Title

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

1993

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Sheldon Greer, Committee Chair

Abstract

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 2.1.1.37)). 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.

Keywords

Biology, Molecular; Biology, Microbiology; Health Sciences, Pharmacology

Link to Full Text

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