The regulation of mRNA synthesis, processing, nucleocytoplasmic transport, and splicing are under investigation using various lentivirus model systems. These studies provide unique insights into the control of eukaryotic gene expression and also into the processes that underlie retroviral pathologies. The research focuses on three fundamental areas of RNA metabolism: cellular transcription factors, viral transcriptional activators (Tats), and viral modulators of mRNA processing (Revs). We have shown that the macrophage-specific transcription factor and proto- oncogene, PU.1/Spi1, binds to and activates the equine infectious anemia virus (EIAV) promoter and is therefore essential for EIAV replication in macrophages. Post-translational modifications of PU.1 that modulate its activity during macrophage differentiation were found. We are continuing to analyze the structure, function, and mechanism of lentivirus trans- regulatory proteins by comparing the cis- and trans-acting components from distantly related viruses. This approach, coupled with mutational and domain exchange experiments, revealed the domain organization of the Tat proteins and suggested their interaction with cellular cofactors. Mutated Tat proteins were constructed that inhibited wild-type Tat function in trans. Tat-Rev fusion proteins were made to analyze RNA- binding properties of Tat in vivo and small-molecule agents, reported to inhibit Tat activity, were tested to define their mode of action. The viral Rev proteins are also under investigation. EIAV was shown to encode a Rev protein whose function and mechanism are similar to, but distinct from, HIV-1 Rev. The Rev effector domains from different viruses were identified and compared with HIV-1 Rev. Finally, a unique Rev-dependant alternative splicing phenomenon was revealed in the EIAV system.
