RSAD2/VIPERIN and CMPK2 Coordinate an Immunometabolic Response to Epstein-Barr Virus

Epstein-Barr Virus (EBV) infection and reactivation in B-lymphocytes is tightly regulated by host antiviral response genes. In the present study, we identify interferon stimulated genes RSAD2 (radical S-adenosyl methionine domain-containing 2) and CMPK2 (Cytidine/Uridine Monophosphate Kinase 2) as key modulators of EBV expression and cellular response during EBV infection and reactivation. EBV primary infection and reactivation lead to a coordinated upregulation of RSAD2 and CMPK2. Depletion of RSAD2 reduced cell viability and limited EBV reactivation, while depletion of CMPK2 led to reactivation of EBV lytic gene expression during latency. Transcriptomic analysis revealed that RSAD2 and CMPK2 have overlapping functions in regulating IFN-signaling pathways, as well as oxidative phosphorylation, protein translation, and unfolded protein response during reactivation. Despite distinct subcellular localizations, RSAD2 at the Endoplasmic Reticulum (ER), and CMPK2 in mitochondria, both genes converge on shared immunometabolic pathways, including control of Gasdermin D (GSDMD) associated pyroptosis and ATF-4 associated unfolded protein response (UPR). EBV reactivation induced formation of antiviral ribonucleotide ddhCTP during lytic EBV reactivation which was strictly dependent on RSAD2. Knockdown of RSAD2 and CMPK2 had significant effects on global metabolites consistent with a remodeling of glycolysis, fatty acid biosynthesis and degradation of superoxides. These observations demonstrate that RSAD2-CMPK2 function in a coordinated ER-mitochondria stress-Interferon signaling axis that shapes EBV reactivation and host immune control, including a novel layer of immunometabolic regulation modulating viral latency and reactivation.