Summary Epstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B-cell compartment and reactivate. Within memory B-cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But, this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic and CRISPR approaches to identify that an abundant methionine supply, and interconnecting methionine and folate cycles, maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes, de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B-cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo . These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B-cell lifecycle and suggest therapeutic approaches. Highlights Methionine metabolism is critical for Epstein-Barr virus B-cell latency Extensive cross-talk enables methionine metabolism to control the EBV epigenome Methionine restriction also impairs EBV-driven human B-cell immortalization Dietary methionine restriction unmasks EBV antigens in Burkitt xenografts in vivo

Methionine Metabolism Controls the B-cell EBV Epigenome and Viral Latency