Rho GTPases and Diaphanous related Formins in HIV-1 replication

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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.