Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro.
The influence of DNA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyl-transferases FnuDII, HhaI and HaeIII had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Addition of TNF-alpha or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/ promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.
Prösch
S
S
Institute of Medical Virology, Humboldt University Medical School, Berlin, Germany.
Stein
J
J
Staak
K
K
Liebenthal
C
C
Volk
H D
HD
Krüger
D H
DH
D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D003587:Cytomegalovirus / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D018922:HL-60 Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000956:Antigens, Viral; D004247:DNA; D015257:DNA-Cytosine Methylases; D015226:Dinucleoside Phosphates; D017874:Immediate-Early Proteins; D013755:Tetradecanoylphorbol Acetate; D014409:Tumor Necrosis Factor-alpha; C044582:immediate-early proteins, cytomegalovirus; C015772:CpG dinucleotide; C081573:DNA modification methylase SssI