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

描述(申请人提供)：HIV-1在感染者中的成功传播取决于病毒颗粒从受感染(生产者)到未受感染(目标)细胞的有效传播。体外繁殖研究已经证明，如果HIV-1粒子萌芽进入产生细胞和目标细胞之间形成的所谓病毒学突触(VS)的裂隙，它们就能最有效地传播到目标细胞。导致这种突触形成、维持和解体的事件还知之甚少。此前，我们和其他实验室已经证实，HIV-1颗粒存在于富含四氢青霉毒素的细胞中。这些细胞膜蛋白通常作为基于质膜的过程的组织者，包括细胞-细胞融合和黏附。我们的初步数据表明，Tetraspanin家族的个别成员不是病毒释放所必需的，但他们指出这些蛋白在调节病毒向靶细胞的转移方面发挥了作用。因此，我们建议评估产生细胞中的Tetraspanins是否是HIV-1颗粒在细胞间有效传播所必需的调节辅助因子。我们还将分析病毒介导的Tetraspanins下调的影响。总之，这些研究将进一步深入了解HIV-1传播的分子机制，从而了解其发病机制。在这项提议的具体目的中，我们建议：1)检验四肽抑制HIV-1诱导的膜融合，从而允许细胞间转移而不融合产生细胞和靶细胞的假设。2)确定HIV-1产生细胞中的Tetraspanins是否通过支持VS的形成、组织和分解而促进病毒颗粒向靶细胞的有效传递。3)研究HIV-1诱导的Tetraspanin在感染细胞中下调的动力学、决定因素和潜在后果。

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.