Dynamics of HIV Packaging and Assembly

摘要，项目3 HIV-1结构蛋白Gag驱动病毒组装，导致感染性病毒。在组装期间 在这个过程中，Gag协调基本病毒成分的掺入，并与宿主因子相互作用， 促进病毒体产生。HIV-1的组装过程是复杂的，而且是精心设计的，但并不完美 机械地和动态地理解。组装传染性颗粒的一个重要步骤是 包装全长HIV RNA基因组（下文称为HIV-1 RNA）。HIV-1 RNA是一种次要RNA， 在感染的细胞中，它是一种病毒，但它被包装到绝大多数病毒颗粒中。填塞物必须锚入 细胞质膜在那里协调粒子的组装。然而，加格之间错综复杂的相互作用， 脂质膜成分和控制颗粒组装的病毒RNA基因组尚不清楚。 HIV-1在病毒复制的多个阶段中篡夺宿主机器。然而，人们对细胞（宿主）的了解甚少。 这些因素是必要的，以及它们在Gag贩运和病毒组装中的作用。 为了研究HIV-1组装的机制和动力学，我们组建了一个优秀的团队， 补充科学知识。我们将使用多学科方法，包括生物物理方法， 结构分析、生物化学和遗传分析、单分子和活细胞成像技术， 传染性病毒是如何组装的以及病毒组装的动力学/动力学。我们的研究集中在三个方面 HIV-1组装中的知识差距。在目标1中，我们将定义Gag选择HIV-1 RNA的机制 通过检查Gag与病毒和非病毒RNA之间的相互作用，我们将描绘 核输出途径对与HIV-1 RNA复合的蛋白质的影响。那个插科打诨的主持人 已知需要蛋白质来促进病毒外出;然而，对于哪些宿主因子 这是非法交易和病毒组装所必需的使用一种新的方法，我们已经确定了多个主机 与加格有关的因素。此外，敲除这些已鉴定的宿主基因中的一些导致 病毒生产的改变，表明它们是组装和/或运输的依赖因素。在Aim中 2我们将阐明这些蛋白质依赖因子如何协助HIV-1组装和促进病毒产生。 在目标3中，我们将使用单分子成像方法和分子建模，定义 Gag：lipid：RNA，以深入了解病毒组装的机制;这些研究将得到补充 通过使用活细胞成像方法检查细胞HIV-1组装的动力学和动力学。 拟议研究的结果将改变我们对HIV-1基因组包装机制的认识， 推进我们对病毒组装所需的宿主-病毒相互作用的理解，并提供对病毒组装的深入了解。 病毒装配的分子机制和这些事件在T细胞中的动力学。

ABSTRACT, PROJECT 3 The HIV-1 structural protein Gag drives virus assembly resulting in infectious virus. During the assembly process, Gag coordinates the incorporation of essential viral components and interacts with host factors to facilitate virion production. The process of HIV-1 assembly is complex and choreographed, but not well understood mechanistically and dynamically. An essential step in assembling infectious particles is the packaging of the full-length HIV RNA genome (referred to as HIV-1 RNA hereafter). HIV-1 RNA is a minor RNA species in infected cells and yet it is packaged into the vast majority of the viral particles. Gag must anchor in the plasma membrane where it orchestrates particle assembly. However, the intricate interplays between Gag, the lipid membrane components, and the viral RNA genome that govern particle assembly are not understood. HIV-1 usurps host machinery during multiple stages of viral replication. Yet, little is known about cellular (host) factors that are necessary for, and their roles in, Gag trafficking and virus assembly. To study the mechanisms and dynamics of HIV-1 assembly, we have assembled an outstanding team with complementary scientific expertise. We will use multidisciplinary approaches including biophysical methods, structural analyses, biochemical and genetic assays, single-molecule and live-cell imaging techniques to study how infectious viruses are assembled and the dynamics/kinetics of virus assembly. Our studies focus on three knowledge gaps in HIV-1 assembly. In Aim 1 we will define the mechanisms whereby Gag selects HIV-1 RNA during virus assembly by examining the interactions between Gag and viral and nonviral RNAs. We will delineate the impact of nuclear export pathway usage on proteins that complexed with HIV-1 RNA. That Gag and host proteins are required to facilitate viral egress is known; however, less is understood which host factors are necessary for Gag trafficking and virus assembly. Using a novel approach, we have identified multiple host factors that associate with Gag. Furthermore, knocking out some of these identified host genes resulted in alteration of viral production, suggesting that they are dependency factors for assembly and/or trafficking. In Aim 2 we will elucidate how these protein dependency factors assist HIV-1 assembly and promote virus production. In Aim 3 using single-molecule imaging approaches and molecular modeling, we will define the interplays among Gag:lipid:RNA to provide insights into the mechanisms of virus assembly; these studies will be complemented by examining the dynamics and kinetics of cellular HIV-1 assembly using live-cell imaging approaches. Findings from the proposed studies will transform our knowledge of HIV-1 genome packaging mechanisms, advance our understanding of host-viral interactions required for virus assembly, and provide insights into the molecular mechanism of virus assembly and the dynamics of these events in T cells.