A Fully 59-CG-39 but Not a 59-CCGG-39 Methylated Late Frog Virus 3 Promoter Retains Activity

Several lines of evidence demonstrate that the DNA of the iridovirus frog virus 3 (FV3) is methylated in all 5*-CG-3* sequences both in virion DNA and in the intracellular viral DNA at late times after infection. The 5-methyldeoxycytidine residues in this viral DNA occur exclusively in 5*-CG-3* dinucleotide positions. We have cloned and determined the nucleotide sequence of the L1140 gene and its promoter from FV3 DNA. The gene encodes a 40-kDa protein. The results of transcriptional pattern analyses for this gene in fathead minnow fish cells document that this gene is transcribed exclusively late after FV3 infection. The L1140 gene and its promoter are fully methylated at late times after infection. We have been interested in resolving the apparent paradox that the methylated L1140 promoter is methylated and active late in FV3-infected cells. Of course, the possibility cannot be excluded that one or a few 5*-CG-3* sequences outside restriction endonuclease sites escaped de novo methylation after FV3 DNA replication. We have devised a construct that places the chloramphenicol acetyltransferase gene under the control of the L1140 promoter. Upon transfection, this construct exhibits activity only in FV3-infected BHK-21 hamster cells, not in uninfected BHK-21 cells. The fully 5*-CG-3* or 5*-GCGC-3* (HhaI) methylated, HpaII-mock-methylated, or unmethylated L1140 promoter-chloramphenicol acetyltransferase gene construct is active in FV3-infected BHK-21 cells, whereas the same construct 5*-CCGG-3* (HpaII) methylated has lost activity. Apparently, complete methylation of the late L1140 promoter in FV3 DNA is compatible with activity. However, a very specific 5*-CCGG-3*methylation pattern that does not naturally occur in authentic FV3 DNA in infected cells abrogates promoter function. These results further support the notion that very specific patterns of methylation are required to inhibit or inactivate viral promoters.