Regulation of EBV lytic replication in epithelial cells
Regulation of the lytic cycle of Epstein-Barr virus (EBV) in epithelial cells is the topic of my thesis research. In this work, I examined the outcome of EBV infection in epithelial cells and characterized cellular factors, particularly C/EBP proteins, that are involved in EBV lytic cycle regulation in the epithelial cell background.
In the first chapter I investigated the controversial association of EBV with epithelial carcinomas such as breast cancer. I examined EBV gene expression in breast carcinoma cells infected in vitro with virus and found evidence for a mixture of latency II and lytic expression. Evaluation of breast carcinoma and normal breast tissues also resulted in detection of lytic ZTA transcripts. Overall, my results suggest that breast epithelial cells can be infected at low frequency and support progression into the EBV lytic cycle. Sporadic lytic EBV infection may contribute to PCR-based detection of EBV in traditionally nonvirally associated epithelial malignancies.
In the second chapter, I evaluated the cellular pathways that are involved in EBV lytic induction in different epithelial and B cell background by treating cells with various chemical inducers. I found that the response to chemical treatment differed from cell line to cell line and there was no common or predictable pattern of response. The inducibility of the promoters for the key lytic regulators, Zp and Rp, was highly dependant on cell type. I conclude that EBV has developed strategies to manipulate multiple cellular pathways depending on which cell type it resides in and which signal it receives to initiate viral replication.
In the third chapter, I evaluated the functional contributions of C/EBP proteins to the transactivation of three regulators of the EBV lytic cycle, the ZTA and RTA proteins and the lytic origin of replication, oriLyt, in epithelial cells. I demonstrated that endogenous C/EBP proteins are associated with EBV lytic promoters in epithelial cells and facilitate a constitutive low level of viral lytic replication.
The ultimate aim of chapter four is to investigate how Epstein-Barr virus establishes and maintains a persistent infection in vivo. I generated and tested the following reagents to determine the feasibility of the proposed approach. (1) generated Lenti/Zp-GFP expression vectors and showed that Lenti/Zp-GFP expression vectors were activated by known lytic-inducing treatments; (2) showed that Lenti/Zp-GFP expression vectors were packaged to make functional lentivirus. In the future, these reagents will be used to transduce mouse bone marrow cells followed by bone marrow transplantation and reconstitution to allow an evaluation of lytic Zp promoter expression in mouse secondary lymphoid tissues such as spleen and tonsil patches.