Exonuclease-deficient polymerase mutant of herpes simplex virus type 1 induces altered spectra of mutations

Abstract

The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.6%, consistent with the tk mutation frequencies reported previously (Y. T. Hwang, B.-Y. Liu, D. M. Coen, and C. B. C. Hwang, J. Virol. 71:7791-7798, 1997). The increased mutation frequencies were 10- to 500-fold higher than those observed for wild-type Pol recombinants. The increased mutation frequencies also were significantly higher than those of supF mutant replicated by exonuclease-deficient Pols in the plasmid-borne assay. Furthermore, characterization of supF mutants demonstrated that recombinants with a defective exonuclease induced types and distributions of supF mutations different from those induced by wild-type Pol recombinants. The types of supF mutations induced by exonuclease-deficient recombinants differed between the plasmid- and genome-based assays. The spectra of supF mutations also differed between the two assays. In addition, exonuclease-defective viruses also induced different spectra of supF and tk mutations. Therefore, both the assay methods and the target genes used for mutagenesis studies can affect the repication fidelity of herpes simplex virus type 1 Pol with defective exonuclease activity.

FIG. 1.

(A) Map of the plasmid pSupF-tk-2. (B) Restriction maps of sequences around the tk locus of recombinant Y7 (panel 1), recombinants Y7/F containing integrated pSupF1 sequences (panel 2), and the plasmid pSupF-tk-2 (panel 3). The relative positions of 300-bp tk and 2.4-kbp pSupF1 probes also are illustrated in panel 3. Enzymes shown in the figure include the following: B, BamHI; P, PstI; R, EcoRI; S, SpeI. Restriction enzymes shown in parentheses indicate that the cutting site is lost due to cloning. ori, ColE1; amp, ampicillin-resistant gene; F, supF gene.

FIG. 1.

(A) Map of the plasmid pSupF-tk-2. (B) Restriction maps of sequences around the tk locus of recombinant Y7 (panel 1), recombinants Y7/F containing integrated pSupF1 sequences (panel 2), and the plasmid pSupF-tk-2 (panel 3). The relative positions of 300-bp tk and 2.4-kbp pSupF1 probes also are illustrated in panel 3. Enzymes shown in the figure include the following: B, BamHI; P, PstI; R, EcoRI; S, SpeI. Restriction enzymes shown in parentheses indicate that the cutting site is lost due to cloning. ori, ColE1; amp, ampicillin-resistant gene; F, supF gene.

FIG. 2.

Southern blots of recombinants containing integrated pSupF1 sequences. The blot was hybridized with the pSupF1 probe (A) and then stripped and reprobed with the 300-bp tk probe (B). The relative locations of probes used are shown in Fig. 1B (panel 3), and the relative sizes of restriction fragments of recombinants Y7 and Y7/F-1 and the plasmid pSupF-tk-2 are depicted in Fig. 1B. Sample orders in panels A and B are as follows: lanes 1 and 4, Y7; lanes 2 and 5, Y7/F-1; lanes 3 and 6, pSupF-tk-2. Samples in lanes 1 to 3 were digested with BamHI, and samples in lanes 4 to 6 were digested with EcoRI. ³²P-labeled lambda DNA digested with HindIII was used as the molecular weight marker in this experiment (not shown), and the relative positions of marker bands are depicted on the left.

FIG. 3.

Analysis of PCR products of the pol fragments of Y7/F recombinants. PCRs were performed to amplify a 967-bp fragment (lanes 1 to 7) encompassing the Exo III motif and a 1,032-bp fragment (lanes 8 to 14) overlapping the C-terminal 1.0-kbp sequence of the pol gene. PCR products then were digested with either ScaI (lanes 1 to 7) or DdeI (lanes 8 to 14) to determine the presence or absence of the corresponding restriction site. The Exo III mutation present in Y7 pol results in the loss of the ScaI recognition (lane2). Mutation at the nucleotides coding for amino acid 1038 results in the resistance to DdeI restriction (lanes 11 and 12). Heterogeneity was found in PCR products in lane 9 in which approximately 10% of samples were resistant to DdeI digestion. BstEII-digested DNA was used as the molecular weight marker, with corresponding sizes depicted on the left. Lanes: 1 and 8, PCR products from wild-type pol gene; 2 and 9, Y7/F-1; 3 and 10, Y7/F-10; 4 and 11, Y7/F-11; 5 and 12, Y7/F-13; 6 and 13, Y7/F-12; 7 and 14, Y7/F-41.

FIG. 4.

Distribution of substituted and deleted bases found in supF mutants isolated from KOS/F recombinants including those reported in Fig. 4 of reference (upper panel) and Y7/F-1 (lower panel). The coding sequence of the supF gene is shown at the top line of each panel from the 5′ end of the sequence. Numbers shown in parentheses refer to the number of mutants identified. Multiple substitutions shown as the shapes of a pyramid or a half-pyramid refer to the same substitution at that particular nucleotide, Δ, base deleted at the nucleotide position shown; underlined bases, mutants containing two or three altered bases.