Mutagenesis occuring following infection with herpes simplex virus and the contribution of virus ribonucleotide reductase

Herpes simplex virus type 2 (HSV-2) has been associated with cervical cancer for many years and certain regions of both the HSV-1 and the HSV-2 genome can induce transformation of tissue culture cells. However, no virus transforming protein has yet been identified and no region of viral DNA appears to be stably retained in transformed cells or tumours. This implies that continued expression of a virus protein is not required and has resulted in the proposal that HSV transforms cells by a "hit and run" mechanism. HSV infection can generate chromosomal breaks together with amplification and rearrangement of cellular genes. Additionally, infection of permissive cells with UV-inactivated HSV-1 and of non-permissive cells with HSV-2 leads to an increase in the mutation frequency of the cellular hypoxanthine-guanine phosphoribosyltransferase gene. HSV-1 was also found to increase the mutagenic frequency of a plasmid-based gene located on the shuttle vector pZ189. These mutagenic effects resemble changes produced by chemical carcinogens and may represent a mechanism for transformation which would not require the retention of viral sequences. The cause of mutations occurring in HSV-infected cells is unknown but could be connected with the destabilization of doxyribonucleoside triphosphate (dNTP) pools seen in infected cells as studies of both prokaryotic and eukaryoti systems have stressed the importance of dNTP concentrations in mediating base substitutions and misincorporations. The HSV enzyme ribonucleotide reductase has a potential role in the destabilization of cellular dNTP pools since, unlike it cellular counterpart, it is not allosterically regulated. This is supported by results which show that a mutant cellular enzyme, which was deficient in allosteric control, raised the cellular mutation rate in transfected cells. In order to study the effect of HSV-2 ribonucleotide reductase on mutagenesis, expression vectors were constructed which contained the large (RRl) and small (RR2) subunits of the HSV-2 ribonucleotide reductase under the control of the inducible mouse metallothionein promoter, which responds at the transcriptional level to heavy metals such as zinc and cadmium. These constructs were used in mutagenesis assays which involved monitoring the mutation frequencies of the cellular adenine phosphoribosyltransferase gene (aprt) in Chinese hamster ovary (CHO) cells and of the plasmid-based suppressor tRNA gene (supF) in human cells. Increasing the amount of enzyme expression by zinc-induction did not increase the mutagenesis of the marker genes in either assay suggesting that virus ribonucleotide reductase expression may not be mutagenic in HSV-infected cells. Experiments using the HSV-1 mutant viruses tsl207 and tsl222, which are temperature sensitive (ts) for RRl and RR2 respectively, support this finding since these viruses were as mutagenic as wild type HSV-1 at both the permissive (P) and the non-permissive (NPT) temperatures. Although expression of the viral ribonucleotide reductase does not appear to be the cause of increased mutagenesis in infected cells, the possibility that enzyme activity is capable of producing a mutagenic effect cannot be ruled out. The mutagenesis assays described above involved only the transient expression of the viral enzyme and the lack of any mutagenic effect might be due to inefficeincies in the transfection procedure and/or insufficient expression of the viral genes. The cellular enzyme would also be present and might counteract dNTP pool imbalances brought about by viral ribonucleotide reductase expression. To address these problems, NIH3T3 and CHO cell lines were constructed using HSV-2 ribonucleotide reductase expressing plasmids that contained the G418-resistance gene (neoR). Mutations causing morphological transformation were assayed in NIH3T3 cells while in CHO cells the aprt gene was assayed. None of the G418-resistant cell lines tested were found to express RRl or RR2 and further lines will be screened for expression before repeating the experiment. The ribonucleotide reductase constructs were used to generate transgenic mice to determine the effect of enzyme expression in an animal model. The mice did not appear to be affected by the presence of a large number (50+) of viral ribonucleotide reductase sequences. Viral ribonucleotide reductase RNA was detected in these animals, however, it did not appear to be inducible and protein could not be detected. Both HSV-1 and HSV-2 increase the spontaneous mutation frequency of the plasmid based supF gene by around 6-fold which is in agreement with values obtained by others in similar studies.