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一个蛋白质。 组装非活性形式，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。作为这种“折叠”构象的结果，衣壳（CA）结构域变成 无法组装。在运输至质膜（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NC的相互作用。 PM组件。该开关释放CA结构域以进行组装，并且该构象转换是通过将CA结构域与CA结构域连接而实现的。 Gag的激活所必需的重排是由特定PM组分的结合亲和力驱动的 对于MA。为了理解这个基本的开关，从分子水平上进行阐明是非常重要的 MA与PM组分的相互作用。有趣的是，密切相关的逆转录病毒的Gag蛋白 例如含有功能基本上与 HIV-1 Gag;然而，PM上Gag组装的机制似乎不同。理解 这种开关的分子基础不仅将揭示HIV-1的组装， 深入了解在PM组装的其他逆转录病毒中的组装控制。组装后 在PM上，Gag蛋白募集包膜（Env）蛋白以掺入病毒颗粒中。有 越来越多的证据表明，新生病毒体中Env蛋白的掺入是由相互作用介导的， 在Gag的MA结构域和gp 41的胞质尾区（CT）之间。加格的机制 介导的Env掺入未知。在这个建议中，我们将使用核磁共振，生物物理，生物化学， 和体内工具，以提供Gag蛋白如何与PM结合的大分子图像， 确定介导Env掺入病毒的Gag-gp 41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。我们提供证据证明 一种新的分子开关在ASV MA结合PM组件。在目标3中，我们将描述 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仿 生理相关的相互作用条件。这些研究将提供分子的细节， 激活PM上Gag组装并帮助Env蛋白募集组装的开关 网站.我们希望这一建议的结果将有助于开发新的抗病毒治疗方法。 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.