Cell heterogeneity contributes to the variable response of HIV-1 to latency-reversing agents.

Transcriptional noise contributes to gene expression variability, but its origin and impact in HIV-1 latency remain incompletely characterized. Here, we combine a dual-copy MS2-tagging system with dedicated mathematical analysis to investigate the variability of HIV-1 transcription in live cells. In the basal state, the transcriptional activity of proviruses located in the same cell was uncorrelated, indicating that expression variability primarily comes from the intrinsic stochasticity of promoter dynamics. Upon stimulation with diverse latency-reversing agents, transcription of viral copies became more correlated within cells than across them, revealing a shift from promoter-driven to cell state-driven variability. Analysis of the molecular drug targets confirmed variable effects across cells. Our findings indicate that cellular heterogeneity can shape the response to latency-reversing agents and demonstrate how quantitative tools can dissect noise sources. This work offers mechanistic insights into HIV-1 latency and informs strategies to target the latent viral reservoir.