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) modulat or/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific meth ylation of the major IE enhancer and/or modulator with the cytosine me thyltransferases FnuDII, HhaI and HaeIII had no significant effect, th e promoter activity was completely repressed by methylation of the cyt osine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Add ition of TNF-alpha or PMA which are strong stimulators of HCMV major I E enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylati on by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicat e that a methyl-CPG binding factor is involved as mediator in the inhi bitory effect of HCMV enhancer/promoter methylation. Taken together, t he HCMV major IE enhancer/promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG d inucleotides, a process that is proposed to play a modulatory role in viral latency.