Mechanisms that determine subcellular sites of HIV-1 assembly

描述(申请人提供)：艾滋病的病原体HIV-1的病毒颗粒组装发生在大多数细胞类型的质膜(PM)，包括自然宿主T细胞。GAG定位到PM是由矩阵(MA)域驱动的。MA通过N-末端肉豆蔻基部分和与酸性脂类结合的高碱性区域(HBR)介导GAG的膜结合。HBr与PM特异性酸性磷脂PI(4，5)P2的结合对PM定位Gag和有效释放病毒至关重要。值得注意的是，体外研究表明，MA HBR也与RNA相互作用，这抑制了GAG与非PI(4，5)P2酸性脂类的结合，表明RNA参与了MA-膜的相互作用。然而，PI(4，5)P2和RNA调节PM特异性GAG定位的机制仍未阐明。一旦到达PM，GAG进一步与称为脂筏的膜微结构域以及更大的PM结构域特异性地结合。在与未感染细胞接触的产生病毒的T细胞中，GAG与其他病毒和细胞蛋白一起特异性地聚集在PM区域，形成被称为病毒学突触(VS)的细胞-细胞连接。VS通过新形成的病毒颗粒的有效转移促进病毒在细胞之间的传播。值得注意的是，在T细胞中，Gag多聚体定位于称为尾足的后端突起，最终构成VS。然而，尽管在病毒传播中很重要，但Gag多聚体定位于尾足类动物并最终定位于VS的机制尚不清楚。我们的长期目标是阐明决定HIV-1组装亚细胞位置的机制。我们在这一应用中的中心假设是，酸性脂质和RNA之间对MA结合的竞争决定了Gag在质膜上的定位，在质膜上，Gag的多聚体和微域结合促进了Gag在尾足的积累，最终形成病毒学突触。为了验证这一假说，我们计划追求以下三个特定目标：[目的1]确定脂质和RNA调节PM结合GAG的机制。使用体外和基于细胞的GAG-膜相互作用的方法，我们将阐明RNA和酸性脂类之间竞争的分子决定因素及其对GAG多聚化的下游影响。[目的2]阐明GAG与膜微区结合的决定因素。使用一种新的体外系统，我们将确定一种独特的MA-PI(4，5)P2相互作用和Gag多聚体对Gag-RAFT结合的贡献。[目的3]确定核衣壳驱动的多聚化将GAG导向尾足的机制。使用生化和高分辨率显微镜方法，我们将分析GAG多聚体与可能将GAG与后向肌动蛋白流动联系在一起的尾足类定向膜蛋白的关联。从本提案中概述的实验中获得的知识可能有助于我们开发药物干预机制，调节PM和VS的GAG定位，从而抑制细胞外病毒的释放和细胞间的传播。

DESCRIPTION (provided by applicant): Virus particle assembly of HIV-1, the causative agent of AIDS, takes place at the plasma membrane (PM) in most cell types including natural host T cells. Gag localization to the PM is driven by the matrix (MA) domain. MA mediates membrane binding of Gag via N-terminal myristoyl moiety and a highly basic region (HBR) that binds acidic lipids. Binding of HBR to a PM-specific acidic phospholipid PI(4,5)P2 is critical for PM localization of Gag and efficient virus release. Notably, in vitro studies showed that MA HBR also interacts with RNA, which suppresses binding of Gag to non-PI(4,5)P2 acidic lipids, suggesting that RNA is involved in MA-membrane interactions. However, mechanisms by which PI(4,5)P2 and RNA regulate PM-specific Gag localization remain to be elucidated. Once at the PM, Gag further associates specifically with membrane microdomains known as lipid rafts as well as larger PM domains. In virus-producing T cells contacting uninfected cells, Gag, along with other viral and cellular proteins, specifically accumulates at the area of the PM forming a cell-cell junction known as the virological synapse (VS). The VS facilitates cell-to-cell virus transmission via efficient transfer of newly formed virus particles. Notably, in T cells, Gag multimers localize to a rear-end protrusion termed the uropod that eventually constitutes the VS. Despite the importance in virus spread, however, the mechanism by which Gag multimers localize to uropods and eventually to the VS is not well understood. Our long-term goal is to elucidate mechanisms that determine subcellular sites of HIV-1 assembly. Our central hypothesis in this application is that competition between acidic lipids and RNA for MA binding determines Gag localization to the plasma membrane, where Gag multimerization and microdomain association facilitate Gag accumulation at the uropod that eventually forms virological synapses. To test this hypothesis, we plan to pursue the following three specific aims: [Aim 1] Determine the mechanisms by which lipids and RNA regulate PM binding of Gag. Using in vitro and cell-based assays for Gag-membrane interactions, we will elucidate molecular determinants for the competition between RNA and acidic lipids and its downstream effect on Gag multimerization. [Aim 2] Elucidate the determinants for association between Gag and membrane microdomains. Using a novel in vitro system, we will determine contributions of a unique mode of MA-PI(4,5)P2 interaction and Gag multimerization to Gag-raft association. [Aim 3] Identify the mechanism by which nucleocapsid-driven multimerization directs Gag to the uropod. Using biochemical and high-resolution microscopy methods, we will analyze association of Gag multimers with uropod-directed membrane proteins that might link Gag to rearward actin flow. The knowledge gained from experiments outlined in this proposal will likely help us develop strategies for pharmacological intervention of mechanisms regulating Gag localization to the PM and the VS, thereby inhibiting extracellular virus release and cell-to-cell transmission.