Abstract The lentiviruses HIV-1 and HIV-2 cause acquired immunodeficiency syndrome (AIDS) in humans. The conventional wisdom that HIV-2 is a more benign cousin to HIV-1 was recently challenged, however, in a large study that found after 23 years of HIV-2 infection, cumulatively over 90% of participants had AIDS and 80% had died. An effective, safe, and scalable cure for HIV should account for any unique features that may characterize HIV-2 latency. During the course of its replication, HIV-2 integrates into human DNA and creates a reservoir that is a barrier to virus eradication - as is the problem for HIV-1. How similar, or different, these HIV reservoirs may be is unknown. The scientific premise of this proposal is that there are unrecognized unique features of HIV-2 biology that may impact virus eradication efforts, relative to HIV-1. As an example, the HIV-2 Vpx protein has recently been shown to counteract the Human Silencing Hub (HUSH), a highly conserved gene silencing complex known to repress HIV-1 proviruses, in ways that may impact virus latency and the reservoir landscape. HIV-2 vpx reactivates latent provirus transcription in primary human CD4+ T cells through degradation of the HUSH complex. To study HIV-2 latency, we have recruited participants with HIV-2 into our longitudinal HIV Eradication and Latency (HEAL) cohort. Our HIV-2 proviral landscape preliminary data has identified differences between the HIV-1 and HIV-2 proviral landscapes and we see phylogenetic evidence that suggests clonal expansion may maintain the HIV-2 reservoir, findings we propose to explore further in this proposal. We hypothesize that HIV-2 latency reversal differs from HIV-1 reactivation, provide data to support this, and propose experiments to investigate the mechanisms that may explain these observations. The goal of this proposal is to use HIV and host genomic information to define the landscape of HIV-2 integration and investigate the mechanisms that control HIV-2 latency and its reversal. Here we propose to leverage cutting-edge techniques to address important knowledge gaps in our understanding of HIV-2 infection biology. We hypothesize that HIV-2 differs from HIV-1 in integration landscape and reversal from latency and that Vpx plays a key role in these differences. Specific Aims of this proposal are to define the HIV-2 integration landscape in vivo, understand the role of HIV-2 vpx in virus latency, and investigate the transcriptional blocks to HIV-2 latency reversal. Our approaches test hypotheses that are central to understand HIV-2 latency and its reversal.