The immediate-early (IE) BZLF1 gene of Epstein-Barr virus (EBV) regulates the switch between latent and lytic infection by EBV. We previously showed that the cellular transcription factor ZEB1 binds to a sequence element, ZV, located at nt −17 to −12 relative to the transcription initiation site of the BZLF1 promoter, Zp, repressing transcription from Zp in a transient transfection assay. Here, we report the phenotype in the context of a whole EBV genome of a variant of EBV strain B95.8 containing a 2-bp substitution mutation in the ZV element of Zp that reduced, but did not eliminate, ZEB1 binding to Zp. Strikingly, epithelial 293 cells latently infected with the EBV ZV mutant spontaneously produced IE-, early-, and late-gene products and infectious virus, while wild-type (WT)-infected 293 cells did not and have never been reported to do so. Furthermore, treatment with the chemical inducers sodium butyrate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to an additional order-of-magnitude production of infectious virus in the ZV mutant–infected 293 cells, but still no virus in the WT-infected 293 cells. Similarly, ZV mutant–infected Burkitt's lymphoma BJAB cells accumulated at least 10-fold more EBV IE mRNAs than did WT-infected BJAB cells, with TPA or sodium butyrate treatment leading to an additional 5- to 10-fold accumulation of EBV IE mRNAs in the ZV mutant–infected cells. Thus, we conclude that ZEB1 binding to Zp plays a central role in regulating the latent-lytic switch in EBV-infected epithelial and B cells, suggesting ZEB1 as a target for lytic-induction therapies in EBV-associated malignancies.
Ninety percent of people in the world become infected with Epstein-Barr virus (EBV). The virus can infect both epithelial and B cells, either making more virus and killing the cell or establishing a latent form of infection where it is stably maintained in the host. EBV infection is associated with the development of some types of cancer. We show here that a mere 2-bp substitution mutation in the silencer element, ZV, of the promoter of EBV's immediate-early BZLF1 gene in the context of a whole EBV genome can lead to spontaneous reactivation of EBV out of latency into lytic replication, with production of infectious virus in some cells. The presence of the mutation also (i) made the virus more responsive to reactivation following treatment with chemical inducers, and (ii) disrupted binding of a cellular transcriptional repressor protein, ZEB1, to the BZLF1 promoter. Our work suggests a method to kill EBV-infected cancer cells by treating them with agents that lower the repressor activity of ZEB1. It also suggests one may be able to generate a vaccine against EBV infection using a constitutively lytic EBV strain made by knocking out the silencer elements of the BZLF1 promoter.