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Shortly after the discovery of EBV and even before it was named, Werne and Gertrude Henle identified that restriction of extracellular arginine induces EBV lytic antigens within Burkitt lymphoma tumor cells. However, for nearly 60 years, it has remained unknown how arginine metabolism supports EBV latency. To gain insights, we performed an amino acid restriction screen in EBV+ Burkitt cell lines. This confirmed that arginine restriction was sufficient to trigger EBV reactivation in Burkitt B cells and in gastric carcinoma models. Arginine restriction strongly impaired\n                      <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                      pyrimidine biosynthesis, and CRISPR- or chemical genetic-blockade of pyrimidine biosynthesis enzymes induced EBV immediate-early and early lytic gene expression. However, arginine restriction blocked EBV lytic DNA replication and, consequently, also late gene expression, suggesting an abortive lytic cycle. By contrast, chemical or CRISPR blockade of the\n                      <jats:italic toggle=\"yes\">de novo<\/jats:italic>\n                      pyrimidine biosynthesis pathway rate-limiting enzyme CAD reactivated a full EBV lytic cycle, suggesting specific arginine restriction roles in support of lytic DNA replication. Arginine restriction caused DNA damage, which was a driver of EBV reactivation. Arginine restriction and DNA hypomethylation additively reactivated EBV. Together, our results highlight arginine and pyrimidine metabolism as potential targets for EBV lytic antigen induction therapy in B-cell and epithelial-cell contexts.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Altered metabolism is a hallmark of cancer, frequently increasing transformed cell dependence on extracellular amino acid supply. Despite current interest in Epstein-Barr virus (EBV) lytic antigen induction therapy, in which viral lytic reactivation sensitizes tumors to the highly cytotoxic effects of the antiviral ganciclovir, there has been no systematic study of extracellular amino acids that control EBV latency. We identified that arginine uptake was important for the maintenance of EBV latency in both Burkitt lymphoma and gastric carcinoma contexts. Metabolic pathway analyses highlighted that arginine uptake and metabolism were required to supply pyrimidines. Disruption of arginine metabolism or de novo pyrimidine synthesis caused DNA damage. Arginine restriction also triggered Burkitt DNA hypermethylation. Building upon this, we provide evidence that the combination of arginine restriction and DNA hypomethylation, either by decitabine or by CRISPR approaches, induced EBV reactivation more strongly than either alone, suggesting a therapeutic approach..<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00933-26","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T13:01:57Z","timestamp":1781528517000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Arginine metabolism supports\n                    <i>de novo<\/i>\n                    pyrimidine biosynthesis to block DNA damage and maintain Epstein-Barr virus 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