Dissecting Epstein-Barr Virus Dependence Across Diverse Infected Cell Models

Epstein-Barr virus (EBV) is maintained clonally in a wide array of tumors and cells of lymphoproliferative disorders, yet the degree to which these infected cells continuously depend on EBV remains unresolved. To directly assess EBV dependence, we used two complementary Cas9-based viral eviction strategies: 1) targeting EBNA1 to promote episome dilution during cell division, and 2) targeting repetitive viral genomic regions to rapidly degrade EBV episomes. Using these approaches, we evaluated EBV-loss sensitivity across newly-derived, phenotypically diverse EBV-positive Burkitt lymphoma lines. We also evaluated cell lines from patients with EBV-associated T/NK and epithelial malignancies. We included the Burkitt Lymphoma cell line, Akata-GFP, as our EBV-loss-tolerant control and a lymphoblastoid cell line as our EBV-loss-sensitive control. We observed EBV-loss sensitivity in all the EBV-positive Burkitt Lymphoma cell lines and the chronic active EBV disease cell line. In contrast, the EBV-positive gastric adenocarcinoma lines were markedly less sensitive to EBV loss. EBV latency type did not predict EBV dependence, indicating that viral gene expression programs alone do not dictate reliance on the virus. These findings demonstrate that EBV dependence is prevalent across multiple EBV-associated disease models and is especially pronounced in early-state or newly derived lymphoid malignancies. AUTHOR SUMMARYEpstein-Barr virus (EBV) infects most people worldwide and is linked to many malignancies. Although EBV is present in cancer cells, it is unclear whether these cells require the virus to survive. To answer this question, we used CRISPR/Cas9-based methods to remove EBV from infected cells. Using these tools, we studied many different EBV-positive cell lines, including new Burkitt lymphoma cell lines. We found that most EBV-positive lymphoid cells required EBV for growth. In contrast, EBV-positive gastric cells were more tolerant of viral loss. These results show that many EBV-associated malignancies, especially early or newly established diseases, rely on EBV to survive.