Background
The replicative polymerase of herpes simplex virus (HSV) is comprised of catalytic (Pol) and processivity subunits (UL42) [
9,
11]. HSV Pol is responsible, in concert with other viral proteins, for regulating the fidelity of DNA replication by selecting the correct nucleotides for incorporation and by proofreading or editing mispaired nucleotides [
20]. The Pol subunit contains intrinsic 3'-5' exonuclease activity [
14‐
16] and the N-terminal region of the polypeptide encoding this function is highly conserved in other polymerases, containing three segments referred to as Exo motifs [
3,
26].
Thymidine kinase polypeptide (TK) is required for the activation of some antiviral nucleoside analogs e.g. acyclovir (ACV) and penciclovir (PCV), in order to inhibit viral DNA replication. HSV with TK mutations may impair activation of these drugs and confer a drug-resistant phenotype. The natural phenomenon of spontaneous mutation, which occurs in the absence of drug selection, results in the accumulation of approximately six to eight TK-deficient variants per 10
4 plaque-forming viruses in virus populations that have never been exposed to selective pressure [
6,
10,
12]. Furthermore, HSV-2 clinical isolates were shown to have a higher frequency of spontaneous mutations resistant to ACV and PCV, approximately 30-fold, compared to HSV-1 isolates, and the majority of these mutations are in
tk. Consistent with this observation, HSV-2 strains also 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]. Therefore, 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.
Since mutations within the viral
pol gene have been previously shown to affect spontaneous viral mutation rates [
10], the higher frequency of errors associated with type 2 viruses may be an inherent property of the type 2 Pol. To assess the direct contribution of polymerases from virus types 1 and 2 to mutation frequency, recombinant HSV expressing a type 2 Pol within a type 1 viral genome were generated by marker transfer and examined along with a panel of other HSV isolates in two mutagenesis assays.
Materials and methods
Cell lines, compounds and viruses
Vero (ATCC, passage 15–20), MRC-5 (ATCC, passage 12–15) and PolB3 (kind gift of D. Coen, Harvard University, Boston MA) cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and incubated at 37°C, 5%CO
2. PolB3 represents a Vero cell line constitutively expressing the HSV-1 Pol [
17]. PCV was synthesized at SmithKline Beecham Pharmaceuticals. ACV used in these studies was obtained from Sigma (St. Louis, MO). For cell culture assays, 10 mg/ml stock solutions were prepared in dimethyl sulphoxide (DMSO) and stored at -20°C. Working dilutions were prepared in assay medium immediately before use as described below. HSV-1 strain SC16 (wild type laboratory virus), HSV-2 SB5 (wild type laboratory virus), HSV-2 6652 (wild type clinical virus), HSV-2 83D (wild type clinical virus), HSV-2 6757 (high error mutant clinical virus) were described previously [
21] and the HSV-1 PAA
r5 (antimutator laboratory virus) utilized in this study was a kind gift of D. Coen (Harvard University, Boston, MA) [
10]. HP66 (HSV-1 genome containing a LacZ insertion within the
pol coding region) viral DNA was kindly provided by D. Coen (Harvard University, Boston, MA) [
17]. HP66 viral stocks were prepared by transfecting HP66 viral DNA into PolB3 cells, which allow for virus propagation by expressing the viral polymerase.
In vitro susceptibility assays
The
tk mutagenesis assay was performed in MRC5 and Vero cells as described previously [
10,
21]. The proportion of resistant virus referred to as mutation frequency or error frequency throughout this study was calculated as follows: (titer in the presence of drug) / (titer in the absence of drug) × 100.
Construction of recombinant viruses
Marker transfer experiments involved co-transfection of HSV-2
pol coding region (HSV-2 SB5, HSV-2 83D, HSV-2 6652 and HSV-2 6757 described in [
21]) with HP66 viral DNA into Vero cells. Recombinant virus was plaque purified to homogeneity in the presence of X-Gal. The
pol coding region from the recombinant viruses was confirmed to be identical to their respective parental virus
pol sequences by terminator cycle sequencing using an automated model 377 DNA sequencer (Perkin Elmer, Applied Biosystems).
Virus growth analysis
To compare the ability of parental viruses and recombinant viruses to replicate in Vero cells with that of the wild type HSV-2 SB5 strain, 3 × 105 Vero cells were infected with each virus at a multiplicity of infection of 5 PFU per cell. Virus progeny were harvested at different time points and titered on Vero cells. All viruses yielded similar PFU/ml (data not shown).
Plasmid construction
A non-HSV gene template (pSV110) was constructed by taking the plasmid pSVβgal (Promega) and inserting the HSV-1 origin of replication (ori
S) SmaI fragment [
5]. The plasmid pTK1 contains HSV-1 SC16 sequence coordinates 45,055–48,634 subcloned into the HindIII / EcoRI site of pUC19 (Invitrogen). The plasmid pTK2 contains HSV-2 SB5 sequence coordinates 44,469–48,184 subcloned into the HindIII / EcoRI site of pUC19. The plasmid pLacZ contains the LacZ coding region from pSVβgal subcloned into the HindIII / EcoRI site of pUC19.
Transient replication/rescue assay
Vero cells were seeded at 3 × 10
5 cells per 2 mL well in a six well plate and incubated overnight at 37°C. The following day, cells were transfected with 1.5 μg of pSV110 and 1 μg of salmon sperm DNA using Lipofectamine reagent according to the manufacturer's recommendations (Invitrogen). Following overnight incubation at 37°C, the medium was removed and virus inoculated at an MOI = 5 in 2 mL volume for 1 h. Following overnight incubation at 37°C, the cells demonstrated CPE and the infected-cell monolayer was rinsed twice with 1X phosphate buffered saline. Next, cell monolayers were lysed by scraping into 1.0 mL of lysis buffer (0.6% SDS, 0.01 M EDTA pH 7.5) per well. The infected cell lysate was adjusted to a final concentration of 1 M NaCl, inverted 10X and stored at 4°C overnight. The samples were centrifuged at 14,000 rpm for 10 minutes at 4°C and the supernatant was collected, treated with 100 μg/ml RNAse at 37°C for one hour. Following that incubation, 100 μg/mL Proteinase K was added and samples were incubated at 50°C for 2 hours. After several phenol-chloroform extractions, the DNA was precipitated, centrifuged at 14,000 rpm for 10 minutes at 4°C and the pellets resuspended in 50 μl water and incubated at 37°C for 2 hours. To remove non-replicated templates, 2.5 μg of infected cell DNA was digested with
DpnI and precipitated, centrifuged at 14,000 rpm for 10 minutes at 4°C and resuspended in 20 μl dH
2O. One-fourth of the
DpnI-treated DNA was transformed into DH5α cells (recombination-deficient bacterial cells; Invitrogen). The transformation mix was plated onto several LB-AMP plates containing Xgal and isopropyl β-D thiogalactoside (IPTG). The number of blue colonies (indicating no mutation in β galactosidase gene) and white colonies (indicating mutation within β galactosidase gene or promoter) were determined. An average of 300,000 colonies were recovered from three independent experiments for each virus infection. Two control samples were included in each experiment to normalize for mutations induced by transfection or superinfection processes. For the transfection control, pSV110 was transfected into Vero cells, mock-infected and directly transformed into bacteria for blue/white colony screening. For the superinfection control, pSV110 was transfected into Vero cells, superinfected with HP66 Pol-mutant virus (which is unable to replicate) and transformed into bacteria for blue/white colony screening. For the controls, both DpnI and non-DpnI treated samples were anlayzed. The values in Table
2 are presented as mutation frequency which is (number of white colonies from test sample/number of blue colonies from test sample) - (number of white colonies from HP66 superinfection sample/number of blue colonies from HP66 superinfection sample) × 100.
Table 2
Mutation frequency in non-HSV DNA.
HSV-2 SB5 | 1.7, 2.0, 1.7c
|
HSV-2 SB5PR | 2.0, 1.8, 2.1 |
HSV-2 6652 | 0.7, 0.9, 1.1 |
HSV-2 6652PR | 1.3, 1.0, 1.5 |
HSV-2 83D | 0.8, 1.1, 1.0 |
HSV-2 83D PR | 1.6, 1.2, 1.8 |
HSV-2 6757 | 4.1, 3.4, 4.9 |
HSV-2 6757PR | 1.2, 1.1, 1.1 |
HSV-1 SC16 | 2.6, 2.2, 2.1 |
PAAr5 | 3.5, 3.6, 4.1 |
S1 nuclease digestion assay
Supercoiled plasmids (5 μg) were treated with 3 units S1 nuclease or mock-treated, in S1 nuclease buffer (Invitrogen) containing 100 mM final concentration of NaCl, for 5 min at 37°C. Reactions were stopped by performing two phenol, and two phenol-chloroform extractions. DNA was precipitated with ethanol, centrifuged and resuspended in dH2O for digestion with restriction enzymes as indicated. Restriction digested samples were electrophoresed on a 0.8% agarose gel and stained with ethidium bromide for visualization. Two separate plasmid purifications were utilized for this study, with similar results.
Discussion
The
tk mutagenesis assay has been utilized by ourselves and others [
10,
13] to determine both frequency and spectrum of mutations in the HSV
tk gene following selection for drug resistance. In this study, we have extended the findings of previous work [
13] by using
pol-recombinant viruses to examine mutation frequency in HSV. Specifically, this study evaluated the contribution of type 2 polymerase to mutation frequency when measured by the
tk mutagenesis and non-HSV DNA mutagenesis assays.
The control viruses, HSV-2 6757 and HSV-1 PAA
r5, demonstrated mutator or antimutator phenotypes, respectively, in the
tk mutagenesis assay, but not in the non-HSV DNA mutagenesis assay. Thus, classification of a
pol gene as mutator (HSV-2 6757) or antimutator (PAA
r5) is not a general characteristic, but is likely dependent upon the assay method and target gene. This observation is consistent with the recent report of high mutation frequency within the
SupF gene upon replication by PAA
r5 [
13].
Since the control viruses described above functioned as expected in the tk mutagenesis assay, recombinant viruses expressing the HSV-2 polymerase within an HSV-1 genome were also examined. Interestingly, three wild type HSV-2 polymerases (6652, 83D and SB5) showed mutation frequencies similar to HSV-1 SC16, rather than their parental virus, when placed within an HSV-1 genome. Only HSV-2 6757 maintained a high error rate (60-fold) over that of HSV-1 SC16, when placed within an HSV-1 genome. However, the mutation frequency of this recombinant virus (HSV-2 6757PR) was not completely penetrant when compared to the parental HSV-2 6757 virus, which showed a 400-fold higher percent of resistant mutants compared to HSV-1 SC16.
An important distinguishing factor between the tk mutagenesis and LacZ test systems is the use of antiviral selectors to measure mutation frequency. Silent mutations within the tk gene that do not impair susceptibility to antiviral agents, or mutations resulting in lethality of the virus, would escape detection as a mutation in the tk mutagenesis assay. Futhermore, the DNA sequence being replicated is unique between these systems and this could contribute to the differences in mutation frequency between assay systems. The demonstration of S1 nuclease-sensitive sites within the type 2 viral genome overlapping the tk coding region, and the absence of such sites in the type 1 viral genome, suggest that local topology may be different between these HSV-1 and HSV-2. Although the potential contribution of this supercoiling-induced phenomenon to modulate mutation frequency during viral replication is unclear, it could partially explain why the type 2 polymerases confer a high mutation frequency to HSV-2 genomes and a low mutation frequency when placed within an HSV-1 genome. Changes in local topology could contribute to polymerase slippage and the incorporation of errors.
The inability of type 2 viruses to confer a higher mutation rate than type 1 viruses in the non-HSV DNA mutagenesis assay is consistent with the absence of S1 nuclease hypersensitive sites in the LacZ gene fragment. Furthermore, the HSV-2 tk sequence used herein contained almost 69% G-C content, compared to the HSV-1 tk which has 65% of bases as G or C. Melting temperature alone may not contribute to the difference in S1 nuclease sensitivity. However, alignment of the tk sequences showed that the type 2 gene had at least eight distinct stretches containing between 7 and 13 G-C bases, whereas the overlapping region in the type 1 tk had four or less G-C bases. This differential in more extensive G-C rich regions supports the observations reported herein.
The data presented herein clearly demonstrate that polymerase alone may not account for the high mutation frequency associated with type 2 viruses in the tk mutagenesis assay. In addition to the potential impact of target sequence and secondary structure topology on mutation frequency, other viral replicative proteins including Pol accessory protein, TK, dUTPase, and uracil-DNA glycosylase may cooperate with Pol to modulate polymerase repair and nucleotide selection [
12,
13,
19,
23].