The transcriptomic landscape of Epstein-Barr virus associated tumors at cellular and single-molecule level

Abstract: Epstein-Barr virus (EBV) was the first oncovirus found in humans. Almost all adults worldwide are asymptomatic carriers of EBV. The latent EBV-infection malignifies in approximately 200,000 individuals each year. The risk of developing certain types of EBV-associated cancer is high in specific regions, for example nasopharyngeal carcinoma in Southeast Asia and Burkitt’s lymphoma in Africa. The overall aim of this thesis was to characterize the EBV gene expression patterns in biopsies and elucidate the function of the expressed viral genes. Bulk transcriptome datasets of 615 tumors from four types of known EBV-associated neoplasms and single-cell transcriptome data from 63 nasopharyngeal samples were screened for EBV expression. The most abundant EBV RNA found at both tissue and single-cell levels, were RPMS1 and the novel co-terminating transcripts which we named BAREs. LMP1/BNLF2a/b and LMP2A/B/BNRF1 were expressed to a lesser extent and large differences were observed between individuals. Single-cell sequencing of B-lymphocytes isolated from the peripheral blood of a patient with a high EBV DNA load showed a similar EBV expression profile as the EBV-positive tumors. Moreover, the highly expressed EBV genes RPMS1 and BAREs were subjected to full-length single-molecule sequencing and all isoforms were characterized using our newly developed bioinformatics tool FLAME. Our results show that available EBV cell models inadequately portray primary tumors with regard to the viral gene expression and/or the propensity for reactivation. We developed an in vitro nasopharyngeal pseudostratified epithelium model which could mimic an EBV infection in the nasopharynx. A donor-dependent susceptibility for EBV infection was observed and both latent and lytic EBV expression patterns were detected in cells from a single donor. Single-cell sequencing data analysis could further distinguish that cells in late lytic stage with virus host shutoff were found amongst the suprabasal cells. The single-cell data from peripheral EBV-transformed B-lymphocytes identified that EBV induces proliferative pathways. In nasopharyngeal carcinoma tissue the EBV-transformed epithelial cells exists in a microenvironment with lymphocytic infiltration and interferon. Single-cell characterization of the nasopharyngeal cancer cells identified that the EBV expression of RPMS1 along with the miR-BARTs encoded in the introns promotes immune evasion by downregulation of interferon responsive genes. The findings suggest that EBV contributes to tumorigenesis in two ways, the first is by host cell reprogramming and induction of proliferation by EBNAs and LMP1, and the second is by immune evasion and escape by RPMS1 and BNLF2a.