RSAD2/VIPERIN and CMPK2 coordinate an immunometabolic response to Epstein-Barr Virus

Epstein-Barr Virus (EBV) infection and reactivation in B-lymphocytes are tightly regulated by host antiviral response genes. In the present study, we identify Interferon Stimulated Genes (ISGs) 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. Despite distinct subcellular localizations, RSAD2 at the endoplasmic reticulum (ER) and CMPK2 in the mitochondria, transcriptomic analysis revealed that both genes functionally converge and exhibit overlapping roles in driving shared immunometabolic pathways, specifically Interferon (IFN) signaling, MAPK signaling, oxidative phosphorylation, mitochondrial function, eukaryotic translation, and ATF-4-associated unfolded protein response (UPR). We show that RSAD2 and CMPK2 knockdown affects IRAK1-TRAF6-TAK1 expression levels, and RSAD2 overexpression downregulates NF-κB signaling by EBV membrane associated oncoprotein LMP1. Depletion of RSAD2 and CMPK2 had significant effects on global metabolites consistent with a remodeling of nucleotide metabolism, glycolysis, fatty acid biosynthesis and degradation of superoxides. EBV reactivation induced formation of antiviral ribonucleotide ddhCTP which was strictly dependent on RSAD2. These observations demonstrate that RSAD2 and CMPK2 function in a coordinated ER-Mitochondria-Interferon signaling axis that shapes EBV reactivation and host immune control, including a novel layer of immunometabolic regulation modulating viral latency and reactivation.