Many viral gene products, such as the HIV-1 transactivator Tat and regulatory Vpr proteins, have an important role in the nucleus during the viral infectious cycle. It has been proposed that both proteins utilize nuclear import pathways that do not appear to be utilised by normal cellular proteins. If confirmed, an objective is to exploit any potential uniqueness within Tat and Vpr nuclear import pathways as specific pharmacological targets to block the HIV-1 infectious cycle or the development of the debilitating symptoms of AIDS. Based upon earlier observations, the proposed study aims to establish a mechanism of (1) ATP-dependent regulation of nuclear import of Tat and (2) the cytoplasmic dependence of nuclear import of Vpr by detecting protein-protein interactions between the two HIV-1 proteins and various transport factors using qualitative and quantitative in vitro and in vivo methods. These methods include the application of yeast two-hybrid screens, native gel electrophoresis coupled with fluorimaging, and fluorescence polarization to examine protein-protein interactions; as well as fluorescence recovery after photobleaching and fluorescence resonance energy transfer to analyze protein transport in living human cells.
