Structural Basis for HIV-1 Gag Interactions with Cellular Constituents

在HIV-1感染的晚期，Gag蛋白被合成并在细胞质中折叠成 组装非活性形式，其中不仅有核衣壳(NC)结构域，而且还有基质(MA)结构域 与RNA相互作用。由于这种“折叠”的构象，衣壳(CA)结构域变成 不能组装的。在运输到组装的质膜(PM)位置期间或之后， 发生构象转换，其中MA与NC的相互作用被交换为MA的相互作用 使用PM组件。此开关释放CA域以进行组装，并且此构象 重排是激活GAG所必需的，它是由特定PM组件的结合亲和力驱动的 为马萨诸塞州。要了解这一关键的开关，在分子水平上阐明是至关重要的。 MA与PM组分的相互作用。有趣的是，与之密切相关的逆转录病毒的Gag蛋白 例如禽肉瘤病毒(ASV)含有结构域，其功能基本上与 HIV-1 Gag；然而，在PM上组装Gag的机制似乎不同。理解 这种开关的分子基础不仅将揭示HIV-1的组装，而且可能提供 洞察在PM组装的其他逆转录病毒的组装控制装配后 在PM上，Gag蛋白招募包膜(Env)蛋白并入病毒颗粒。的确有 越来越多的证据表明，新生病毒粒子中包膜蛋白的掺入是通过相互作用来调节的 在GAG的MA结构域和gp41的胞质尾部(CT)之间。打喷嚏的机制 调解环境公司的合并尚不清楚。在这个提案中，我们将使用核磁共振、生物物理、生化 以及活体工具，以提供GAG蛋白如何与PM结合的大分子图像，并 确定介导env进入病毒的gag-gp41相互作用的分子元件 粒子。在目标1中，我们将确定HIV-1 Gag与PM结合的确切分子机制 我们设计了创新的方法来表征MA和膜模拟物之间的相互作用。 在目标2中，我们将确定PM上ASV Gag组装的分子元件我们拿出证据证明 ASV MA与PM组分结合后的一种新的分子开关。在目标3中，我们将描述 HIV-1MA与gp41 CT的相互作用我们设计了一种新的战略来重建和模仿 相互作用的生理相关条件。这些研究将提供有关分子的详细信息 激活PM上的GAG组装并帮助环境蛋白募集到组装的开关 网站。我们希望这项提议的结果将有助于开发新的抗病毒疗法。 抑制组装和病毒产生的试剂。

During the late phase of HIV-1 infection, the Gag protein is synthesized and folds in the cytoplasm into an assembly inactive form in which not only the nucleocapsid (NC) domain but also the matrix (MA) domain interacts with RNA. As a consequence of this "folded" conformation, the capsid (CA) domain becomes incapable of assembling. During or upon transport to the plasma membrane (PM) site of assembly, a conformational switch occurs in which the interaction of MA with NC is exchanged for an interaction of MA with PM components. This switch liberates the CA domains for assembly and this conformational rearrangement, essential for activation of Gag, is driven by the binding affinity of specific PM components for MA. To understand this essential switch, it is fundamentally important to elucidate at the molecular level the interaction of MA with PM components. Interestingly, the Gag proteins of closely related retroviruses such as avian sarcoma virus (ASV) contain structural domains with functions essentially identical to those of HIV-1 Gag; however, the mechanisms for Gag assembly on the PM appear to be different. Understanding the molecular basis of this switch will not only shed light on the assembly of HIV-1 but is likely to provide insight into the control of assembly in other retroviruses that assemble at the PM. Subsequent to assembly on the PM, the Gag proteins recruit the envelope (Env) protein for incorporation into virus particles. There is mounting evidence that incorporation of the Env protein in nascent virions is mediated by interactions between the MA domain of Gag and the cytoplasmic tail (CT) of gp41. The mechanism by which Gag mediates Env incorporation is not known. In this proposal, we will employ NMR, biophysical, biochemical and in vivo tools to provide a macromolecular picture of how the Gag protein binds to the PM, and to determine the molecular elements for Gag-gp41 interactions that mediate Env incorporation into virus particles. In Aim 1, we will determine the precise molecular mechanism for HIV-1 Gag binding to the PM. We have devised innovative approaches to characterize interactions between MA and membrane mimetics. In Aim 2, we will identify the molecular elements of ASV Gag assembly on the PM. We present evidence for a novel molecular switch in ASV MA upon binding to PM components. In Aim 3, we will characterize the interactions between HIV-1 MA and gp41 CT. We devised a new strategy to reconstitute and mimic the physiologically relevant conditions for interactions. These studies will provide details on the molecular switches that activate Gag assembly on the PM and help in the recruitment of the Env protein to assembly sites. We hope that the outcome of this proposal will help in the development of new antiviral therapeutic agents that inhibit assembly and virus production.