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多聚蛋白靶向血浆 膜（PM）用于组装、未成熟颗粒的形成和病毒释放。Gag-PM结合是介导的 通过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）蛋白被募集到PM中以掺入到细胞内。 病毒颗粒Env的募集，以及gp 41的募集，对新生病毒体的下游感染性至关重要。 没有gp 41，就没有融合，也就没有感染性。几条证据表明，Env掺入是 由gp 41的胞质尾区（gp 41 CT）和Gag的MA结构域之间的相互作用介导。已经 长期认识到只有少数（< 10）gp 41分子嵌入MA层中。gp 41 CT和一个很好的- 形成的MA晶格对于掺入和感染性是必不可少的。看来，仅仅嵌入 gp 41 CT的成熟，但是对于MA层来说，需要经历裂解诱导的成熟步骤， GP 41完全激活。gp 41的掺入和活化是一个长期存在的问题， 解决问题需要一种结构性的方法。最近的低分辨率冷冻电子断层扫描（cryo-ET）研究 提出了一个模型，在该模型中，MA域经历了结构转换，形成不同的MA晶格 在组装期间和成熟时。在目前的资助期间，我们的实验室已经表明，MA形成了一个 具有中心孔的三聚体晶格的六聚体，被认为容纳gp 41 CT以促进掺入 病毒粒子我们还表明，PI（4，5）P2能够结合到MA上的替代位点，与一种新的 以及在未成熟颗粒的组装期间和组装后可能不同的MA-膜结合机制。 成熟然而，MA晶格的结构细节（未成熟和成熟）与膜结合， 控制MA构象转换的因素，MA晶格（去）稳定的因素，以及MA晶格的结构基础。 MA-gp 41 CT在组装过程中和成熟时的相互作用仍然缺乏。这项建议的目的是 旨在通过研究gp 41 CT如何与HIV感染相关的分子机制来阐明HIV感染的分子机制。 嵌入MA层。我们设计了创新的“控制装配”方法，使我们能够 产生未成熟和成熟gp 41 CT-MA复合物的生物学上真实的亚结构。研究这些 亚结构，我们已经开发出冷冻电子显微镜（cryo-EM）的方法，这将使我们能够使用 单粒子，而不是低温ET技术，使近原子结构的测定。我们的目标是 (1)确定在未成熟颗粒的组装过程中MA晶格形成的结构基础， 成熟，（2）确定MA晶格形成和Env掺入的关键因素，以及（3）确定 MA-gp 41 CT-膜复合物的结构，通过单颗粒冷冻-EM。拟议的研究将填补 HIV复制和传染性方面的一个主要空白，这可能有助于开发新的抗病毒治疗方法 抑制组装、Env掺入和最终病毒产生的试剂。

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.