SUMMARY The persistence of latent, replication-competent HIV-1 proviruses in resting CD4+ T cells represents a major barrier to curing HIV-1 infection. To date, efforts to eradicate this viral reservoir via the shock and kill approach have not led to complete, long term viral suppression in either cell and/or animal models. Thus, we need to consider alternate approaches that could lead to a sterilizing or functional cure for HIV-1 infection. The block and lock approach seeks to silence the transcriptional activity of latent proviruses, such that when antiretroviral therapy (ART) is removed viral rebound is significantly delayed or, better yet, prevented. Several research groups have identified small molecule inhibitors that target different factors of the HIV-1 transcription machinery, leading to a block and lock phenotype. However, blocking only one transcription pathway may not be sufficient to silence all proviruses, and thus it is likely that successful implementation of this strategy will require a combination of inhibitors. In this regard, there is a critical need to identify new molecules with different mechanisms of action. Our laboratory recently discovered that the p21-activated kinase (PAK) inhibitor PF- 03758309 is an exceptionally potent inhibitor of HIV-1 latency reactivation (IC50 in the pM to low nM range) with a huge selectivity index (> 3,000). (The discovery of PF-03758309 as an inhibitor of HIV-1 latency reversal is described in: Vargas B, Giacobbi NS, Sanyal A, Venkatachari NJ, Han F, Gupta P, Sluis-Cremer N. Antimicrob Agents Chemother. Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency. 2019 Jan 29;63(2). pii: e01744-18.) In the long term, we anticipate that PF-03758309 alone, or in combination with other drugs, could be used to facilitate a “block and lock” sterilizing cure in HIV-infected individuals. However, we do not know the mechanism(s) by which this inhibitor abrogates the reactivation of latent HIV-1 infection in CD4+ T cells. Indeed, preliminary studies in our laboratory revealed that its inhibition of HIV-1 latency reversal is not due to inhibition of the PAKs. Accordingly, the primary goal of this R21 proposal is to elucidate the mechanism(s) by which PF-03758309 silences HIV-1 proviruses.