The HIV-1 Rev protein regulates the nucleocytoplasmic distribution of viral precursor mRNAs that encode HIV-1 structural proteins. Biochemical features of Rev and its essential cis-acting binding element, the RRE, have been defined using functional in vivo assays. Yet, the molecular details of how Rev mediates nuclear viral mRNA export remains poorly understood. The major focus of this proposal is to understand the molecular basis of Rev-mediated nuclear RNA export. Here, we propose in vitro and in vivo studies for defining the cellular factors and molecular interactions essential for Rev function. These studies will provide additional insights into the mechanism of Rev action, as well as the cellular nuclear RNA export machinery. Specifically, we will analyze functional interactions between Rev and hCRM1, a beta-importin like protein, and the small nuclear GTPase Ran. Biochemical and immunological experiments will address whether hCRM1 mediates a direct interaction between Rev and hRIP/Rab. We will also explore whether hRIP/Rab interacts directly or indirectly with Ran-GTP, alpha importin, NTF2 or other cellular factors. In collaboration with established investigators, we will determine whether hRIP/Rab plays a role in modulating GTPase activity, NPC targeting and translocation or signal transmission between the nuclear envelope and nucleocytoplasmic transport components. Two genetic strategies are proposed to define interactions between Rev, hCRM1, and Ran during Rev-mediated nuclear RNA export. These genetic analyses will also be used to characterize the normal cellular function of hRIP/Rab. Lastly, we have established Rev assays based on microinjection in Xenopus oocytes and isolated germinal vesicles (GVs). We will use these systems to identify and characterize components of the Rev-mediated nuclear RNA pathway and test whether Rev function requires sequential interactions similar to nuclear protein import. Additionally, we will use factor competition and immuno-inhibition strategies to investigate the relationship between the Rev-mediated nuclear RNA export pathway and the nuclear RNA export pathway(s) used by different classes of cellular RNA.
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