Methylation of the EBV genome and establishment of restricted latency in low-passage EBV-infected 293 epithelial cells

Abstract

Epstein-Barr virus (EBV) encodes multiple latency programs: a growth-transforming program (type III) latency program and restricted-latency (types I and II) programs. During type III latency, EBV expresses six nuclear antigens, all of which are encoded by a single complex transcriptional unit driven by two linked promoters, Cp and Wp, while restricted viral latency is characterized by the expression of a single nuclear antigen, EBNA1, whose expression is driven from a distinct transcription unit under the control of the Qp promoter. EBV infection of the 293 epithelial cell line frequently leads to the establishment of a type I/II latent infection. Here we report that during the initial stages of virus infection of the 293 cell line, both Cp and Wp are active. However, analysis of four established, low-passage EBV-infected 293 cell lines revealed that three of these exhibited Qp-driven transcription of the EBNA 1 gene and little or no detectable Cp and Wp activity, while the fourth cell line exhibited Cp activity. Notably, all four cell lines contained the necessary transcription factors to drive transcription initiation from Cp and Wp when transiently transfected with unmethylated reporter constructs. Furthermore, in the cell lines exhibiting restricted EBV latency the viral genomes were extensively methylated around Cp and Wp, but not Qp. In contrast, in the cell line exhibiting Cp activity the viral genomes were hypomethylated around Cp, Wp, and Qp. Taken together, these results provide evidence that the establishment of a restricted latent infection in the 293 epithelial cell line is not due to a failure to initiate the growth-transforming (type III) latency program, but rather may arise from a selection against the type III latency program. Furthermore, these results are consistent with the hypothesis that methylation of Cp and Wp is required for entry into the type I or II latency programs.