ZEB1 and c-Jun Levels Contribute to the Establishment of Highly Lytic Epstein-Barr Virus Infection in Gastric AGS Cells

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Abstract
The induction of lytic infection has been proposed as a therapeutic strategy for treating Epstein-Barr virus (EBV)-positive malignancies. To succeed, efficient methods are needed for activating the EBV immediate-early (IE) promoters, Zp and Rp. Here we compared factors which regulate Zp and Rp in AGS gastric carcinoma cells that support a remarkably high level of persistently lytic EBV infection with HeLa cervical cells that permit only tightly latent infection. We found that the level of Zp activity assayed by transient transfection assays with reporter plasmids was high in AGS cells but low in HeLa cells. The level of Rp activity was low in both cell types. Mutational analysis indicated that sequences within Zp located between -70 and +27 relative to the transcription initiation site were sufficient to confer a high level of Zp activity in AGS cells. The Zp CRE motif was necessary for this constitutive activity, while the ZIA and ZIB MEF2D motifs were not. Consistent with these findings, immunoblot analysis indicated that phosphorylated c-Jun, which activates Zp through the CRE motif, was expressed at a much higher level in EBV-infected AGS cells than in EBV-infected HeLa cells. In contrast, ZEB1, which represses Zp via the ZV motif located near the transcription initiation site, was abundant in HeLa cells, while it was absent from AGS cells. Exogenous addition of ZEB1 led to the repression of Zp in AGS cells. We conclude that the unusually high Zp activity level in AGS cells is due to the high abundance of positively acting transcription factors such as c-Jun combined with the low abundance of negatively acting factors such as ZEB1.