Inhibition of ALKBH5 demethylase of m(6)A pathway potentiates HIV-1 reactivation from latency.

BACKGROUND: 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 (m(6)A) 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 m(6)A pathway, specifically investigating the inhibition of ALKBH5 and its effect on latent HIV-1 reactivation mediated by the LRA romidepsin. METHODS: 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 m(6)A methylation levels on HIV-1 RNA upon ALKBH5 inhibition. RESULTS: 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 m(6)A-methylated HIV-1 RNA as shown by meRIP and immuno-RNA FISH. CONCLUSION: Our study provides a proof-of-concept for the modulation of the m(6)A 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.