DDX54 drives ALKBH5-mediated demethylation of selected transcripts to suppress interferon antiviral response.

DEAD-box (DDX) proteins are currently reported to shape host innate immunity by regulation of N⁶-methyladenosine (m⁶A) modification of transcripts for host factors involved in antiviral signaling. However, which DDX proteins are involved in antiviral response remains incompletely understood. Here, we identified DDX54 as an inhibitor of type I interferon antiviral response by facilitating m⁶A demethylation of selected transcripts. In the presence of VSV infection, DDX54 overexpression downregulated the interferon response and consequently promoted VSV replication, while DDX54 depletion yielded the opposite effects. Knockout of ALKBH5 revealed that DDX54 downregulated the interferon antiviral response through ALKBH5. VSV infection enabled DDX54 relocation from the nucleolus to the nucleoplasm, where DDX54 and ALKBH5 independently bound to the selected m⁶A-modified transcripts forming m⁶A RNA/protein complexes. Although the total enzymatic activity of cellular ALKBH5 was severely impaired in response to VSV infection, DDX54 interaction with ALKBH5 on the selected transcripts promoted the enzymatic activity of ALKBH5, which, in turn, demethylated these selected transcripts, including mavs mRNA. Consequently, these selected transcripts were largely retained in the nucleus to limit their translation in the cytoplasm, thereby impairing the interferon antiviral response. ATPase activity was essential for DDX54 to bind the selected transcripts and also to promote ALKBH5 demethylase activity. In the absence of VSV infection, DDX54 failed to recognize cellular m⁶A-modified RNAs and barely promoted the enzymatic activity of ALKBH5. Altogether, we conclude that VSV infection activates a DDX54/m⁶A/ALKBH5 axis to fine tune cellular interferon antiviral response by regulating m⁶A modification of the selected transcripts.IMPORTANCEThe m⁶A methylation modification on cellular mRNAs affects many cellular processes, including the innate antiviral response. In this study, we reported that VSV infection facilitated RNA helicase DDX54 to relocate from the nucleolus to the nucleoplasm, where DDX54, together with the m⁶A eraser ALKBH5, bound to a common subset of m⁶A-modified transcripts for host factors involved in antiviral signaling. Such binding promoted the enzymatic activity of ALKBH5 to demethylate the m⁶A modification on these transcripts, therefore limiting their protein translation and consequently impairing interferon antiviral response. Our results reveal an inhibitory function of DDX54 on host innate antiviral response.