Project Summary/Abstract HIV remains a major global health issue; strict adherence to combined antiretroviral therapy can reduce a patient's active virus to undetectable levels, but the presence of latent viral reservoirs necessitates lifelong adherence to antiretrovirals. HIV-1 reverse transcriptase (RT) is a major target in the treatment of HIV as it is responsible for producing a double stranded DNA copy of the viral genome, which can be integrated into the host. RT inhibitors fall into two major classes: nucleoside RT inhibitors (NRTIs) which are incorporated into the growing DNA chain but usually lack the 3' hydroxyl group needed to continue reverse transcription, and non- nucleoside RT inhibitors (NNRTIs) which bind to an allosteric pocket 10Å away from the active site, causing a conformational change which alters the rate of chemical catalysis. These two classes of inhibitors are typically combined when treating patients to provide more protection against inevitable drug-resistant mutants. In cells, NNRTIs have shown synergy with NRTIs, but some in vitro experiments suggest that NRTI incorporation and NNRTI binding are mutually exclusive. The molecular mechanism which underlies these drug interactions remains unclear. Additionally, recent studies have revealed that a subset of NNRTIs can enhance RT homodimerization and induce HIV-specific pyroptosis by prematurely activating the viral protease. This subset of compounds may be promising components of new “shock and kill” therapies, which aim to cure HIV by activating viral reservoirs and killing HIV-infected cells. While these dual function NNRTIs would represent an exciting advancement in HIV treatment, the mechanistic and structural features of this process are not yet understood. The proposed project will provide insights into the allosteric interactions underlying these two key processes using complementary kinetic, structural, and dynamic methodologies. In Aim 1, I will investigate the mechanism for crosstalk between the active site and the NNRTI-binding site. To do this, I will use transient kinetic analyses to determine whether incoming nucleotides can be incorporated in the presence of a covalent NNRTI, which cannot be displaced. Additionally, I will identify long-range interactions responsible for allosteric inhibition with HDX MS experiments. In Aim 2, I will identify key structural characteristics of dimerizing NNRTIs and the underlying mechanism by which they mediate this dimerization effect. To do this, I will use complementary X-ray crystallography and cryoEM techniques to determine the structures of NNRTIs bound to the p66/p66 homodimer and p66 monomer and identify the interactions between dimerizing NNRTIs and their binding site(s) which are responsible for this effect. I will also monitor the progression from p66 monomer to p66 homodimer in the presence of dimerizing NNRTIs by HDX MS to determine the mechanism by which they enhance dimerization. The insights gleaned from t...