，并调节一系列编码疏水性多肽的细胞和病毒蛋白的核输出，称为核输出信号(Ness)。HIV-1REV蛋白编码富含亮氨酸的NES，并将CRM1招募到编码顺式作用REV反应元件(RRE)的病毒mRNAs中，以促进它们的核输出。CRM1的S活动在上下文中是物种受限的 REV的突变导致了关于CRM1的S蛋白进化和细胞特有的作用模式的重要问题。首先，人CRM1(HCRM1)在做什么，而小鼠CRM1(MCRM1)不在做什么？我们已经定位了调节REV活性的hCRM1物种特异性决定因素。在目标1中，我们将研究这个决定因素在Rev/gRNA复合体形成中的作用，并确定mCRM1对HIV-1 Rev功能的阻断是否可以在人类细胞中表现出来。第二，为什么HIV-1 Rev适应优先利用hCRM1？在目标2中，我们将鉴定新发现的HIV-1Rev和gRNA突变体，它们在缺乏hCRM1的情况下拯救小鼠细胞中病毒粒子的产生，并测试hCRM1或这些病毒突变体是否能够在小鼠细胞培养中提供活跃的HIV-1复制。最后，在目标3中，我们将建立一个基于系统的活细胞成像策略，以研究影响HIV-1 gRNA核输出的扰动如何影响gRNA运输、Gag翻译、gRNA包装和有效的病毒颗粒组装的下游阶段。

DESCRIPTION (provided by applicant): Human mmunodeficiency virus type 1 (HIV-1) causes the acquired immunodeficiency syndrome (AIDS). The lack of an HIV-1 vaccine, multi-drug resistance and complications from (and cost of) treatment emphasize a continuing need to identify new virus-host interfaces with the potential for targeting with antiviral strategies. We recently identified a species-specific attribute of the cellular CRM1 nuclear export receptor that suppresses the nucleocytoplasmic transport of HIV-1's intron-containing mRNAs (including gRNAs) in murine cells. CRM1 is remarkably well conserved among vertebrates and regulates the nuclear export of a broad range of cellular and viral proteins encoding hydrophobic peptides known as nuclear export signals (NESs). The HIV-1 Rev protein encodes a leucine-rich NES and recruits CRM1 to viral mRNAs that encode the cis- acting Rev response element (RRE) in order to facilitate their nuclear export. That CRM1's activities are species limited in the context of Rev leads to important questions regarding CRM1's protein evolution and cell- specific modes of action. First, what is human CRM1 (hCRM1) doing that murine CRM1 (mCRM1) is not? We have mapped the hCRM1 species-specific determinant that regulates Rev activity. In Aim 1 we will study the role of this determinant in Rev/gRNA complex formation and determine if the mCRM1 block to HIV-1 Rev function can be made manifest in human cells. Second, why is HIV-1 Rev adapted to preferentially exploit hCRM1? In Aim 2, we will characterize newly identified HIV-1 Rev and gRNA mutants that rescue virion production in murine cells in the absence of hCRM1, and test if hCRM1 or these viral mutants can provide for active HIV-1 replication in murine cell culture. Finally, in Aim 3 we will establish a systems- based live cell imaging strategy to study how perturbations affecting HIV-1 gRNA nuclear export can influence the downstream stages of gRNA trafficking, Gag translation, gRNA packaging and efficient virus particle assembly.