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Background: O-GlcNAcylation modification affects multiple physiological and pathophysiolocal functions of cells. Altered O-GlcNAcylation was reported to participate in antivirus response. Stimulator of interferon genes (STING) is an adaptor mediating DNA virus-induced innate immune response. Whether STING is able to be modified by O-GlcNAcylation and how O-GlcNAcylation affects STING-mediated anti-DNA virus response remain unknown. Methods: Metabolomics analysis was used for detecting metabolic alterations in HSV-1 infection cells. Succinylated wheat germ agglutinin (sWGA), co-immunoprecipitation, and pull-down assay were employed for determining O-GlcNAcylation. Mutagenesis PCR was applied for the generation of STING mutants. WT and Sting1−/− C57BL/6 mice (KOCMP-72512-Sting1-B6NVA) were infected with HSV-1 and treated with O-GlcNAcylation inhibitor for validating the role of STING O-GlcNAcylation in antiviral response. Results: STING was functionally activated by O-GlcNAcylation in host cells challenged with HSV-1. We demonstrated that this signaling event was initiated by virus infection-enhanced hexosamine biosynthesis pathway (HBP). HSV-1 (or viral DNA mimics) promotes glucose metabolism of host cells with a marked increase in HBP, which provides donor glucosamine for O-GlcNAcylation. STING was O-GlcNAcylated on threonine 229, which led to lysine 63-linked ubiquitination of STING and activation of antiviral immune responses. Mutation of STING T229 to alanine abrogated STING activation and reduced HSV-1 stimulated production of interferon (IFN). Application of 6-diazo-5-oxonorleucine (DON), an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAcylation, markedly attenuated the removal of HSV-1 in wild type C57BL/6 mice, leading to an increased viral retention, elevated infiltration of inflammatory cells, and worsened tissue damages to those displayed in STING gene knockout mice. Together, our data suggest that STING is O-GlcNAcylated in HSV-1, which is crucial for an effective antiviral innate immune response. Conclusion: HSV-1 infection activates the generation of UDP-Glc-NAc by upregulating the HBP metabolism. Elevated UDP-Glc-NAc promotes the O-GlcNAcylation of STING, which mediates the anti-viral function of STING. Targeting O-GlcNAcylation of STING could be a useful strategy for antiviral innate immunity. [ABSTRACT FROM AUTHOR]