Landscape of Epstein-Barr virus gene expression and perturbations in cancer.

Epstein-Barr virus (EBV) is the causative agent for multiple neoplastic diseases of epithelial and lymphocytic origin1-3. The heterogeneity of the viral elements expressed and the mechanisms by which these coding and non-coding genes maintain cancer cell properties in vivo remain elusive4,5. Here we conducted a multi-modal transcriptomic analysis of EBV-associated neoplasms and identified that the ubiquitously expressed RPMS1 non-coding RNAs support cancer cell properties by disruption of the interferon response. Our map of EBV expression shows a variable, but pervasive expression of BNLF2 discerned from the overlapping LMP1 RNA in bulk sequencing data. Using long-read single-molecule sequencing, we identified three new viral elements within the RPMS1 gene. Furthermore, single-cell sequencing datasets allowed for the separation of cancer cells and healthy cells from the same tissue biopsy and the characterization of a microenvironment containing interferon gamma excreted by EBV-stimulated T-lymphocytes. In comparison with healthy epithelium, EBV-transformed cancer cells exhibited increased proliferation and inhibited immune response induced by the RPMS1-encoded microRNAs. Our atlas of EBV expression shows that the EBV-transformed cancer cells express high levels of non-coding RNAs originating from RPMS1 and that the oncogenic properties are maintained by RPMS1 microRNAs. Through bioinformatic disentanglement of single cells from cancer tissues we identified a positive feedback loop where EBV-activated immune cells stimulate cancer cells to proliferate, which in turn undergo viral reactivation and trigger an immune response.