Mechanisms that determine subcellular sites of HIV-1 assembly

描述(申请人提供)：HIV-1颗粒的产生是由病毒结构蛋白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聚集在晚期的内小体中。决定病毒组装位置的分子机制仍有待阐明。观察到的细胞类型差异与一种细胞因子(S)参与确定HIV-1组装的亚细胞位置的可能性一致。我们最近报道，HeLa细胞质膜脂质PI(4，5)P2的耗尽会使Gag从质膜重新定位到晚期内小体，并显著减少病毒颗粒的产生。初步结果表明，PI(4，5)P2耗竭时，GAG与总膜的结合也受到损害。最近的结构数据表明，PI(4，5)P2直接与GAG的基质结构域相互作用。这些结果表明，Gag-PI(4，5)P2相互作用是病毒在质膜上组装所必需的。我们在这个方案中的目标是阐明PI(4，5)P2调节不同细胞类型的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依赖的GAG区域。这将通过a)GAG衍生物的鉴定和b)适应低PI(4，5)P2水平的病毒的分离和鉴定来实现。[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依赖的细胞转运途径调节GAG定位，或两者兼而有之。为了解决第一种可能性，我们将开发体外Gag-脂质结合分析。为了检验第二种可能性，我们将分析抑制膜转运途径的影响，并将其与细胞PI(4，5)P2耗尽的影响进行比较。[目的3]研究PI(4，5)P2在巨噬细胞内晚期内体Gag定位和病毒颗粒产生中的作用。我们将对PI(4，5)P2-Gag共定位以及在有或没有PI(4，5)P2干扰的情况下巨噬细胞释放病毒进行定量分析。病毒组装的亚细胞位置对病毒的生产效率和病毒的持久性起着关键作用。因此，对决定组装部位的因素的分析对干预HIV-1感染的策略具有重要意义。这里提出的实验将确定PI(4，5)P2在HIV-1颗粒生产中所起的作用，并将有助于未来针对HIV-1组装和释放的抗病毒药物的开发。

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.