Investigating the Heterogeneity of Epstein-Barr Virus Lytic Reactivation

Abstract

Epstein-Barr virus (EBV) is a y-herpesvirus that infects up to 95% of the world’s population by adulthood and persists through the life of the host. Primary infection is often benign in immune-competent individuals but can lead to infectious mononucleosis. However, in immune-suppressed backgrounds, EBV is associated with a variety of more severe malignancies including gastric cancer, nasopharyngeal carcinoma, and various lymphomas. In EBV-associated lymphomas, the virus can be difficult to target within the cancer cells since it is maintained primarily in a latent state. EBV has two main life cycles: latency and lytic reactivation. Latency is defined by the expression of very few viral genes with the goal of maintaining the genome in the infected cell. Lytic reactivation is characterized by expression of all ~80 viral genes with the goal of replicating the genome and synthesizing new viral particles. To develop more targeted therapies for EBV-associated lymphomas, kick and kill strategies have entered clinical trials. This approach utilizes pharmaceuticals like HDAC inhibitors to reactivate the virus so that infected cancer cells can be better targets for downstream antivirals. However, it is well known that this reactivation is often inefficient. In this dissertation, I characterize the effect of HDAC inhibitors on several models of EBV-associated malignancies through multiple single-cell approaches. We observed that there is a careful balance between cell cycle arrest, cell death, and potential EBV lytic reactivation. We also observed that there is incomplete progression through the lytic cycle. To investigate this further, we completed two single-cell RNA sequencing experiments to better characterize both host and viral gene expression patterns during this process. We found that abortive -or incomplete- lytic replication is characterized by high NF-B activity as well as expression of host immune signaling genes. Further validation and mechanistic follow up revealed that CD137 signaling has a direct effect on lytic reactivation. Overall, the results of our research highlight the complex viral-host interactions that occur during lytic reactivation. Additionally, this work has important implications for the future success of kick and kill therapies.