Despite the success of antiretroviral therapy (ART) in suppressing plasma viremia in people living with HIV, the long-term persistence of integrated HIV genome copies in a small pool of long-lived CD4 T cells is a major hurdle to an HIV cure. A growing body of evidence suggests that a certain fraction of the reservoir cells is capable of producing transcripts and viral proteins despite ART, challenging the initial definition of latency. Studying the active HIV-1 reservoir is crucial, as we hypothesize it may be the first contributor to viral rebound following treatment interruption. This grant proposal aims to comprehensively characterize the transcriptionally active reservoir in the peripheral blood and tissues such as lymph nodes and gut biopsies, its contribution to viral rebound, and the host and viral drivers of HIV-1 expression. Therefore, we propose to develop a novel single cell technique that simultaneously assesses the proviral genome, the integration site, the T cell receptor and the transcriptional landscape of infected cells expressing viral transcripts. Factors underlying HIV-1 expression will be further studied using a physiologically relevant in vitro system, enabling a controlled assessment of the effects of T cell receptor engagement and the viral integration site landscape on HIV gene transcription in engineered primary CD4 T cells. Ultimately, this research should contribute to the development of therapeutic interventions.