Single-Cell Profiling of Latently SIV-Infected CD4⁺ T Cells Directly Ex Vivo to Reveal Host Factors Supporting Reservoir Persistence

HIV-1 cure strategies aiming to eliminate persistent infected cell reservoirs are hampered by a poor understanding of cells harboring viral DNA in vivo. We describe a novel method to identify, enumerate, and characterize in detail individual cells infected in vivo using a combination of single-cell multiplexed assays for integrated proviral DNA, quantitative viral and host gene expression, and quantitative surface protein expression without any in vitro manipulation. Latently infected CD4⁺ T cells, defined as harboring integrated provirus in the absence of spliced viral mRNA, were identified from macaque lymph nodes during acute, chronic, and combination antiretroviral therapy (cART)-suppressed simian immunodeficiency virus (SIV) infection. Latently infected CD4⁺ T cells were most abundant during acute SIV (~8% of memory CD4⁺ T cells) and persisted in chronic and cART-suppressed infection. Productively infected cells actively transcribing viral mRNA, by contrast, were much more labile and declined substantially between acute and chronic or cART-suppressed infection. Expression of most surface proteins and host genes was similar between latently infected cells and uninfected cells. Elevated FLIP mRNA and surface CD3 expression among latently infected cells suggest increased survival potential and capacity to respond to T cell receptor stimulation. These findings point to a large pool of latently infected CD4⁺ T cells established very early in acute infection and upregulated host factors that may facilitate their persistence in vivo, both of which pose potential challenges to eliminating HIV-1 reservoirs. IMPORTANCE Effective combination antiretroviral therapy controls HIV-1 infection but fails to eliminate latent viral reservoirs that give rise to viremia upon treatment interruption. Strategies to eradicate latently infected cells require a better understanding of their biology and distinguishing features to promote their elimination. Tools for studying these cells from patients are currently limited. Here, we developed a single-cell method to identify cells latently infected in vivo and to characterize these cells for expression of surface proteins and host genes without in vitro manipulation, capturing their in vivo state from SIV-infected macaques. Host factors involved in cell survival and proliferation were upregulated in latently infected cells, which were abundant in the earliest stages of acute infection. These studies provide insight into the basic biology of latently infected cells as well as potential mechanisms underlying the persistence of HIV-1/SIV reservoirs to inform development of novel HIV-1 cure strategies.