PROJECT SUMMARY The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells that escape immune-mediated clearance. Due to the lack of transcriptional activity, latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies. Therefore, understanding how HIV-1 transcription is regulated will open doors to novel therapeutic strategies targeting the latent reservoir. Pharmacological approaches to reactivate the latent reservoir in vivo have been considered as potential eradication therapies for more than a decade. However, the effectiveness of these approaches has been disappointing as only a minority of latently infected cells appear to be responsive to latency-reversing agents. A deeper understanding of the transcriptional regulation of the HIV-1 promoter is necessary before we can design novel and effective LRAs. Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein with multiple functions both in cellular and viral biology. Our recent finding that CPSF6 controls the stability and activity of PP2A, a phosphatase directly implicated in the de-phosphorylation and inactivation of CDK9 and RNA Pol II, reveals an exciting new line of investigation into potential cure strategies. This proposal will focus on the following innovative directions: (a) to further understand the factors regulating HIV-1 transcription, with a focus on CPSF6 and PP2A; (b) to explore the possibility of using PP2A inhibition as a method to reactivate latent viruses; and (3) to further explore the unknown functions of CPSF6 based on a recent proteomics experiment from our group yielding several exciting hits.