Methylation of transcription factor binding sites in the Epstein-Barr virus latent cycle promoter Wp coincides with promoter down-regulation during virus-induced B-cell transformation

Two Epstein-Barr virus latent cycle promoters for nuclear antigen expressio n, Wp and Cp, are activated sequentially during virus-induced transformatio n of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously pub lished restriction enzyme studies have indicated hypomethylation of CpG din ucleotides in the Wp and Cp regions of the viral genome in established LCLs , whereas these same regions appeared to be hypermethylated in Burkitt's ly mphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in e stablished LCLs with the typical pattern of dominant Cp usage; surprisingly , this showed substantial methylation in the 400-bp regulatory region upstr eam of the Wp start site. This was not an artifact of long-term in vitro pa ssage, since, in cultures of recently infected B cells, we found progressiv e methylation of Wp (but not Cp) regulatory sequences occurring between 7 a nd 21 days postinfection, coincident with the period in which dominant nucl ear antigen promoter usage switches from Wp to Cp. Furthermore, in the equi valent in vivo situation, i.e., in the circulating B cells of acute infecti ous mononucleosis patients undergoing primary EBV infection, we again frequ ently observed selective methylation of Wp but not Cp sequences. An effecto r role for methylation in Wp silencing was supported by methylation cassett e assays of Wp reporter constructs and by bandshift assays, where the bindi ng of two sets of transcription factors important for Wp activation in B ce lls, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylatio n of their binding sites.