The Cell Biology of HIV-1 Genome Trafficking

项目概要/摘要 截至2017年，全世界有超过3600万人感染HIV-1，艾滋病毒/艾滋病导致 每年约有100万人死亡。HIV-1是一种终身的持续感染。目前还没有治疗方法 能够在急性感染或潜伏期内永久抑制病毒基因表达 反弹该项目的长期目标是阐明HIV-1 RNA的细胞机制 亚细胞运输，翻译和基因组包装，以开发治疗方法， 在体内选择性地消除这些阶段。在当前的资助期内，我们阐明了细胞内在的障碍， 在小鼠和其他啮齿类动物细胞中的HIV-1基因组核输出。我们还开发了切割- 用于研究HIV-1病毒RNA（vRNA）运输和病毒颗粒的边缘定量成像策略 组装动力学。总的来说，这些研究表明， 离散的顺式作用病毒RNA结构元件和确定的RNA结合蛋白程序之间的关系 vRNA在细胞核和细胞质中具有显著的转运行为。例如，我们发现艾滋病毒- 1的Rev反应元件（RRE），由病毒Rev蛋白和细胞XPO 1核输出调节 受体，决定了以前未预料到的3步vRNA转运途径，其特征在于瞬时 亚核区室化事件、“爆发”核输出动力学和向外周部位的扩散 在细胞质中的翻译和基因组包装。在这里，我们测试的首要假设， HIV-1适应于利用XPO 1介导的“爆发”输出，以确保快速、非线性增加， 病毒晚期基因表达和促进病毒基因组向病毒体装配的有效递送 位于细胞外围。具体目标1的目标是定义核膜事件， 使用先进的高分辨率成像模式支持XPO 1指导的“爆发”vRNA核输出。 具体目标2应用比较视觉和生化方法来定义保守的功能， 广谱抗病毒靶向背景下的XPO 1连接的vRNA输出模块。具体目标3 使用基于细胞的分析和新的HIV-1报告病毒来研究在细胞中的“爆发”输出之间的联系。 细胞核和细胞质事件，包括Gag/Gag-Pol翻译、基因组包装和病毒颗粒 组装件.总的来说，这些详细的研究旨在揭示新的细胞生物学， 研究病毒的工具，并确定新的病毒-宿主相互作用相关的发展， 抑制体内HIV-1病毒体产生的疗法。

PROJECT SUMMARY / ABSTRACT More than 36 million people worldwide are living with HIV-1 infection as of 2017, with HIV/AIDS causing ~1 million deaths per year. HIV-1 establishes a life-long, persistent infection. There are no therapies yet capable of permanently suppressing viral gene expression in the context of acute infection or latency rebound. This project’s long-term goal is to elucidate the cellular mechanisms that underpin HIV-1 RNA subcellular trafficking, translation, and genome packaging toward the development of therapies to selectively abrogate these stages in vivo. In the current funding period we elucidated cell-intrinsic barriers to HIV-1 genome nuclear export in cells derived from mice and other rodents. We also developed cutting- edge, quantitative imaging strategies for studying HIV-1 viral RNA (vRNA) trafficking and virus particle assembly dynamics in human cells. Collectively, these studies revealed that cooperative interactions between discrete cis-acting viral RNA structural elements and defined RNA binding proteins program vRNAs for striking transport behaviors both in the nucleus and cytoplasm. For example, we found that HIV- 1’s Rev response element (RRE), regulated by the viral Rev protein and cellular XPO1 nuclear export receptor, dictates a previously unanticipated 3-step vRNA transport pathway characterized by transient subnuclear compartmentalization events, “burst” nuclear export kinetics, and diffusion to peripheral sites of translation and genome packaging in the cytoplasm. Herein we test the overarching hypothesis that HIV-1 is adapted to exploit XPO1-mediated “burst” export in order to ensure rapid, non-linear increases to viral late stage gene expression and to promote the efficient delivery of viral genomes to virion assembly sites at the cell periphery. The goal of Specific Aim 1 is to define the nuclear membrane events that underpin XPO1-directed “burst” vRNA nuclear export using advanced high-resolution imaging modalities. Specific Aim 2 applies a comparative visual and biochemical approach to define conserved features of XPO1-linked vRNA export modules in the context of broad-spectrum antiviral targeting. Specific Aim 3 uses cell-based assays and new HIV-1 reporter viruses to study the links between “burst” export at the nucleus and cytoplasmic events including Gag/Gag-Pol translation, genome packaging, and virus particle assembly. Collectively, these detailed studies are intended to expose new cell biology, deliver innovative tools for studying viruses, and identify novel virus-host interactions relevant to the development of therapies to suppress HIV-1 virion production in vivo.