Structural Basis for HIV-1 Gag Interactions with Cellular Constituents

 描述(申请人提供)：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不活跃的形式，其中不仅核衣壳(NC)结构域，而且基质(MA)结构域与RNA相互作用。由于这种“折叠”的构象，衣壳(CA)结构域变得不能组装。在运输到组装的质膜(PM)位置时或在运输到质膜(PM)位置时，发生构象开关，其中MA与NC的相互作用交换为MA与PM成分的相互作用。这种开关解放了CA结构域进行组装，这种对激活GAG至关重要的构象重排是由特定PM组分与MA的结合亲和力驱动的。为了理解这一重要的开关，在分子水平上阐明MA与PM组分的相互作用是非常重要的。有趣的是，密切相关的逆转录病毒如禽肉瘤病毒(ASV)的Gag蛋白含有结构域，其功能基本上与HIV-1 Gag相同；然而，在PM上组装Gag的机制似乎不同。了解这种开关的分子基础不仅有助于阐明HIV-1的组装，而且可能为控制其他组装的逆转录病毒的组装提供洞察力 在首相会议上在PM上组装后，Gag蛋白招募包膜(Env)蛋白并入病毒颗粒。越来越多的证据表明，TH env蛋白在新生病毒粒子中的掺入是通过GAG的MA结构域和gp41的细胞质尾部(CT)之间的相互作用来介导的。GAG介导Env整合的机制尚不清楚。在这个提案中，我们将使用核磁共振、生物物理、生化和体内工具来提供Gag蛋白如何与PM结合的大分子图，并确定介导Env进入病毒颗粒的Gag-gp41相互作用的分子元件。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提出了在ASV MA中与PM组分结合时存在新的分子开关的证据。在目标3中，我们将描述HIV-1 MA和gp41 CT之间的相互作用。我们设计了一种新的策略来重建和模拟相互作用的生理相关条件。这些研究将提供激活PM上的GAG组装的分子开关的细节，并有助于将Env蛋白招募到组装位置。我们希望这项提议的结果将有助于开发抑制组装和病毒产生的新的抗病毒治疗剂。

 DESCRIPTION (provided by applicant): 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 th 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.