ABSTRACT As of 2021, over 38 million individuals are living with human immunodeficiency virus type 1 (HIV-1). Currently, there is no cure for HIV, and these individuals are subjected to life-long, oral drug therapy. Highly active antiretroviral therapy (HAART) is the standard treatment regimen comprising a cocktail of several antiretroviral drugs. HAART has made remarkable strides in controlling viral loads, reducing transmission, and preventing the onset of acquired immunodeficiency syndrome (AIDS) for patients with access to treatment. However, the long- term effectiveness of HAART remains threatened due to drug resistance. Therefore, developing novel antiretroviral drugs with different mechanisms of action (MOA) is required to improve therapeutic options. Of all approved antiretrovirals, the HIV-1 reverse transcriptase (RT) is one of the most frequently targeted enzymes in the virus. The primary role of RT is to convert the single-strand viral RNA genome into double-strand DNA (dsDNA) for host-genome integration. Non-nucleoside RT inhibitors (NNRTI) are one of the FDA-approved classes of antiretroviral often included in HAART. It stops the formation of the viral dsDNA by binding to RT at a distinct hydrophobic pocket (NNIBP) in proximity but not overlapping the enzyme’s polymerase active site. JT- 4-173 (JT) is a novel potent HIV-1 specific inhibitor previously identified in the Sarafianos lab. A recent structural study in our lab revealed that JT binds to the NNIBP, yet it does not inhibit normal polymerization. Our preliminary data showed that JT has no significant impact on early nor late-stage RT products, but the number of integrated proviruses significantly reduced. This behavior was not observed in any of the currently known NNRTIs. I hypothesize that JT inhibits the polymerization activity required for strand displacement synthesis, an essential step in late-stage reverse transcription for the complete formation of the dsDNA. In the proposal, I aim to determine the impact of JT on early-, intermediate- and late-reverse transcription products along with 1- and 2- long terminal repeat (LTR) circles and integrated provirus (Aim 1.1), the exact stopping site(s) of reverse transcription by JT using sequencing techniques (Aim 1.2), and the impact of JT on HIV-1 RT in strand displacement synthesis using a primer-extension biochemical assay (Aim 1.3). To determine the molecular mechanism of JT, I will use cryogenic electron microscopy (cryo-EM) to solve the HIV-1 RT/JT structures in complex with dsDNA substrates relevant to sites of strand displacement (Aim 2.1), then identify and mutate the key interactive residues on RT to confirm its effect on strand displacement synthesis using the primer-extension assay (Aim 2.2). These studies will reveal JT’s exact impact on HIV-1 reverse transcription and elucidate the unique MOA of a novel NNRTI. I will take full advantage of our lab’s background in RT biology and our collaborators’ specialties in structural, sequencing, and biochemical studies to complete these proposed experiments and advance my training. These planned studies will not only lay the foundation for developing a novel antiretroviral but will also improve our understanding of HIV-1 RT biology.

PROJECT NARRATIVE Tens of millions of individuals living with HIV-1 are taking highly active antiretroviral therapy (HAART), a long- term and life-saving treatment comprising the use of multiple antivirals that prevent HIV-1 infection by targeting essential enzymes involved in the viral replication cycle. Our lab previously reported a potent non-nucleoside reverse transcriptase inhibitor (NNRTI), JT-4-173, that binds at the typical NNRTI-binding site but inhibits the virus through a previously undescribed process that is unique to this compound. Here, I aim to characterize the novel mechanism of action of JT-4-173, which can both further our understanding of HIV-1 reverse transcriptase biology and aid in the development of future generations of HAART.