Background <p>Current latency-reversing agents (LRAs) employed in the “shock-and-kill” strategy primarily focus on relieving epigenetic and transcriptional blocks to reactivate the latent HIV-1. However, their clinical efficacy is limited, partly due to their inability to fully reverse latency and the lack of LRAs specifically targeting post-transcriptional mechanisms. N6-methyladenosine (m⁶A) modification in HIV-1 RNA is emerging as an important post-transcriptional regulator of HIV-1 gene expression, yet its role in latency and reactivation remains largely unrecognized. Here, we explored the potential of small chemical compounds targeting the m⁶A pathway, specifically investigating the inhibition of ALKBH5 and its effect on latent HIV-1 reactivation mediated by the LRA romidepsin.</p> Methods <p>We used four in vitro cellular models of latency, primary model of CD4⁺ T cells HIV-1 infection and ex vivo cultures of CD8⁺-depleted PMBCs from ART-treated HIV⁺ patients. We measured latent viral reactivation by evaluating the expression of reporter protein GFP by flow cytometry, viral production by CA-p24 ELISA, and viral transcripts by RT-qPCR. CRISPR/Cas9 method was used to deplete ALKBH5. MeRIP and immuno-RNA FISH were used to address the m⁶A methylation levels on HIV-1 RNA upon ALKBH5 inhibition.</p> Results <p>We showed that ALKBH5 inhibitor 3 (ALKi-3) potentiated romidepsin-mediated viral reactivation in in vitro models of latency, primary model of CD4⁺ T cells infected with HIV-1 as well as in ex vivo cultures of CD8⁺-depleted PBMCs from ART-treated HIV⁺ patients. CRISPR/Cas9-mediated depletion of ALKBH5 mimicked the effects of ALKi-3. ALKi-3 increased levels of m⁶A-methylated HIV-1 RNA as shown by meRIP and immuno-RNA FISH.</p> Conclusion <p>Our study provides a proof-of-concept for the modulation of the m⁶A pathway in enhancing HIV-1 reactivation. This approach represents a promising adjunct to existing reactivation protocols and provides a concept of “dual-kick”, aiming to target transcriptional and post-transcriptional steps in HIV-1 reactivation from latency.</p>