The occurrence of a latent reservoir of replication-competent virus remains a major obstacle in the development of more effective antiretroviral therapies aimed at eliminating or ameliorating FIIV-1 infections. The long-half life of latently infected cells suggests that current regimens of highly active antiretroviral therapy (HAART), which inhibit the spread of virus but do not specifically eliminate infected cells, cannot achieve the ultimate goal of viral eradication. A demonstrated pattern of Tat-limited transcriptional control in HIV-infected individuals is consistent with patterns of viral gene expression found among HAART-treated patients. Similar patterns of Tat-limited transcription have also been found in cell line models of latency and activation where the molecular basis of latency can be correlated with genetic polymorphisms in viral sequences controlling the processivity of viral transcription. We propose to characterize the molecular basis and pathologic significance of Tat-limited transcriptional processivity in HAART-treated patients. Our approach will involve the quantitative determination of levels of transcriptional processivity among individuals receiving HAART. We will test the genetic basis for Tat-limited processivity by determining sequences of proviral DNA and viral genomic RNA encoding the viral processivity apparatus in PBMC obtained from HAART patients, before and after mitogenic stimulation of these cells in culture. Tat and/or TAR encoding regions obtained from patients demonstrating Tat-limited processivity will also be tested for relative levels of transactivation potency using reporter gene constructs. Finally, the molecular basis for selected mutants demonstrating defects in transcriptional processivity will be further probed by characterizing the interaction of viral mutants with the host cyclinTl/cdk9 complex. These experiments will provide critically needed information on the nature of the HAART-resistant viral latent reservoir and may contribute to the development of more effective modalities of therapy.
