Regulation of the latent-to-lytic switch of Epstein-Barr Virus by ZEBs and miRNAs

Epstein-Ban Virus (EBV) is a human gammaherpesvirus that infects approximately 90% of the global population. Latent infection of EBV is associated with several malignancies of epithelial and B-lymphocytic cells. The Mertz laboratory previously reported that the cellular protein ZEB1 maintains EBV latency, repressing expression of the EBV latent-to-lytic switch BZLF1 gene by directly binding the ZV element of its promoter, Zp. Here, I definitively show using si- and shRNA technologies that ZEB1 plays a role in maintenance of EBV latency in some cell lines. I additionally show that another zinc-finger E-box-binding protein, ZEB2/SIP1, can also bind to and repress Zp via binding the ZV element. In EBV-positive cells containing only ZEB1, knockdown of ZEB1 leads to viral reactivation out of latency. However, in EBV-positive cells containing both ZEBs, ZEB2, not ZEB1, is the key repressor binding to and repressing lytic gene expression via the ZV element of Zp. Thus, I conclude that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell type-dependent manner.;Given their key roles in maintenance of EBV latency, I next sought to identify factors that regulate ZEB expression. Others have shown that the 200 family of cellular miRNAs can downregulate both ZEB1 and ZEB2 protein synthesis. Here, I investigated the role of cellular miR-200 family members in regulating the latent-lytic switch of EBV via their effects on ZEB1 and ZEB2 expression levels. Analysis of multiple EBV-positive epithelial and B-lymphocytic cell lines revealed that the levels of these miRNAs negatively correlate with the levels of the ZEBs and positively correlate with EBV lytic-gene expression. Over-expression of either miRNA 200b or 429 leads to lytic reactivation of wild-type EBV, but not of an EBV mutated in the ZV site Inhibition of these miRNAs leads to decreased wild-type EBV lytic-gene expression. Thus, I conclude that miRNAs 200b and 429 can function as key regulators of the latent-lytic switch of EBV. Furthermore, I identify ZEBs as potential targets for lytic induction therapy in EBV-associated malignancies, with miRNAs being potential agents with which to knock them down.