The ability to infect cells latently helps HIV to establish persistent infections despite strong humoral and cellular immune responses against the viral proteins and remains the single greatest obstacle to the development of therapies that result in the elimination of HIV from infected individuals. This proposal builds on some remarkable observations we have recently made demonstrating that the activation of HIV transcription is an extraordinarily dynamic process. We have found using high resolution chromatin immunoprecipitation (ChIP) experiments that the recruitment of RNA polymerase;TFIIH and numerous other components of the transcription machinery to the proviral promoter rapidly rises and falls as NF-kappaB enters and exits the nucleus. The discovery that the transcriptional responses of HIV to cellular activation signals is intermittent, and not simply an irreversible response to an initial signaling event, was completely unexpected. In complementary studies, we also plan to investigate the molecular mechanisms utilized by HIV to enter latency. We have recently found that transcription from integrated lentiviral vectors that carry Tat in cis progressively diminishes over a period of 4 to 6 weeks until the virus enters a latent state. The shutdown in transcription appears to be associated with a loss of Tat activity since mutations in Tat that reduce its activity lead to more rapid viral shutdown. These observations lead to the hypothesis that HIV enters a latent state once initiation rates have fallen to a level where threshold levels of Tat are no longer produced. Activation of NF-kappaB and related transcription factors restores transcription initiation and re-establishes the Tat feedback mechanism leading to high levels of viral RNA synthesis. Because reactivation of HIV transcription in response to cellular stimulation is a highly dynamic process in which multiple transcription factors are rapidly recruited and exchanged at the promoter, multiple signaling pathways can be used to regulate both the timing and magnitude of the transcriptional response of HIV to external stimuli. We plan to rigorously test this hypothesis using a combination of biochemical studies of HIV promoter activation in model cellular systems, and the development of primary cell models for proviral quiescence and latency. These studies promise to lead to a detailed new understanding of the specific factors that drive HIV latency and suggest new avenues for antiviral research.
Showing the most recent 10 out of 14 publications
