Single-molecule RNA-FISH analysis reveals stochasticity in reactivation of latent HIV-1 regulated by Nuclear Orphan Receptors NR4A and cMYC

HIV-1 eradication strategies require reactivation of all latent cells. Current methods lack effectiveness due to incomplete knowledge of proviral reactivation. We employed a single-molecule RNA-FISH (smRNA-FISH) analysis and found that proviral reactivation is highly variable, stochastic, and occurs in bursts and waves, with different kinetics in response to diverse LRAs. Approximately 1-5% of latent cells displayed stochastic reactivation even without stimulation. Through single-cell RNA-seq analysis, we identified NR4A3 and cMYC as extrinsic factors selectively associated with HIV-1 reactivation. Concomitant with HIV-1 reactivation cMYC was downregulated and NR4A3 was upregulated in both latent cell lines and primary CD4+ T-cells from aviremic patients. By inhibiting cMYC using SN-38, an active metabolite of irinotecan, we induced NR4A3 and HIV-1 expression. Our results suggest that inherent stochasticity in proviral reactivation contributes to cell-to-cell variability, which could potentially be modulated by drugs targeting cMYC and NR4A3. scRNA-seq analysis of uninduced ACH2, J1.1, or J-Lat cells. The frozen cells were revived and allowed to grow for two passages and then the ongoing cultures were submitted to genomics facility for scRNA-Seq (1 million cells/ml).