Demalonylation of DDX3 by Sirtuin 5 promotes antiviral innate immune responses

Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnβ transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnβ transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.