Department of Virology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan1
Author for correspondence: Kimiyasu 4. Fax +81 76 434 5020. e-mail kshiraki{at}toyama-mpu.ac.jp
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Abstract |
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In this study, we determined the nucleotide changes in the DNA polymerase gene of ACV-resistant VZV mutants. These mutants were analysed for their susceptibility to anti-herpetic drugs. In addition, we have demonstrated identical amino acid substitutions and a discrete discrepancy in the drug susceptibility between VZV and herpes simplex virus type 1 (HSV-1) DNA polymerase mutants.
Human embryonic lung (HEL) cells were grown and maintained in Eagles minimum essential medium supplemented with 10% foetal bovine serum (FBS) for growth or 2% FBS for maintenance. We used the VZV parent Kawaguchi strain which was serially plaque-purified six times using cell-free virus (Shiraki et al., 1983 , 1985
, 1992
). The ACV-resistant virus strains A1A6 and A8, each with mutations in the DNA polymerase gene, were isolated in the presence of increasing concentrations (4·5, 11·3 and 22·5 µg/ml) of ACV (100 µM). Mutants were passaged three times at each concentration until the appearance of typical cytopathology. After two plaque purification steps in the presence of 100 µM ACV, the plaque-purified viruses were used as ACV-resistant mutants (Shiraki et al., 1983
, 1990
).
VZV DNA was prepared from cell cultures infected with the parent Kawaguchi strain or from the various mutants (Shiraki et al., 1991a , b
). The 3·6 kb fragment encoding the VZV DNA polymerase gene was amplified by PCR and sequenced using the Auto Sequencer Core kit (Toyobo) with Cy5-labelled primers designed according to the sequence of VZV Dumas strain (Davison & Scott, 1986
). The cycle sequencing reaction products were run on the ALF DNA Sequencer (Pharmacia).
The susceptibility of each ACV-resistant VZV mutant to ACV (Sigma), phosphonoacetic acid (PAA) (Sigma), 9--D-arabinofuranosyladenine (Ara-A) (ICN) and aphidicolin (Aph) (Wako Pure Chemical Industries) was determined by plaque reduction assays in HEL cells (Ida et al., 1999
; Shiraki et al., 1983
, 1992
). Briefly, confluent monolayers of HEL cells in 60 mm plastic Petri dishes (in duplicate) were inoculated with 100 p.f.u. per dish of cell-free virus in 0·2 ml SPGC medium (PBS supplemented with 5% sucrose, 0·1% sodium glutamate and 10% FBS). After incubation for 1 h to permit adsorption, 5 ml of maintenance medium and the various concentrations of drug were added. After 5 days of incubation, cells were fixed and stained, after which plaques were counted. The 50% effective concentration (EC50) was defined as the concentration that reduced plaque formation by 50%.
Genotypic and phenotypic characterizations of ACV-resistant mutants are summarized in Table 1. Sequence analysis of the DNA polymerase gene of ACV-resistant mutants showed that each mutant had a single nucleotide substitution. All seven ACV-resistant mutants showed only a single amino acid change in the whole protein. The ACV-resistant mutants A1, A3 and A4 showed a G
T change at nucleotide position 48224, which resulted in the amino acid substitution G805C. These three mutants were resistant to ACV and PAA, sensitive to Ara-A and hypersensitive to Aph. The ACV-resistant mutants A2 and A5 showed a G
A change at nucleotide position 48074, which resulted in the amino acid substitution V855M. These two mutants were resistant to ACV, PAA and Ara-A and hypersensitive to Aph. The ACV-resistant mutants A6 and A8 showed an A
G change at nucleotide position 48301, which resulted in the amino acid substitution N779S. These two mutants were resistant to ACV, sensitive to Aph and hypersensitive to PAA and Ara-A. Thus, seven ACV-resistant mutants were classified into three groups according to their genotypic and phenotypic characterizations. Mutations in the TK gene of ACV-resistant mutants were observed near the guanosine homopolymer in HSV (Sasadeusz et al., 1997
) or downstream from two sequential guanosine nucleotides in VZV (Ida et al., 1999
), but the nucleotide changes in the VZV DNA polymerase gene were not related to these sequences, thus indicating that these mutations were probably not induced by ACV.
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We compared the mutations of the VZV DNA polymerase gene with those of the HSV-1 DNA polymerase gene, as shown in Fig. 1. The positions of the three mutations at amino acid residues 779, 805 and 855 found in the ACV-resistant VZV corresponded to amino acid residues 815, 841 and 890 of HSV-1, respectively, which were within the conserved regions III and I of the HSV-1 DNA polymerase gene. The mutations found at residues 779 and 805 of VZV were identical to residues 815 and 841 in HSV-1, respectively, as reported previously (Larder et al., 1987
; Chiou et al., 1995
). Region III of the VZV DNA polymerase gene may interact directly with drugsubstrate binding, in a manner similar to that of the conserved region III of the HSV-1 DNA polymerase gene (Larder et al., 1987
; Gibbs et al., 1988
). Marcy et al. (1990)
have reported that one of the HSV-1 DNA polymerase mutations has a V892M change. Although V855 of VZV corresponds to I890 of HSV-1, as shown in Fig. 1
, the V855M mutation in VZV may correspond to the V892M mutation in HSV-1 by way of biochemical behaviour.
We compared the drug susceptibility of the VZV DNA polymerase mutants with those reported for the HSV-1 DNA polymerase mutants (Table 2) containing identical amino acid substitutions (Larder et al., 1987
; Chiou et al., 1995
; Marcy et al., 1990
). This indicates that the role of the conserved regions is identical between the two DNA polymerases. The HSV-1 mutants with the N815S mutation were susceptible to PAA and Ara-A and more resistant to ACV, similar to the VZV mutants with the N779S mutation. Although HSV-1 N815S mutants were resistant to Aph, VZV N779S mutants were as sensitive to Aph as wild-type VZV. The mutants with a G to C change at amino acids 841 and 805 of HSV-1 and VZV, respectively, showed similar susceptibility to Ara-A and were resistance to both ACV and PAA. The mutants with a V to M change at 892 of HSV-1 and 855 of VZV showed similar susceptibility to Aph and similar resistance to PAA. Among the four anti-herpetic drugs, the VZV V855M mutant was more resistant to ACV than the HSV-1 V892M mutant. In spite of both the identical and similar amino acid substitutions, ACV-resistant mutants of VZV and HSV-1 showed a different susceptibility to ACV and Aph. It was suggested that the discrepancy in drug susceptibility between HSV-1 and VZV might be reflected from the non-conserved regions, which may have caused a minute difference in the affinity of the DNA polymerase to ACV and Aph.
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Acknowledgments |
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References |
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Received 24 April 2001;
accepted 13 July 2001.