Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Abstract

Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10 ؊4 was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA r 5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three-to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA r 5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.DNA replication in mammalian cells is a complicated process (reviewed in references 2 and 25). It requires intricate DNA replication machinery that both duplicates chromosomal DNA and maintains genomic integrity. As the pivotal enzymes, DNA polymerases (Pols) are critical for ensuring the accuracy of DNA synthesis via their roles in the selection of nucleotides to be incorporated during DNA replication (polymerization) and in the removal of misinserted nucleotides from the 3Ј-OH end of the primer strand (proofreading) (reviewed in references 20 and 23

DNA Replication Fidelity of Herpes Simplex Virus

“…Figure 1 illustrates the supF mutagenesis assays used for the study of HSV-1 replication fidelity. Studies of recombinant viruses derived from the HSV-1 KOS strain and the transient replication assay using the modified shuttle plasmid have revealed that the KOS virus replicates the supF genes at a mutation frequency (Hwang, Liu, and Hwang, 2002;Hwang et al, 1999), which is consistent with that observed for the supF amplicon propagated in mammalian cells (Seidman et al, 1985).…”

“…Although tk mutants are easily identified in a simple plaque assay, this method may not be sensitive enough to detect all mutants, such as silent mutations and mutations that maintain enzyme activity. It is estimated that the TK mutagenesis assay can detect less than 50% of all possible substitutions of tk mutations (Hwang, Liu, and Hwang, 2002). Despite this, an extensive study (Lu, Hwang, and Hwang, 2002a) of tk mutants derived from wild-type HSV-1 strain KOS found that HSV replicates DNA with a genomic mutation rate similar to those of other DNA-based microbes (Drake and Hwang, 2005).…”

“…This mutant Pol induces differences in the type and distribution of base substitutions relative to those by wild-type Pol (Hwang, Liu, and Hwang, 2002), suggesting that the amino acid change in PAA r 5 Pol results in alterations in its replication fidelity. However, this mutant Pol has a modest mutator activity in replicating the supF gene (Hwang, Liu, and Hwang, 2002), which is in contrast to the anti-mutator activity observed by the TK mutagenesis assay (Hall et al, 1984;Hall et al, 1985;Hwang and Chen, 1995). Perhaps the sequence contents of the target genes contribute to the differences, similar to those observed for other Pols (reviewed in (Goodman et al, 1993)).…”

“…The TK mutagenesis study demonstrates that Y577H and Y577H/D581A recombinant viruses are highly mutagenic relative to the parental wild-type virus (Hwang et al, 1997). On the other hand, the supF mutagenesis assay reveals the modest effects of the exonuclease activity on replication fidelity (Hwang, Liu, and Hwang, 2002). It is possible that different sequence content of the target genes could contribute to the difference of mutation frequency.…”

“…We have modified and inserted the supF amplicon into the tk locus, allowing measurement of the supF mutation frequency in the context of the viral genome during viral replication Hwang, Liu, and Hwang, 2002;Hwang et al, 2004). Furthermore, the supF amplicon can be modified to contain the origin (ori) sequences required for HSV-1 DNA replication and applied as a shuttle plasmid for transient DNA replication in HSV-infected cells for the mutagenesis assay , an assay similar to that applied by Seidman et al (Seidman et al, 1985).…”