Selective methylation of DNA in HSV-1 infected cells as a mechanism of viral inhibition; studies of an analogue of methyldeoxycytidine: Trifluoromethyldeoxycytidine (F3mdC)
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
First Committee Member
Sheldon Greer, Committee Chair
Although several hypomethylating agents such as 5-azadeoxycytidine and 5-fluorodeoxycytidine have been 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 (mdC) analogue trifluoromethyldeoxycytidine (F3mdC) showed significant antiviral activity against herpes simplex virus type 1 (HSV-1). This analogue of both dC and dT is selectively incorporated into the DNA of herpesvirus-infected cells, but not the DNA of uninfected cells, due to the unique specificity of the herpesvirus thymidine kinase (TK). Furthermore, F3mdC is selectively phosphorylated by HSV-1 TK in preference to dC. In contrast, the deoxycytidine kinase of uninfected cells preferentially phosphorylates dC and has a poor affinity for F3mdC.F3mdC hemisubstituted M13 DNA did not act as a substrate for methylation-sensitive restriction enzymes but acted as an efficient template for eukaryotic DNA methyltransferase (S-adenosyl-L-methionine DNA ((cytosine-S) methyltransferase: Enzyme Commission Classification Number 18.104.22.168)). Using the persistently-infected CEM cell model system, percent DNA methylation was shown to increase in a dose-CEM cell model system, percent DNA methylation was shown to increase in a dose-dependent manner when HSV-1-infected CEM cells were treated with increasing concentrations of F3mdC. 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 methylation-sensitive restriction enzymes at a time when either analogue was present in culture or virus production was decreased. We infer from these results that the methylation-like properties of the incorporated F3mdC occur concomitantly with, and appear to be involved in the mechanisms of inhibition by the analogue of HSV-1 replication.
Biology, Molecular; Biology, Microbiology; Health Sciences, Pharmacology
Barletta, Janet M., "Selective methylation of DNA in HSV-1 infected cells as a mechanism of viral inhibition; studies of an analogue of methyldeoxycytidine: Trifluoromethyldeoxycytidine (F3mdC)" (1993). Dissertations from ProQuest. 3159.