Targeting the Super Elongation Complex to Regulate HIV Transcription and Latency

About This Grant

PROJECT SUMMARY The persistence of Human Immunodeficiency Virus (HIV) in long-lived latent reservoirs remains one of the major barriers to a functional cure. The latent reservoir consists of cells harboring replication-competent, but transcriptionally inhibited proviruses that evade immune clearance even in patients on long-term antiretroviral therapy (ART). Spontaneous reactivation of these proviruses results in viral rebound upon treatment cessation, thus mandating life-long treatment. Furthermore, the production of viral transcripts and antigens from these cells, even in the absence of productive infection, is thought to contribute to persistent inflammation and increased rates of co-morbidities among people with HIV (PWH). Therefore, the development of small molecule regulators of viral transcription as ART adjuvants to reign in chronic inflammation and/or to manage the reservoir en route to a functional cure remains an NIH HIV/AIDS high priority research topic. The licensing of transcriptional elongation by the cyclin-dependent kinase complex known as P-TEFb is one of the most heavily regulated steps in mammalian gene expression. P-TEFb is normally recruited to sites of nascent transcription by a series of context-dependent complexes, including transcription factors, epigenetic regulators, and super elongation complexes (SECs). HIV-1 circumvents this regulatory step through its viral Tat protein, which directly recruits P- TEFb to sites of nascent proviral transcription through recognition of the TAR RNA stem loop produced at the 5’ end of viral RNA transcripts. Biochemical purifications of Tat have found that it also binds a specific cellular SEC, which has been shown to facilitate viral transcription in some cell line models. However, we recently found that this SEC is dispensable for viral replication in primary CD4+ T cells. To validate this finding, we characterized a series of small molecule SEC inhibitors (iSECs) that block the interaction of P-TEFb with these SEC complexes. Treatment of primary CD4+ T cells with iSECs increased viral replication and viral transcript production, suggesting that the SEC is not only dispensable for viral replication, but that it may serve as a reservoir for P- TEFb release. Several small molecules that enhance the availability of P-TEFb (e.g., BRD4 inhibitors) have been shown to stimulate viral transcription and reactivation from latency. Indeed, we found that iSECs synergize with latency reversing agents to enhance viral transcription in peripheral blood mononuclear cells from PWH on suppressive ART. Based on our preliminary data showing that the SEC is not required for viral transcription in primary CD4+ T cells and that it can be targeted as a reservoir of P-TEFb by iSECs for latency reversal, here we seek to address three critical questions: 1) What P-TEFb-containing complex is required for HIV transcription in primary CD4+ T cells?; 2) Does HIV use the SEC in other cellular contexts?; and 3) What is the potential of iSECs as latency reversing agents compared to other P-TEFb release strategies? Ultimately, these studies will clarify the transcriptional elongation complexes that regulate HIV replication and latency towards the development of next-generation ART adjuvants designed to manage the reservoir.