Transcriptomic HIV-1 reservoir profiling reveals a role for mitochondrial functionality in HIV-1 latency

Identifying cellular mechanisms maintaining HIV-1 latency in the viral reservoir is crucial for devising effective cure strategies. Here we developed a flow cytometry-fluorescent in situ hybridization (flow-FISH) approach using a combination of probes that detects abortive and elongated HIV-1 transcripts for ex vivo isolation and characterization of viral reservoir cells in peripheral blood from people with HIV-1. Following the isolation of three distinct cell populations from CD4+ T cells (i.e. cells harboring transcriptionally latent HIV-1, cells harboring transcriptionally active HIV-1, or uninfected cells), we determined their transcriptomic profile by RNA sequencing (RNAseq). Supervised gene expression analysis identified several differentially expressed mitochondrial genes in infected cell populations compared to uninfected cells, but also in latently infected compared to productively infected CD4+ T cells. Our transcriptomic profiling data shows an association between diminished mitochondrial functioning and the transcriptional activity of the viral reservoir. These findings underline the relevance of metabolic regulation in HIV-1 infection, and support the development of strategies modulating immunometabolism to target viral latency. Overall design: To investigate the mechanisms underlying HIV-1 latency in the viral reservoir, we designed probes targeting abortive TAR and elongated Gag HIV-1 transcripts for flow-FISH detection and isolation of transcriptionally latent or active HIV-1-infected CD4+ T cells. Using this method, we isolated transcriptionally latent HIV-1-infected cells (TAR+Gag-), transcriptionally active HIV-1-infected cells (TAR+Gag+), and uninfected cells (TAR-Gag-) from the CD4+ T cell population in peripheral blood mononuclear cells (PBMCs) from people with HIV-1 (N=5) by cell sorting. We then performed 3' RNAseq and gene expression profiling analysis to assess the transcriptomic profiles of the three isolated populations.