The persistence of an HIV latent reservoir remains a major obstacle in the development of more effective antiretroviral therapies Although current regimens of highly active antiretroviral therapy (HAART) offer long-term suppression of HIV plasma viremia, they cannot eradicate infection Studies on viral dynamics have shown that a population long-lived cryptically-infected cells endure HAART while cells producing high levels of virus are rapidly cleared Based on the central role played by the viral transactivation complex in regulating viral gene expression, we have proposed that HAART may mediate the accumulation of viral mutations that attenuate viral gene expression and thereby facilitate escape from immune detection A variety of polymorphisms in Tat and TAR sequences have been observed in a cohort of HAART-treated individuals and preliminary evidence has suggested that HAART responders demonstrate reduced levels of transactivation activity The experiments described in this proposal will characterize patterns of genetic variation in Tat and TAR sequences from HAART responders and examine the correspondence between proviral and expressed viral sequences The relative transactivation potencies of molecular clones of Tat proteins and TAR sequences isolated from these individuals will be determined using an inducible mammalian expression system that permits control over intracellular levels of Tat expressed in transfected cells Viral mutants will be tested at varying levels of induction for their ability to transactivate expression of an HIV LTR-reporter gene fusion Transactivation deficient mutations will be further probed by examining the interactions of the viral Tat protein and TAR RNA sequences with the cellular cofactors cyclin T1 and CDK9 Since single nucleotide substitutions in the transactivation machinery may make the difference between cryptic and productive infection, further characterization of such HAART-associated mutants can help elucidate the events mediating the establishment and maintenance of an HIV latent reservoir Moreover, the characterization of these in vivo mutations can provide valuable insights into the structure/function relationships of the HIV transactivation complex.
