Impact of HIV-1 5' Leader Defects on plasma viremia, proviral dynamics, and immune responses

About This Grant

PROJECT SUMMARY Antiretroviral therapy (ART) effectively suppresses HIV replication but fails to eliminate the viral reservoir—a heterogeneous population of infected cells that persist despite treatment. While most proviruses are defective, many retain the ability to express RNA, proteins, and even viral particles, contributing to persistent viremia, chronic immune activation, and complicating the interpretation of viral load measurements. Among these, proviruses with defects in the 5’ leader (5’L) region are common, and they decay more slowly than intact proviruses, are translation-competent, and are often implicated in persistent non-suppressible viremia (NSV), making them a critical component of HIV persistence and a significant source of residual antigenic stimulation. The long-term goal of this project is to define the mechanisms by which 5’L-defective proviruses evade negative selection, characterize their contribution to persistent viremia, and determine their impact on HIV-specific immune responses. Our central hypothesis is that 5’L-defective proviruses retain the ability to express viral genes while evading immune-mediated killing and cytopathic effects. To test this, we will (1) apply a novel digital PCR assay that distinguishes intact from 5’L-defective RNA without requiring sequencing to quantify their contribution of defective proviruses to plasma HIV RNA; (2) isolate and phenotype clonally expanded CD4+ T cells harboring 5’L-defective proviruses using two complementary approaches—stimulation with Tat-LNP followed by p24+ cell sorting, and limiting dilution culture ex vivo—to assess viral transcription and translation through multi-omic analyses (SMARTseq, Ribo-seq); (3) investigate susceptibility of 5’L defective proviruses to HIV-specific CTL, and leverage longitudinal samples from individuals with NSV to evaluate how antigen expression from defective proviruses shapes HIV-specific immune responses and clonotype dynamics over time. This research is innovative in developing a first-in-class assay to parse 5’L-defective RNA without sequencing and applying cutting-edge single-cell and immunologic profiling to study the interplay between defective proviral expression and host immunity. The proposed research is significant because it is expected to address critical gaps in our understanding of HIV persistence, improve the interpretation of HIV cure clinical trials, and inform new strategies aimed at targeting cells with intact and defective proviruses. Ultimately, this project has the potential to offer new tools to improve the clinical outcome of people living with HIV and opportunities for the development of innovative strategies to achieve HIV remission.