Project Summary Human immunodeficiency virus type 1 (HIV-1) infection affects more than 38 million people worldwide and remains incurable due to the early establishment of reservoirs where the virus remains latent. HIV-1 enters the brain within the first two weeks of infection, and neurologic symptoms have been observed with accompanying central nervous system (CNS) biomarkers in acute HIV disease. The seven billion microglial cells in the CNS are the primary cell type infected by HIV in the adult human brain, and in the central nervous system represents a large potential reservoir site. Additionally, the brain is one of the organs with the highest burden of HIV-associated disease. HIV-associated neurocognitive disorder (HAND) affects 20-50% of people with HIV (PWH), with the milder forms of HAND predominating in the era of combined antiretroviral therapy (cART). Importantly, intact HIV proviruses persist in the brain despite viral suppression with cART. Despite this, little is known about the unique regulatory mechanisms governing HIV activation and latency in the brain. According to our recent cell studies in human postmortem brain, inflammation-associated reprogramming of microglial transcriptomes and 3D genomes (chromosomal conformations) is a key factor linked to viral infection and integration in brain cells during advanced stages of infection associated with encephalitis. However, non-encephalitic infected human brain, other than showing transcriptomic signatures indicative for disrupted interactions of microglia with the neuronal synapse, provides little information about the epigenomic and other determinants governing viral activation and latency in the brain. Here, as a first step towards understanding molecular mechanisms governing HIV latency in the humanized mouse brain, we will explore an extremely innovative molecular toolbox differentiating, on the single cell level, infected microglia and other myeloid cells actively expressing HIV, and separating them from infected cells not expressing HIV (latent). We will use this toolbox for advanced experimental approaches to quantitatively test molecular, epigenetic, and pharmacological interventions aimed at reducing the reservoir of humanized HIV+ brain, spleen, and blood. We will employ a novel genetic approach called enhanced HIV- induced lineage tracing (E-HILT) to reveal the frequency and kinetics of the establishment of latency in the CNS at the single cell resolution. We will define, in cell culture, and in humanized mouse brain and spleen/blood at single cell level resolution, the proportional representation of productively infected versus latently infected microglia and lymphocytes and other peripheral myeloid cells subject to genetically or pharmacologically induced disruption of chromatin-bound silencers, including the Human Silencing Hub (HUSH)/CTIP2- KAP1/KMT1E/SETDB1 repressive histone methyltransferase complex and more, broadly, histone H3-lysine 9 methylation (H3K9me)-associated repressive chromatin remodeling. The study will uncover the degree of latency in primary microglial cells in vivo, explore the transcriptome, epigenomic and chromatic 3D architecture that supports latency and explore the effects of chromatin modulating drugs on these cell states.

Project Narrative HIV infection of microglial cells in the brain causes HIV-associated neurocognitive disorder and represents a viral reservoir that is a barrier to cure the infection. This study exploits a novel experimental model system where primary human microglial cells and immune cells are xenografted into mice and altered to carry a genetic reporter system to identify actively and latently HIV-infected cells. The system will illuminate at single cell resolution the transcriptomics, epigenomics, and 3D genomic state of the HIV infected cells in the brain in response to antiretroviral treatment and after treatment with latency reversal agents.