The persistence of HIV in infected individuals on HAART remains an insurmountable obstacle to the treatment and cure of AIDS. HIV establishes reservoirs in many cells and tissues. Integrated proviruses are not transcribed and infected cells are not recognized by the immune system. Nevertheless, upon cellular activation, growth and proliferation, these latent proviruses can direct the synthesis of fully infectious virions that infect other cells. Thus, the uneasy symbiosis between HIV and the host persists. In this study, the hypothesis is that much of this proviral latency is maintained at the levels of elongation of viral transcription. Studies of infected individuals at seroconversion and of patients on HAART, revealed that a substantial proportion of HIV in circulating PBMC is transcribing only short transcripts that contain the transactivation response (TAR) RNA stem loop. Thus, transcription has initiated but not elongated. Upon cellular activation and/or the addition of Tat, which can enter cells freely, full-length viral transcripts accumulate and HIV replication is observed. Factors that contribute to this lack of transcriptional elongation are called negative transcription elongation factor (N-TEF). They are opposed by the positive transcription elongation factor b (P-TEFb) that phosphorylates the C-terminal domain of RNA polymerase II and subunits of N-TEF to modify the transcription complex for productive elongation. Tat and NF-kB are central players that recruit P-TEFb to the HIVLTR. In this study, the contributions of N-TEF and P-TEFb to proviral latency will be dissected in great detail, using approaches with dominant negative proteins and RNAi. Moreover, secreted Tat and P-TEFb.Tat fusion proteins will be used to try to abrogate this transcriptional arrest on the HIVLTR. Although these studies will be carried out first in cell lines and PBMC from the rhesus macaque, future therapeutic intervention using gene therapy with Tat and P-TEFb.Tat chimeras are contemplated. During these studies, our human model of proviral latency will find resonance with SIV in monkeys. Similar studies to those that had been carried out in infected humans will be carried out in rhesus macaques, i.e. we shall look for short and long transcripts from the SIVLTR, examine effects of blocking N-TEF or supplying P-TEFb to monkey PBMC, and finally, develop strategies to secrete abundant Tat and P-TEFb.Tat chimeras in monkeys before infection with SIV and later, after seroconversion and optimal treatment of the rhesus macaques. These studies should reveal once and for all what role transcriptional elongation plays in the latency of HIV, what effects, if any Tat has on the organism, and if manipulating P-TEFb could play a role in eliminating the reservoir of HIV from the host.
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