The impact of HIV LTR intra- and inter-individual sequence variability on HIV latency and reactivation dynamics

The National Institute of Allergy and Infectious Diseases awarded Boston Medical Center Corporation $765,050 on September 1, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to investigate mechanisms of HIV-1 latency establishment and reactivation across diverse viral isolates and clades.

The research explores how long-terminal repeat (LTR) sequence variability within and across HIV-infected individuals influences proviral expression, latency establishment, and transcriptional reactivation. The project builds on preliminary massively parallel reporter assays showing extensive transcriptional activity variability of LTRs across approximately 6,000 HIV-1 isolates and clades. Work encompasses generating a library of viruses carrying 158 representative LTRs reflecting different transcription factor configurations and clades to examine how LTRs and local chromatin environment influence proviral transcription and latency establishment. The research addresses current limitations of shock-and-kill therapeutic strategies targeting persistent latent HIV-1 reservoirs, which have been only partially successful due to their failure to effectively target broad genetic and transcriptional proviral variability.

Performance occurs in Roxbury, Massachusetts. The period of performance runs September 1, 2026, through August 31, 2027. The assistance type is a Project Grant.

Abstract

Persistent latent HIV-1 reservoirs pose a major challenge to the eradication of HIV as they enable rapid viral rebound upon interruption of antiretroviral therapies. Multiple mechanisms contribute to limited proviral expression, HIV-1 latency, and HIV-1 proviral transcriptional reactivation, including recruitment of cellular transcription factors, epigenetic regulation, and RNA polymerase II pause release. Current shock and kill strategies, aimed at reactivating and then eliminating these reservoirs, have been only partially successful due to their failure to effectively target the broad genetic and transcriptional proviral variability within latent reservoirs. To address these challenges, we propose a strategy for exploring the mechanisms of HIV-1 latency establishment and reactivation across assorted HIV isolates from multiple clades, with the ultimate goal of improving the effectiveness of therapeutic interventions targeting the persistent reservoir. Our preliminary studies show extensive transcriptional activity variability of long-terminal repeats (ltrs) across ~6,000 HIV-1 isolates and clades using massively parallel reporter assays (mpras). These initial studies have identified ltrs from clades with higher activity, LTR regions responsible for the variable activity, and transcription factors contributing to LTR function. The proposed research builds upon this work and is structured around three specific aims. The first aim will determine how LTR activity across HIV-1 isolates impacts provirus expression and the establishment of HIV-1 latency. We will generate a library of viruses that have 158 representative ltrs reflecting the different transcription factor configurations and clades identified by mpras to examine how ltrs and local chromatin environment influence proviral transcription and the establishment of latency. The second aim will focus on mechanisms that reactivate ltrs that represent different clades in response to different latency reversal agents, testing the hypothesis that differential responses to latency reversal agents reflect LTR and the chromatin environment of integration sites. The goal of the third aim is to develop predictive models of LTR activity and utilize clinical samples from people with HIV to test these models. Aim 3 will provide insights into how sequence features and kinetic parameters evolve during infection and treatment. Successful completion of this proposed work will provide a better understanding of how HIV transcription is regulated across a diverse array of HIV-1 clinical isolates and clades, as well as provide new approaches to evaluate compounds designed to target the HIV reservoir.