Exploiting Metabolism to Uncloak Epstein-Barr Virus Immunogens in Latently Infected B-cells

Project Summary Epstein-Barr virus (EBV) is spread through the saliva, where it establishes oropharyngeal infection, invades the tonsils and colonizes the B-cell compartment. Orally transmitted EBV is the cause of infectious mononucleosis and of multiple B-cell and epithelial cancers, including Burkitt lymphoma (BL) and nasopharyngeal carcinoma. Much remains to be learned about how epigenetic regulation of the viral genome enables it to colonize the oropharynx and tonsils to establish persistent infection of >95% adults and to cause 200,000 cancers per year. In tonsillar memory B-cells, EBV expresses a single, poorly immunogenic antigen. BL, which can present as tumors of the jaws, face and orbit, use a similar viral program to evade anti-EBV T-cell responses. By contrast, EBV expresses nearly 80 viral proteins in AIDS patients with oral hairy leukoplakia of the tongue, and as many as immunogenic eight latency proteins in post- transplant lymphoma. During the initial post-doctoral period, the applicant used two CRISPR screens to characterize B-cell factors important for EBV latency in BL. These provided insights into epigenetic pathways that restrict expression of lytic cycle and latency genes in B-cells and highlighted epigenetic enzymes that initiate and maintain a high level of DNA methylation of the EBV genome in this highly restricted form of latency. Perturbation of EBV DNA methylation de- represses EBV antigen expression and sensitizes Burkitt cells to CD8+ T-cell recognition. Yet, little is known about cross-talk between infected B-cell metabolism and epigenetic pathways, which must occur to supply methyl groups for DNA and histone modification. The applicant hypothesizes that latently EBV-infected B cells subvert methionine and folate metabolism pathways to drive EBV genome hypermethylation necessary for silencing of immunogenic EBV gene products and T-cell immunoevasion. During the mentored K99 phase, the applicant will 1) identify how methionine metabolism contributes to EBV genome hypermethylation and silencing in latently infected B-cells and EBV oncoproteins, and 2) characterize the interplay between the folate cycle and EBV genome methylation in latently-infected cells. During the R00 phase, the applicant will perform independent research to identify how latent EBV programs alter B-cell metabolic pathways to reinforce viral genome epigenetic states at the level of DNA and histone methylation. Collectively, these studies will open a new area of EBV biology and promise to support novel therapeutic approaches. The career development plan will prepare the applicant for transition to independence as an investigator with a multi-disciplinary approach to study metabolomic control of virus/host interactions.