Genomic sequencing reveals absence of DNA methylation in the major late promoter of adenovirus type 2 DNA in the virion and in productively infected cells.

By using methylation-sensitive restriction endonucleases, we have previously provided evidence that adenovirus type 2 (Ad2) virion DNA or free intranuclear Ad2 DNA in productively infected hamster or human cells is not methylated. We have now chosen a different experimental approach and have investigated the major late promoter (MLP) sequence of Ad2 DNA for the presence of 5-methyldeoxycytidine (5-mC) residues with the genomic sequencing technique. This study has been prompted by the finding that the MLP of Ad2 DNA can be inactivated by sequence-specific methylation in experiments in which a MLP-chloramphenicol acetyltransferase construct has been transcribed in a cell-free system from HeLa cell nuclear extracts. Virion Ad2 DNA and Ad2 DNA isolated from productively infected human or hamster cells between 1 and 48 h post-infection (p.i.) have now been analyzed. There is no evidence for the presence of 5-mC in the cytidine positions in the MLP of any of these Ad2 preparations. We conclude that DNA methylation does not seem to play a role in the early-late control of this viral promoter. The sensitivity of the genomic sequencing technique does not permit us to exclude the unlikely presence of 5-mC in a few Ad2 DNA molecules.