Abstract
BackgroundThe thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1.MethodsA panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases.ResultsAlthough HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture.ConclusionsThis study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.
Highlights
The thymidine kinase mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations
HSV-2 clinical isolates were shown to have a higher frequency of spontaneous mutations resistant to ACV and PCV, approximately 30fold, compared to HSV-1 isolates, and the majority of these mutations are in tk
Pol-recombinant viruses HSV-2 pol (SB5, 83D, 6652 and 6757 described in [21]) or HSV-1 pol (SC16 and PAAr5) coding regions were cotransfected with replication defective HP66 viral DNA containing a LacZ insertion within pol[17] into Vero cells to generate recombinant viruses
Summary
The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. HSV-2 clinical isolates were shown to have a higher frequency of spontaneous mutations resistant to ACV and PCV, approximately 30fold, compared to HSV-1 isolates, and the majority of these mutations are in tk. Consistent with this observation, HSV-2 strains exhibited approximately a 20- to 80-fold higher spontaneous mutation rate to cidofovir (HPMPC), an inhibitor of HSV Pol, resistance compared with HSV-1 [21] These naturally-occuring mutations are not unique to tk and TK substrates as detected with antiviral agents ACV and PCV, but were extended with the direct Pol inhibitor, HPMPC, to other loci, most likely pol
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