Current treatment for HIV infection, using multiple combined antiviral drugs to different molecular targets, can achieve maximal suppression of viral replication for extended periods. Nevertheless, this success lasts only as long as adherence to the treatment regimen is maintained. For the vast majority of infected patients, discontinuation of treatment results in viral rebound within weeks, requiring tchronic, lifelong administration of antiretroviral therapy. The obstacle to drug induced eradication of HIV infection is viral latency. Many studies indicate that resting CD4 T cells, primarily those with central memory function, compose a major reservoir for latent HIV infection. Because memory T cells have long life spans, such reservoirs are predicted to be extremely stable and therefore, not amenable to depletion through natural cellular decay. Recent efforts in HIV treatment have turned towards the development of strategies to target the viral reservoir and interfere with control of latent infection. However, before effective therapeutic approaches can be designed, a better understanding of the basic biologic mechanisms underlying the establishment and maintenance of persistent, nonproductive HIV infection is needed. Our knowledge of the interactions between viral and cellular elements that are required to maintain HIV latency is limited at present. A major hurdle to advancing research in this area is the extremely low frequency at which such latently infected cells are found in the peripheral circulation of infected individuals (approximately 1 - 10 per million CD4 cells). In addition, such latently infected CD4 cells cannot be isolated for study, because they persist mainly in tissues in a quiescent cell state and cannot be phenotypically discerned from uninfected resting T cells. These impediments to in vivo and ex vivo study of latent infection have necessitated the use of in vitro cell models. Although the majority of these models have consisted of repressed HIV replication in immortalized human T cell lines and clones, continuous culture systems of primary T cells with silenced viral infection have become available in recent years. Results generated from these models indicate various potential molecular blocks in viral transcription initiated from the long terminal repeat (LTR). Because all of these models use continuously cycling T cells, it remains unclear how much of the findings are relevant to the actual biologic state of HIV latency in vivo. We propose a unique approach to study molecular interactions that regulate persistent/latent HIV infection using a well-defined primary cell model of infected, non-dividing resting CD4 T cells. We hypothesize that conserved epigenetic mechanisms control the transcriptional state of the HIV provirus and regulate viral latency. Work will focus on two major components of epigenetic control: chromatin structure at the site of HIV integration, and sequence specific DNA methylation in the LTR promoter region.
The specific aims of the project are to: 1) determine the contribution of histone deacetylase (HDAC) function to the maintenance of HIV latency, using targeted iRNA knockdown of selected HDACs;2) determine specific histone modifications associated with a repressed vs. active state of viral transcription, using ChIP of histones with select acetylated and methylated residue modifications;and 3) determine HIV LTR sequence specific methylation patterns associated with a repressed vs. active state of viral transcription, using bisulfite driven methylcytosine sequence analysis. Knowledge derived from these studies will add significantly to our understanding of the molecular mechanisms controlling HIV latency --- which is a critical step in the path to developing new treatment strategies.
More than 40 million people worldwide are infected with HIV, and the number continues to increase as new infections outpace deaths each year. It is estimated that over 1 million adults in the U.S. are infected with HIV;and the VA Healthcare System has become the single largest provider of care to HIV-infected patients in the nation. Although combined antiretroviral therapy has dramatically reduced HIV morbidity and mortality, the presence of latently infected cell reservoirs necessitates chronic, lifelong administration of drug therapy. Extended long-term treatment to suppress HIV disease is costly, both in terms of dollars spent and associated drug toxicity to major organ systems of the body over time. There is an urgent need to elucidate mechanisms of HIV latency that will lead to develop of new innovative treatment strategies that can target and eliminate the residual, smoldering pockets of HIV infection, which elude current therapy designs, and require continual drug induced viral suppression.