Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells

Cells of the A11 lineage of Rat-1 contain a single complete Rous sarcoma provirus. Variation in the activity of this provirus accompanies fluctuations in the lineage between normal and transformed phenotypes. Increased proviral cytosine methylation of the doublet CpG in the tetranucleotide CCGG correlates with transcriptional inactivity and this pattern of cytosine hypermethylation is stable, even when the cells are transformed by another virus. However, transformation can also be induced by 5-azacytidine (but not by other mutagens) and in these transformants reduced proviral cytosine methylation is accompanied by increased proviral transcription. Differences in CCGG methylation between normal and transformed cells are found mainly in the 3' half of the provirus; sites near and within the src gene are heavily methylated only when the provirus is transcriptionally inactive. On the other hand, both transformed and normal A11 derivatives show little, if any, cytosine methylation of CCGG sequences in and flanking the 5' portion of the provirus.