Location, dynamics and expression of functional proteins in the HIV viral reservoir

After HIV infection, the virus quickly spreads throughout the body and establishes a viral reservoir. Although effective Antiretroviral therapy (ART) can inhibit viral replication, functional virus persists in CD4+ T cells and is difficult to clear in large numbers in lymphoid organs. To better characterize these CD4+ T cells carrying replication-competent viruses, we established the HIV Gag and Envelope Reactivation co-Detection Assay (GERDA) and analyzed the tissue homing properties of cells that transiently appear in circulating blood. The presence and function of HIV in key body compartments was confirmed by GERDA in combination with proviral DNA and poly-A transcripts, with low viral activity in circulating cells early after diagnosis. CD4+ T cells carrying replication-competent virus were more likely to be lymph node homing, with TN and TCM being the primary viral reservoirs. We then performed an in- depth analysis of the env regions of circulating PBMCs and T cell subsets, and we found that individual immune system function was negatively correlated with the diversity of viral reservoirs, with archived viral sequences often containing inactivating mutations and showing higher genetic diversity in TTM and TEM.
HIV Tat can activate viral transcription by interacting with transactivation response element (TAR), Rev is needed for expression of late genes (eg Env, Gag). With a series of Tat and Rev mutants, we revealed a regulatory role for Rev in the early events of viral infection, and we found that premature expression of Rev in an artifact produced a dominant negative phenotype that interfered with wild-type viral production, suggesting that the non- complementary negative effects of Rev when expressed prior to HIV infection or prior to Tat expression suggest that i) Tat and Rev functions can be decoupled (Env expression without Tat) and ii) strict kinetic dependence is critical for a productive HIV infection in vitro. Our results provide an overall analysis of not only the location and dynamics of the viral reservoir but also the protein function of the virus, which provides a theoretical basis for understanding and clearing HIV latent reservoirs.