Rho GTPases and Diaphanous related Formins in HIV-1 replication

作为专性细胞寄生虫，人类免疫缺陷病毒的复制依赖于其目标宿主细胞的多种机制，包括那些调节细胞骨架结构和动力学的机制。以前的研究表明，在HIV-1复制周期的各个步骤中，存在小的Rho GTP酶(Rho、Rac和CDC42)以及肌动蛋白和微管细丝系统。然而，HIV-1与宿主细胞Rho GTP酶及其下游效应物的精确功能相互作用在很大程度上是未知的。我们这项研究计划的最初目标将是利用病毒细胞-细胞传播的同步细胞系统，在单轮HIV-1复制过程中对Rho GTP酶活性进行动力学和定量分析。这些研究旨在将内源性GTP酶总活性的生化测量与成像方法相结合，以定位HIV-1感染细胞中激活的GTP酶。其次，我们将描述控制小GTP酶下游肌动蛋白聚合的细胞机制，如透明相关的Formins或HIV-1复制中的Arp2/3复合体。这些研究将使用HIV允许的细胞系，在这些细胞系中，单个肌动蛋白核仁的表达可以通过Tet诱导的RNAi表达而被抑制。随后将对已确定在病毒复制中发挥作用的成分进行分析，以确定它们在艾滋病毒生命周期中的作用及其潜在的分子机制。总之，这些研究将产生HIV-1感染的功能相互作用的详细图谱，以及宿主细胞小GTP酶及其肌动蛋白聚合效应的调节。

As obligate cell parasites replication of Human Immunodeficiency Viruses depends on multiple machineries of their target host cells including those regulating cytoskeletal architecture and dynamics. Previous studies implied small Rho GTPases (Rho, Rac and Cdc42) as well as actin and microtubule filament systems in individual steps of the HIV-1 replication cycle. However, the precise functional interactions of HIV-1 with host cell Rho GTPases and their downstream effectors are largely unknown. Our initial goal of this research proposal will be the kinetic and quantitative analysis of Rho GTPase activities over the course of a single round of HIV-1 replication using a synchronized cell system of viral cell-cell transmission. These studies aim at combining biochemical measurements of overall endogenous GTPase activities with imaging approaches to localize activated GTPases in HIV-1 infected cells. Secondly, we will characterize the involvement of cellular machineries that control actin polymerization downstream of small GTPases, such as Diaphanous related Formins or the Arp2/3 complex in HIV-1 replication. These studies will employ HIV permissive cells lines in which expression of individual actin nucleators can be suppressed via Tet-inducible RNAi expression. Components identified to play a role in virus replication will subsequently be analyzed for the localization of their action in the HIV life cycle as well as their underlying molecular mechanism. Together, these studies shall yield a detailed map of the functional interplay of HIV-1 infection and the regulation of host cell small GTPases and their actin polymerizing effectors.