METHYLATION OF HSV-1 DNA AS A MECHANISM OF VIRAL INHIBITION - STUDIES OF AN ANALOG OF METHYLDEOXYCYTIDINE - TRIFLUOROMETHYLDEOXYCYTIDINE (F3MDCYD)

Although several hypomethylating agents such as 5-azadeoxycytidine and 5-fluorodeoxycytidine have shown to activate transcription after incorporation into viral or cellular DNA, agents which selectively affect the methylation status of virus-infected cells have not been described. Studies on the antiviral effect of the methyldeoxycytidine (mdCyd) analogue trifluoromethyldeoxycytidine (F3mdCyd) showed significant antiviral activity against herpes simplex virus type 1 (HSV-1). This analogue of both dCyd and dThd is selectively incorporated into the DNA of herpesvirus infected cells due to the unique specificity of the herpesvirus thymidine kinase (TK) because the HSV-1 TK is both a dCyd and dThd kinase. In contrast, the deoxycytidine kinase of uninfected cells preferentially phosphorylates dCyd and has a poor affinity for F3mdCyd. F3mdCyd hemisubstituted M13 DNA displayed the same properties as mdCyd-substituted M13 DNA with respect to cleavage by restriction enzymes, and acted as an efficient template for eukaryotic DNA methyltransferase (S-adenosyl-L-methionine DNA (cytosine-5) methyltransferase: EC 2.1.1.37). Using the persistently infected CEM cell model system, the extent of DNA methylation was shown to increase in a dose-related manner when HSV-1-infected CEM cells were treated with increasing concentrations of F3mdCyd. Higher levels of methylation correlated with significant decreases in HSV-1 titers. Isoschizomer analyses followed by Southern blotting and hybridization with genomic HSV-1 DNA showed that DNA from HSV-1-infected, analogue-treated Vero cells was resistant to cleavage by restriction enzymes at a time when productive virus was not present in culture. We infer from these results that the methylation-like properties of the incorporated F3mdCyd occur concomitantly with, and appear to be involved in, the mechanisms of the analogue's antiviral effect towards HSV-1.