Structural Basis for HIV-1 Gag Interactions with Cellular Constituents

 描述（由申请人提供）：在HIV-1感染的晚期，Gag蛋白合成并在细胞质中折叠成组装失活形式，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”构象，衣壳（CA）结构域变得不能组装。在运输到质膜（PM）组装位点期间或之后，发生构象转换，其中MA与NC的相互作用被交换为MA与PM组分的相互作用。这种开关释放CA结构域进行组装，并且这种对于Gag的活化至关重要的构象重排由特定PM组分对MA的结合亲和力驱动。为了理解这一基本开关，它是从根本上重要的是在分子水平上阐明MA与PM组件的相互作用。有趣的是，密切相关的逆转录病毒，如禽肉瘤病毒（ASV）的Gag蛋白含有与HIV-1 Gag功能基本相同的结构域;然而，PM上Gag组装的机制似乎是不同的。了解这种开关的分子基础不仅有助于了解HIV-1的组装，而且可能有助于了解其他逆转录病毒的组装控制。 在PM。在PM上组装后，Gag蛋白募集包膜（Env）蛋白以掺入病毒颗粒中。越来越多的证据表明，新生病毒体中Env蛋白的掺入是由Gag的MA结构域和gp 41的胞质尾区（CT）之间的相互作用介导的。Gag介导Env掺入的机制尚不清楚。在这个建议中，我们将采用NMR，生物物理，生物化学和体内工具，以提供一个大分子的图片如何Gag蛋白结合到PM，并确定介导Env纳入病毒颗粒的Gag-gp 41相互作用的分子元素。在目标1中，我们将确定HIV-1 Gag与PM结合的精确分子机制。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，我们将鉴定PM上ASV Gag组装体的分子元件。我们提出的证据表明，一种新的分子开关在ASV MA结合PM组件。在目标3中，我们将表征HIV-1 MA和gp 41 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。这些研究将提供有关激活PM上Gag组装的分子开关的细节，并有助于将Env蛋白募集到组装位点。我们希望这项提案的结果将有助于开发新的抗病毒治疗剂，抑制组装和病毒生产。

 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.