Molecular regulation of HIV-1 assembly, release and cell-to-cell transmission

描述（由申请方提供）：HIV-1在感染个体中的成功传播取决于病毒颗粒从感染（生产者）到未感染（靶）细胞的有效传播。体外繁殖研究已经确定，如果HIV-1颗粒在生产细胞和靶细胞之间形成的所谓病毒学突触（VS）的间隙中发芽，则它们最有效地传播到靶细胞。导致这种突触的形成、维持和分解的事件知之甚少。以前，我们和其他实验室已经确定，HIV-1颗粒在富含四跨膜蛋白的位点从细胞中退出。这些细胞膜蛋白通常作为基于质膜的过程的组织者起作用，包括细胞-细胞融合和粘附。我们的初步数据表明，四跨膜蛋白家族的个别成员不是病毒释放所必需的，但它们指出了这些蛋白质在调节病毒转移到靶细胞中的作用。因此，我们建议评估生产细胞中的四跨膜蛋白是否是HIV-1颗粒有效细胞间传播所必需的调节辅因子。我们还将分析病毒介导的四跨膜蛋白下调的影响。总之，这些研究将提供进一步深入了解HIV-1传播的分子机制，从而发病机制。在本提案的具体目的中，我们提出：1）检验四跨膜蛋白抑制HIV-1诱导的膜融合，从而允许细胞间转移而不使生产细胞和靶细胞融合的假设。2)确定HIV-1生产细胞中的四跨膜蛋白是否通过支持VS的形成、组织和分解来促进病毒颗粒向靶细胞的有效传播。3）检查感染细胞中HIV-1诱导的四跨膜蛋白下调的动力学、决定因素和潜在后果。公共卫生相关性：拟议的分析旨在阐明HIV-1从细胞到细胞的传播，这是病毒复制周期中的一个步骤，目前仍知之甚少。因此，这些结果可能揭示了干预HIV-1在感染者中传播的新靶点。

DESCRIPTION (provided by applicant): Successful dissemination of HIV-1 in infected individuals depends on efficient transmission of viral particles from infected (producer) to uninfected (target) cells. In vitro propagation studies have established that HIV-1 particles are most effectively transmitted to target cells if they bud into the cleft of the so-called virological synapse (VS) that forms between producer and target cells. The events leading to the formation, maintenance and disassembly of this synapse are poorly understood. Previously, we and other laboratories have established that HIV-1 particles exit from cells at sites that are enriched in tetraspanins. These cellular membrane proteins normally function as organizers of plasma membrane-based processes, including cell-cell fusion and adhesion. Our preliminary data suggest that individual members of the tetraspanin family are not required for virus release, but they point to a role of these proteins in regulating virus transfer to target cells. We thus propose to evaluate if tetraspanins in producer cells are regulatory cofactors necessary for efficient cell-to-cell transmission of HIV-1 particles. We will also analyze the effects of virus-mediated downregulation of tetraspanins. Altogether, these studies will provide further insight into the molecular mechanisms underlying HIV-1 spread and thus pathogenesis. In the Specific Aims of this proposal we propose: 1) To test the hypothesis that tetraspanins inhibit HIV-1-induced membrane fusion, thus allowing cell-to-cell transfer without fusion of producer and target cell. 2) To determine if tetraspanins in HIV-1 producer cells promote efficient transmission of viral particles to target cells by supporting the formation, the organization and the disassembly of the VS. 3) To examine the kinetics, determinants and potential consequences of HIV-1-induced tetraspanin downregulation in infected cells. PUBLIC HEALTH RELEVANCE: The proposed analyses are aimed at elucidating HIV-1 transmission from cell-to-cell, a step in the viral replication cycle that remains poorly understood. Hence, the results may reveal novel targets for intervention with HIV-1 spread in infected individuals.