Structural Basis for HIV-1 Gag assembly and Env incorporation

在HIV-1感染周期的晚期，病毒编码的Gag多聚蛋白被靶向血浆。 用于组装、形成未成熟颗粒和释放病毒的膜(PM)。GAG-PM结合是介导的 N-末端肉豆蔻酰化基质(MA)结构域与磷脂酰肌醇4，5-二磷酸的相互作用 (PI(4，5)P2)。在GAG组装的同时，包膜(Env)蛋白被招募到PM以并入 病毒颗粒。环境病毒招募，因此gp41，到一个新生的病毒粒子，是下游传染性的关键。 没有gp41，就没有融合，也没有传染性。有几条证据表明，Env公司是 Gp41(Gp41CT)的细胞质尾部与GAG的MA结构域相互作用。一直以来 Long认识到，只有少数(&lt；10)gp41分子嵌入MA层。Gp41CT和一口井- 形成的MA晶格对于整合和感染性是必不可少的。看来，仅仅嵌入 Gp41CT在MA层，但MA层需要经历一个切割诱导成熟步骤 Gp41变得完全活跃。Gp41的加入和激活一直是一个长期存在的问题，其 解决方案需要一种结构性方法。低分辨率冷冻电子断层扫描(CRYO-ET)研究进展 提出了一个模型，在该模型中，MA域经历结构转换以形成不同的MA格 在组装过程中和成熟后。在目前的资助期内，我们的实验室表明，MA形成了一个 带有中心孔的三聚体晶格的六角体，被认为容纳gp41CT以促进整合到 病毒粒子。我们还证明了PI(4，5)P2能够与MA上的替代位点结合，这与一个新的 可能在未成熟颗粒的组装过程中和在 成熟。然而，MA晶格的结构细节(不成熟和成熟)结合在膜上，因素 控制MA构象开关的因素，(去)稳定MA晶格的因素，以及 MA-gp41CT在组装和成熟时的相互作用仍然缺乏。这项提议的目的是 旨在通过研究gp41CT是如何 嵌入在MA层中。我们设计了创新的“受控组装”方法，使我们能够 产生未成熟和成熟的gp41CT-MA复合体的生物真实亚结构。为了研究这些 子结构，我们已经开发出低温电子显微镜(CRYO-EM)方法，它将使我们能够使用 单粒子而不是冷冻技术，以实现近原子结构的确定。我们的目标是 (1)确定未成熟粒子组装过程中MA晶格形成的结构基础 成熟，(2)确定MA晶格形成和环境掺入的关键因素，以及(3)确定 用单粒子冷冻EM法研究MA-gp41CT-膜复合体的结构。拟议的研究将填补 艾滋病毒复制和传染性方面的重大差距，这可能有助于开发新的抗病毒疗法 抑制组装、环境病毒掺入并最终抑制病毒生产的药物。

During the late phase of HIV-1 infection cycle, the virally encoded Gag polyproteins are targeted to the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag–PM binding is mediated by interactions of the N-terminally myristoylated matrix (MA) domain with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Concurrent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Env recruitment, and hence gp41, to a nascent virion is essential for downstream infectivity. Without gp41, there is no fusion and no infectivity. Several lines of evidence suggest that Env incorporation is mediated by interactions between the cytoplasmic tail of gp41 (gp41CT) and the MA domain of Gag. It has been long recognized that only a few (< 10) gp41 molecules are embedded in the MA layer. Both gp41CT and a well- formed MA lattice are essential for incorporation and infectivity. It appears that it is not sufficient to only embed gp41CT in the MA layer, but it is necessary for the MA layer to undergo a cleavage induced maturation step for gp41 to become fully active. The incorporation and activation of gp41 has been a long-standing problem whose solution requires a structural approach. Recent low-resolution cryo-electron tomography (cryo-ET) studies proposed a model in which the MA domain undergoes a structural transformation to form distinct MA lattices during assembly and upon maturation. During the current funding period, our lab has shown that MA forms a hexamer of trimers lattice with a central hole, thought to accommodate gp41CT to promote incorporation into virions. We have also shown that PI(4,5)P2 is capable of binding to alternate sites on MA, consistent with a novel and perhaps distinct MA–membrane binding mechanisms during assembly of the immature particle and upon maturation. However, the structural details of the MA lattice (immature and mature) bound to membrane, factors that govern the MA conformational switch, factors that (de)stabilize the MA lattice, and the structural basis for MA–gp41CT interaction during assembly and upon maturation, are still lacking. The aims of this proposal are designed to elucidate the molecular mechanisms that render HIV infectious by studying how gp41CT is embedded in the MA layer. We devised innovative “controlled assembly” approaches which will enable us to generate biologically authentic substructures of the immature and mature gp41CT–MA complex. To study these substructures, we have developed cryo-electron microscopy (cryo-EM) approaches which will allow us to use single particle rather than cryo-ET techniques to enable near atomic structural determination. Our aims are to (1) determine the structural basis for MA lattice formation during assembly of the immature particle and upon maturation, (2) determine factors critical for MA lattice formation and Env incorporation, and to (3) determine the structure of the MA–gp41CT–membrane complex by single-particle cryo-EM. The proposed studies will fill a major gap in HIV replication and infectivity, which may help in the development of new antiviral therapeutic agents that inhibit assembly, Env incorporation, and ultimately virus production.