Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies. Overall design: The scRNA-Seq analysis of HC69 was performed as follows: immortalized human microglia HC69 cells were left untreated or treated with dexamethasone [DEXA, 1µM (Sigma-Aldrich, St. Louis, MO, USA)] for 72 h. After harvesting, a minimum of 600,000 viable cells per condition was subjected to Drop-Seq as described in (Macosko et al, 2015). After capturing individual cells in the oil droplets with barcoded beads (Chemgenes, Inc. Wilmington, MA, 01887 USA), droplets were broken, and cDNA libraries were generated using Illumina Nextera XT kit (Illumina, Inc., 5200 Illumina Way San Diego, CA, 92122 USA). Next Generation Sequencing and quality control were performed at Medgenome Inc (Foster City, CA, 94404 USA). Drop-Seq Tools v.1.0 and STAR-2.5.1b alignment tools were used to process and map the sequences to the hg18 human reference genome and to the retroviral portion of the HIV-1pNL4-3 construct. As a result, we built digital gene expression matrices (DGE) containing the read counts for human and HIV-1 genes.