Mechanisms that determine subcellular sites of HIV-1 assembly

描述（由申请人提供）：HIV-1颗粒的产生是一个由病毒结构蛋白Gag驱动的过程，主要发生在包括T细胞在内的细胞类型的质膜上，而在巨噬细胞中，大多数病毒颗粒似乎在晚期核内体中聚集。决定病毒组装位点的分子机制仍有待阐明。观察到的细胞类型差异与细胞因子参与确定HIV-1组装亚细胞位置的可能性是一致的。我们最近报道了HeLa细胞中质膜脂PI(4,5)P2的消耗将Gag从质膜转移到后期的核内体，并显著减少病毒颗粒的产生。初步结果表明，当PI(4,5)P2缺失时，Gag与总膜的结合也会受损。最近的结构数据表明PI(4,5)P2直接与Gag的矩阵结构域相互作用。这些结果表明Gag-PI(4,5)P2相互作用对于病毒在质膜上的组装至关重要。我们的目标是阐明PI(4,5)P2在不同细胞类型中调控HIV-1组装的分子机制。我们的具体目标是：[目标1]确定负责PI(4,5)P2依赖性的Gag区域。这将通过a) Gag衍生物的表征和b)适应低PI(4,5)P2水平的病毒的分离和表征来实现。[目的2]确定PI(4,5)P2是否通过直接与Gag结合或通过PI(4,5)P2依赖的细胞运输途径，或两者兼而有之来调节Gag定位。为了解决第一种可能性，我们将开发体外gag -脂质结合试验。为了检验第二种可能性，我们将分析抑制膜运输途径的影响，并将其与细胞PI(4,5)P2耗竭的影响进行比较。[目的3]探讨PI(4,5)P2在巨噬细胞Gag定位到晚期核内体和病毒颗粒产生中的作用。我们将定量分析PI(4,5)P2- gag共定位和巨噬细胞在PI(4,5)P2干扰或不干扰下的病毒释放。病毒组装的亚细胞位点在病毒生产效率和病毒持久性中起着关键作用。因此，分析决定组装位点的因素对HIV-1感染的干预策略具有重要意义。本文提出的实验将确定PI(4,5)P2在HIV-1颗粒产生中的作用，并将有助于未来开发针对HIV-1组装和释放的抗病毒药物。

DESCRIPTION (provided by applicant): HIV-1 particle production, a process driven by the viral structural protein Gag, takes place predominantly at the plasma membrane in cell types including T cells, whereas in macrophages a majority of virus particles appears to assemble in the late endosomes. The molecular mechanisms that determine the sites of virus assembly remain to be elucidated. The observed cell type difference is consistent with the possibility that a cellular factor(s) is involved in determining the subcellular location of HIV-1 assembly. We recently reported that depletion of the plasma membrane lipid PI(4,5)P2 in HeLa cells relocates Gag from the plasma membrane to late endosomes and markedly reduces virus particle production. Preliminary results show that Gag binding to total membrane is also impaired upon PI(4,5)P2 depletion. Recent structural data suggest that PI(4,5)P2 interacts directly with the matrix domain of Gag. These results suggest that a Gag-PI(4,5)P2 interaction is essential for virus assembly at the plasma membrane. Our goal in this proposal is to elucidate the molecular mechanisms by which PI(4,5)P2 regulates HIV-1 assembly in different cell types. Our specific aims are: [Aim 1] To identify Gag regions that are responsible for PI(4,5)P2 dependence. This will be achieved by a) characterization of Gag derivatives and b) isolation and characterization of viruses adapted to low PI(4,5)P2 levels. [Aim 2] To determine if PI(4,5)P2 regulates Gag localization by directly binding to Gag or through a PI(4,5)P2-dependent cellular trafficking pathway, or both. To address the first possibility, we will develop in vitro Gag-lipid binding assays. To examine the second possibility, we will analyze the impact of inhibition of membrane transport pathways and compare it with the effects of cellular PI(4,5)P2 depletion. [Aim 3] To examine, the role of PI(4,5)P2 in Gag localization to late endosomes and virus particle production in macrophages. We will perform quantitative analyses of PI(4,5)P2-Gag colocalization and virus release from macrophages with or without PI(4,5)P2 perturbation. The subcellular sites of virus assembly play key roles in the efficiency of virus production and viral persistence. Therefore, analyses of factors that determine the assembly sites have major implications in strategies for intervention in HIV-1 infection. The experiments proposed here will determine the roles played by PI(4,5)P2 in HIV-1 particle production and will contribute to the future development of antiviral drugs that target HIV-1 assembly and release.