Basic Science Award

One of the remaining questions in HIV research is how the virus establishes a dormant (latent) state and thereby escapes eradication by current antiretroviral therapy. Latently infected T cells do not produce significant amounts of viral genomes or proteins due to the silencing of a specific step in the virus life cycle?viral transcription. Viral transcription can be reactivated in latently infected cells, a process that rekindles HIV infection after antiretroviral therapy is discontinued and remains a major barrier to eradication. A key regulator of viral transcription is the viral Tat protein. We have a longstanding interest in elucidating the regulatory mechanisms that govern Tat function. A critical conserved residue in Tat is lysine at position 41 (K41) as K41A mutations abrogate Tat transcriptional functions. We recently performed a proteomics analysis of cellular Tat immunoprecipitated from latently infected T cells after reversion of latency. This analysis identified a modification of 42 Da at K41, indicating that this residue is either trimethylated or acetylated. The goal of the proposed study is to generate and characterize modification-specific antibodies for Tat-K41 and to conclusively determine if Tat is acetylated or trimethylated in cells. Subsequently, using a set of shRNAs directed against trimethyltransferases or acetyltransferases, we will determine which of these enzymes regulate Tat function in a K41-specific manner. Using mass spectrometry of synthetic Tat, we will search for specific enzymes that trimethylate or acetylate Tat-K41 in vitro. We anticipate that our findings will provide novel insight into the mechanisms of HIV transcription with a specific focus on the regulation of viral latency