Molecular mechanisms of the CA-SP1 switch in HIV assembly and maturation

项目总结/摘要 HIV最初是作为一种非传染性的、不成熟的颗粒组装起来的，它必须经历一个成熟过程， 变得具有传染性。组装和成熟涉及病毒衣壳的大规模重排。这些 反过来，大规模的重排与衣壳中程序化的局部构象变化有关， 称为分子开关的蛋白质片段。在这些研究中，我们将使用结构，生物物理和其他 互补的方法来了解不成熟的HIV-1衣壳的结构，以及分子 跨越HIV-1 Gag蛋白CA-SP1区域的开关控制未成熟病毒装配， 成熟我们提出：（1）检验CA-SP1连接折叠成6螺旋束的假设， 在未成熟病毒体内进行可逆的和合作的解折叠，以调节病毒的进入。 成熟过程中的病毒蛋白酶。我们还将确定小分子成熟抑制剂，如贝韦瑞 通过结合和稳定6-螺旋束来破坏这一过程。我们还将开展实验， 了解Gag-Gag相互作用在成熟的早期阶段如何变化。(2)详细说明 未成熟HIV-1 Gag晶格的分子组织。特别是，我们将确定高分辨率 Gag-Gag相互作用（由CA-NTD结构域介导）的最外层如何组织的详细信息 在未成熟的颗粒中。我们还将测试的假设，CA-NTD层之间的相互作用， 下面CA-CTD层对于正确组装是重要的，且可以是 连接两个位于CA区侧翼的分子开关。最后，我们还将分析未成熟的Gag格， 其他逆转录病毒，以阐明开关介导的Gag组装的一般性质。如果成功， 这些研究将导致对逆转录病毒的未成熟衣壳结构的彻底理解， 在逆转录病毒装配过程中，构象转换转化为大规模衣壳重组， 成熟，以及如何使用小分子抑制剂来破坏衣壳重排。

Project Summary/Abstract HIV initially assembles as a non-infectious, immature particle that must undergo a maturation process to become infectious. Assembly and maturation involve large-scale rearrangements of the viral capsid. These large-scale rearrangements, in turn, are associated with programmed local conformational changes in capsid protein segments termed molecular switches. In these studies, we will use structural, biophysical, and other complementary approaches to understand the structure of the immature HIV-1 capsid and how a molecular switch spanning the CA-SP1 region of the HIV-1 Gag protein controls immature virus assembly and maturation. We propose to: (1) Test the hypothesis that the CA-SP1 junction folds into a 6-helix bundle that undergoes reversible and cooperative unfolding and folding within immature virions to regulate access of the viral protease during maturation. We will also determine how small molecule maturation inhibitors like bevirimat disrupt this process by binding and stabilizing the 6-helix bundle. We will also initiate experiments to understand how Gag-Gag interactions change during the early stages of maturation. (2) Elucidate the detailed molecular organization of the immature HIV-1 Gag lattice. In particular, we will determine the high resolution details of how the outermost set of Gag-Gag interactions (mediated by the CA-NTD domain) are organized within the immature particle. We will also test the hypotheses that interactions between the CA-NTD layer and the underlying CA-CTD layer are important for proper assembly and may be part of a structural network that links two molecular switches that flank the CA region. Finally, we will also analyze the immature Gag lattices of other retroviruses, in order to elucidate the general nature of switch-mediated Gag assembly. If successful, these studies will lead to a thorough understanding of the immature capsid structure of retroviruses, how local conformational transitions translate into large-scale capsid rearragenements during retroviral assembly and maturation, and how small molecule inhibitors may be deployed to disrupt capsid rearrangement.