Laboratory of Antiviral Chemotherapy1 and Laboratory of Molecular Immunology2, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Author for correspondence: Graciela Andrei. Fax +32 16 337340. e-mail graciela.andrei{at}rega.kuleuven.ac.be
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Abstract |
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Introduction |
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Acyclovir (ACV) has been shown to be effective in the prophylaxis and treatment of mucocutaneous HSV infections in immunocompromised patients (Whitley & Gnann, 1992 ). The development of resistance to ACV has been most extensively studied (Coen, 1996a
; Collins & Darby, 1991
). ACV is phosphorylated to its monophosphate form by the virus-encoded thymidine kinase (TK) and, after further phosphorylation to the triphosphate form by cellular enzymes, it inhibits herpesvirus DNA polymerases specifically by competing with the binding of natural triphosphates and their subsequent insertion into growing DNA strands. Three different mechanisms are recognized that render HSV resistant to ACV. Most of the acyclovir-resistant (ACVr) strains that have been isolated, either from cell culture or from patients, lack a functional TK (TK- mutant) and are resistant because of the inability to monophosphorylate ACV (McLaren et al., 1985
; Chatis & Crumpacker, 1991
; Hill et al., 1991
). A few isolates have been found to have a TK with altered substrate specificity, resistant viruses producing a TK enzyme that is functional but that lacks the ability to phosphorylate ACV (Ellis et al., 1987
; Nugier et al., 1991
). Finally, resistance to ACV due to mutations that alter binding and utilization of ACV by the viral DNA polymerase has been described (Parker et al., 1987
; Collins et al., 1989
; Sacks et al., 1989
).
Foscarnet (PFA) is an alternative therapeutic modality for the treatment of TK-, ACVr HSV infections, since it does not require activation by the viral TK (Safrin et al., 1991 ). Unfortunately, administration of PFA often causes renal failure and alterations in the plasma calcium and phosphorus levels. In addition, resistance to both ACV and PFA has been described in the clinic (Safrin et al., 1994
; Safrin, 1996
).
A new approach to the therapy of TK+ as well as ACVr and ACVr/PFAr mucocutaneous HSV infections is based on the use of cidofovir [HPMPC; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], which does not depend on the viral TK for its activity (Hitchcock et al., 1996 ; Naesens et al., 1997
). HPMPC has proven to be effective in the treatment of progressive mucocutaneous infections due to ACVr and ACVr/PFAr HSV in immunocompromised patients (Snoeck et al., 1993
; Lalezari et al., 1997
). It is one of the leading compounds of a new series of antiviral molecules, the acyclic nucleoside phosphonate (ANP) analogues. Within this class of compounds, (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) and 2-phosphonylmethoxyethyl (PME) derivatives of both purines and pyrimidines have been synthesized (De Clercq et al., 1986
, 1987
). The HPMP derivatives have potent and selective activity against a broad spectrum of DNA viruses, including herpes-, hepadna-, irido-, pox-, adeno-, papilloma- and polyomaviruses (Hitchcock et al., 1996
; Naesens et al., 1997
). HPMPC has been approved for the treatment of cytomegalovirus retinitis in AIDS patients (Safrin et al., 1997
) and is being evaluated for the treatment of other herpesviruses including HSV, varicella-zoster virus and EpsteinBarr virus, as well as adeno-, papilloma- and polyomaviruses. In contrast to the HPMP derivatives, the PME derivatives show marked and selective activity against retroviruses. Like the HPMP derivatives, the PME derivatives are also active against herpes-, hepadna- and iridoviruses, but unlike the HPMP derivatives they do not inhibit adeno- or poxviruses. PME-adenine (PMEA), as its oral prodrug form (adefovir dipivoxil), has proceeded to phase III clinical trials in both the USA and Europe for the treatment of human immunodeficiency virus.
Phosphonylmethoxyalkyl derivatives of purines and pyrimidines, which can be considered as analogous to the monophosphate forms of acyclic nucleosides, are further phosphorylated to their active metabolites by cellular enzymes. Thus, they circumvent the need for activation by the virus-specified TK and hence retain their activity against TK-deficient or -altered strains of HSV (Hitchcock et al., 1996 ; Naesens et al., 1997
). Furthermore, it appears that HSV mutants with deficient or altered TK activity are more susceptible to HPMPC, due to a reduction in the dCTP pools, which normally depend on TK (Mendel et al., 1995
). The antiviral effect of the ANP analogues is the result of selective inhibition of the viral DNA polymerase by their diphosphate metabolites. Based on the structural resemblance to natural deoxynucleoside triphosphates, the diphosphate metabolites act both as competitive inhibitors and as alternative substrates during the DNA polymerase reaction (Foster et al., 1991
; Xiong et al., 1996
, 1997
). These diphosphate forms inhibit HSV DNA polymerase at concentrations that are 50- to 600-fold lower than those needed to inhibit human
,
and
DNA polymerases (Merta et al., 1990
; Ho et al., 1992
).
The isolation and characterization of drug-resistant mutants of HSV DNA polymerase can be useful in elucidating the mechanism(s) of selective drug action, in assessing the potential for drug resistance in the clinic and its possible avoidance and in evaluating the structurefunction relationship of the polymerase gene domains (Coen, 1996a ).
The aim of the present study was to determine the molecular basis of HSV-1 resistance to both HPMP and PME derivatives by the identification of the nucleotide changes that occurred in the DNA polymerase genes of HSV-1 mutants selected under drug pressure in vitro. Also, the neurovirulence of the different mutants was evaluated in a mouse model. The relationship between cross-resistance for the different drug classes in vitro and neurovirulence in vivo may form a biochemical basis for combination chemotherapy with far-reaching implications in clinical practice.
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Methods |
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Compounds.
The sources of the compounds were as follows: acyclovir [ACV, 9-(2-hydroxyethoxymethyl)guanine], Wellcome Research Laboratories, Research Triangle Park, NC, USA; foscarnet [phosphonoformate sodium salt, PFA] and phosphonoacetic acid (PAA), Sigma; PMEA [9-(2-phosphonylmethoxyethyl)adenine] and HPMPC [(S)-1-(3-hydroxy-2-phosponylmethoxypropyl)cytosine], Gilead Sciences, Foster City, CA, USA; HPMPA [(S)-9-(3-hydroxy-2-phosphonymethoxypropyl)adenine] and PMEDAP [9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine], A. Holy, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Selection of drug-resistant strains.
The drug-resistant virus strains were obtained by serial passage of the reference HSV-1 KOS strain in the presence of increasing concentrations of the compounds. The virus was passaged in Vero cells in the presence of the compounds, starting at the IC50. The cell cultures were incubated until virus CPE was maximal, and the drug concentration was increased 2-fold with every subsequent passage of the virus. After reaching the highest possible concentration for each compound (100 µg/ml for PMEA and PMEDAP, 200 µg/ml for PFA and 40 µg/ml for HPMPC and HPMPA), a last passage was done in drug-free medium in order to obtain the virus stock. The various drug-resistant HSV-1 strains, denoted PFAr, PMEAr, PMEDAPr, HPMPCr and HPMPAr, were titrated and subsequently tested for their sensitivity in vitro to a broad range of antiviral compounds. Drug resistance was defined taking into consideration the potency of each anti-HSV compound. Thus, drug resistance corresponded to an increase in IC50 of at least 10-fold for the HPMP derivatives, at least 5-fold for the PME derivatives and at least 3-fold for the pyrophosphate analogues. In the case of ACV, a 5- to 10-fold increase in IC50 was regarded as indicative of resistance due to alterations at the DNA polymerase level.
Plaque purification.
Each drug-resistant strain was plaque-purified by standard procedures. Several individual PFAr, PMEAr, PMEDAPr, HPMPCr and HPMPAr clones were collected, amplified and tested for their sensitivity in vitro to various antiviral compounds.
Antiviral assays.
Drug sensitivity of the different drug-resistant HSV strains was determined by virus CPE reduction assays in HEL cells. Confluent HEL cells were inoculated with the different virus strains at an input of 100 CCID50 (1 CCID50 corresponds to the virus dose infective for 50% of the cell cultures). The IC50 was defined as the concentration required to reduce virus CPE by 50%. The IC50 values for the individual compounds represent the means from at least three independent experiments.
Cloning and sequencing of the DNA polymerase gene variants.
HSV DNA was prepared directly from virus produced after infecting Vero cell cultures with the KOS strain or the different mutants. Two HPMPCr clones (clone A4 and clone C3), two HPMPAr clones (clone B5 and clone D1), one PFAr clone (clone A), one PMEAr clone (clone B) and one PMEDAPr clone (clone C) were selected for cloning and sequencing of the DNA polymerase genes. To avoid the introduction of mutations by PCR amplification, all cloning experiments were executed directly on viral DNA. Viral DNA from the different strains was digested with BamHI and fragments were separated by electrophoresis in agarose gels. A 3·4 kb BamHI fragment that contains about 87% of the HSV-1 DNA polymerase gene coding region was purified and further digested with SacI. The two resulting fragments of 2·0 and 1·4 kb from each virus were purified and ligated to pUC18 cleaved with BamHI and SacI. The ligation mixtures were used to transform Escherichia coli (strain DH5) made competent by the method of Hanahan (1985)
. Ampicillin-resistant colonies were identified and screened for plasmids containing the appropriate fragments of 2·0 and 1·4 kb. Plasmid DNA was prepared and aliquots of either 6 µg (for T7 DNA polymerase sequencing) or 500 µg (for cycle sequencing) were used. The total insert of the plasmid DNAs was sequenced by the dideoxynucleotide chain termination method starting with fluorescent universal forward and reverse M13 primers, followed by primer walking with internal fluorescent primers (AutoRead sequencing kit, Pharmacia Biotech) designed according to the wild-type HSV-1 strain KOS sequence. The sequencing reaction products were run on an automated laser fluorescent DNA sequencer under standard conditions (Chen & Seeburg, 1985
).
Screening of identified mutations in various plaque-purified virus clones.
To determine the presence of specific mutations in various plaque-purified drug-resistant HSV-1 isolates, cycle sequencing of total viral DNA was performed (Thermo sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP, Pharmacia Biotech). We used specific primers that allowed the determination of the nucleotide sequence of the region of the viral DNA polymerase in which the mutations had occurred.
Determination of the in vivo pathogenicity of the plaque-purified drug-resistant strains.
Virus stocks of the plaque-purified drug-resistant strains were titrated in HEL cells by plaque formation and the virus titre was expressed in p.f.u./ml. In parallel, adult NMRI mice were inoculated intracerebrally with 10-fold dilutions of each virus stock. Ten mice were used per dilution. Mortality was recorded over a period of 20 days and virus titre was expressed in LD50/ml. The pathogenicity index for the different strains was calculated as the log (p.f.u./LD50).
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Results |
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The presence of the Ser-724 to Asn mutation was confirmed in three PMEAr, three PMEDAPr and two PFAr plaque-purified mutants after screening of the 724 mutation by direct cycle sequencing on the total viral DNA. All the different PMEAr, PMEDAPr and PFAr virus isolates presented the Ser-724 to Asn mutation (Table 2).
Phenotypic and genotypic characterization of HPMPCr and HPMPAr HSV-1 clones
The HPMPCr and HPMPAr clones proved to be resistant only to HPMPC and HPMPA, remaining sensitive to ACV, the PME derivatives and the pyrophosphate analogues PFA and PAA (Tables 1 and 2
). Indeed, some of the HPMPCr clones appeared to be hypersensitive to the pyrophosphate analogues.
Two HPMPCr virus isolates (clone A4 and clone C3) and two HPMPAr isolates (clone B5 and clone D1) were selected for molecular cloning and sequencing of the DNA polymerase genes. As shown in Table 1, the HPMPAr clones B5 and D1 and the HPMPCr clone C3 possessed single amino acid changes in the DNA polymerase. The HPMPAr clones B5 and D1 showed an Ile-1028 to Thr change and a Leu-1007 to Met change, respectively, while the HPMPCr clone C3 presented a Val-573 to Met mutation. The HPMPCr clone A4 contained two mutations, Ala-136 to Thr and Arg-700 to Met.
Several HPMPCr and HPMPAr mutant viruses were selected for the determination of their drug-susceptibility profiles and were tested by direct cycle sequencing for the presence of specific mutations at positions 136, 573 and 700 (for the HPMPCr clones) and at positions 1007 and 1028 (for the HPMPAr clones). The Arg-700 to Met mutation was confirmed in four HPMPCr clones and, in contrast to the HPMPCr clone A4, these clones did not present the Ala-136 to Thr change (Table 3). Three of the phenotypically HPMPCr viruses (clones E, J and N) did not present mutations at positions 136, 573 or 700 (Table 3
), suggesting that these isolates may contain other (new) mutations.
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Locations of the mutations in the HSV-1 DNA polymerase that confer resistance to acyclic nucleoside analogues
The Ser-724 to Asn mutation, which confers resistance to ACV in addition to PMEA, PMEDAP and PFA, is mapped to conserved region II of the HSV-1 DNA polymerase (Fig. 1). The serine at position 724 is highly conserved in all herpesvirus DNA polymerases and in almost every other polymerase that shares this region of sequence similarity.
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The mutations found in the HPMPCr clone C3 and the HPMPAr clones B5 and D1 (at positions 573, 1028 and 1007, respectively) are located in non-conserved regions within the DNA polymerase. Position 573 is located just before two well-conserved residues, Tyr-577 and Asp-581, which are within conserved region A (Fig. 1). HSV-1 DNA polymerase has a C-terminal UL42-binding domain. Positions 1007 and 1028 lie beyond the last region of conserved sequence (region V) and before the UL42-binding domain.
Neurovirulence
Neurovirulence of the different drug-resistant HSV-1 mutants was evaluated by intracerebral inoculation of the viruses into mice. The LD50 values for the different isolates were determined, and the ratio p.f.u./LD50 was monitored as a parameter of neurovirulence. A TK- ACVr KOS mutant with a reduced neurovirulence (increase in the ratio of p.f.u. to LD50) was included as control (Fig. 2). The strains resistant to PFA, PMEA and PMEDAP showed high neurovirulence similar to, or slightly lower than, that of the parental neurovirulent KOS strain. In contrast, the HPMPCr strains (clone C3, clone A4 and clone D) and HPMPAr strains (clone A3 and clone B5) showed a reduction in neurovirulence (ratio of p.f.u. to LD50
1·9). Two of the HPMPAr clones (clone D1 and clone B5) showed high neurovirulence similar to that of the KOS strain.
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Discussion |
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HSV-1 DNA polymerase is a 1235 amino acid multifunctional enzyme with a 5'3' exonuclease/RNase H function, a 3'
5' exonuclease editing function, a deoxyribonucleotide polymerizing (catalytic) function and a UL42-binding domain (Earl et al., 1986
; Larder et al., 1987
; Coen, 1996b
). This polymerase belongs to a large family of polymerases with seven regions having somewhat conserved nucleotide sequences. The regions are numbered I to VII on the basis of degree of conservation among the DNA polymerase genes, with region I being the most conserved. The recent report of the crystal structure of one member of this family of polymerases, that of bacteriophage RB69 (Wang et al., 1997
), allows these conserved regions to be correlated with structural and functional roles. Regions I and II are parts of the polymerase palm subdomain and contain the three Asp residues (717, 886 and 888 in HSV-1) essential for polymerase activity. Regions III and VI are located at the base of the fingers subdomain and regions V and VII are at the base of the thumb subdomain. These four regions appear to flank the catalytic site in the palm subdomain and may play a role in positioning the template and primer strands. Region IV is part of the 3'
5' exonuclease editing domain and these residues may play a more structural role. Finally, a further region, A, has been identified. Region A is now recognized as being part of a larger region called
-region C that is shared by polymerases related to eukaryotic DNA polymerase
(Coen, 1996b
). Although there is only low sequence conservation, this region appears to be part of the 3'
5' exonuclease editing domain that includes the conserved catalytic residues Tyr-577 and Asp-581 (Fig. 1
).
Most work concerning mutations in the HSV DNA polymerase has been done with ACV and PFA, and the vast majority of drug-resistant mutations have been mapped to regions I, II and III and the additional region, A (Fig. 1). Mutations involving reduced susceptibility to nucleoside analogues and pyrophosphate analogues should reflect regions of the enzyme that are important for the binding of dNTP and pyrophosphate. Regions II and III contain the greatest clustering of mutations and are therefore considered most likely to interact directly with drugs and natural ligands (Coen, 1996a
). HSV-1 strains that were selected for resistance against either PFA, PMEA or PMEDAP share a single amino acid change (Ser-724 to Asn) in conserved region II of the DNA polymerase. Furthermore, this amino acid change has previously been shown to confer resistance to PFA (Larder et al., 1987
; Gibbs et al., 1988
). Therefore, our results indicate that the PME compounds interact at a site on the DNA polymerase that overlaps with the pyrophosphate-binding site. In contrast, HPMPAr and HPMPCr strains were found to contain mutations either in non-conserved regions or in non-conserved positions within conserved regions of the viral DNA polymerase. The significant mutations found in the HPMPCr clones (i.e. excluding Ala-136 to Thr) are located within
-region C (Val-573 to Met) and within region II (Arg-700 to Met). Based on the structure of the DNA polymerase from bacteriophage RB69 (Wang et al., 1997
), residue 573 appears to be located within an
-helix in the 3'
5' exonuclease editing domain. It is one turn of the helix before Tyr-577 and two turns before Asp-581, both of which are catalytically important. Thus, position 573 is very close to the catalytic site of this domain. Interestingly, position 700 is not conserved among DNA polymerases but an Arg-700 to Gly change has been reported in a PFAr strain (Gibbs et al., 1988
). The bacteriophage RB69 structure further suggests that position 700 is located within a
-strand (named
14) adjacent to the strands bearing the catalytic triad of Asp residues (717, 886 and 888 in HSV-1). Mutations at this position may affect the positioning of the base moiety of the incoming nucleotide.
The product of the UL42 gene is an accessory protein of HSV polymerase, a DNA-binding protein with an apparent molecular mass of 65 kDa, which functions as a processivity factor. The carboxy-terminal 35 amino acids of HSV DNA polymerase have been shown to be crucial for UL42-binding activity (Digard et al., 1993 ). The mutations in the HPMPAr clones mapped to residues 1007 and 1028, beyond the last region of conserved residues in the catalytic domain and upstream the UL42-binding domain. On the basis of weak sequence similarity to the bacteriophage RB69 polymerase (Wang et al., 1997
), we suggest that these residues are in the thumb subdomain of the catalytic domain. Our sequence alignment against the crystal structure suggests that the nearby (positively charged) Arg residues at positions 1019, 1020, 1026 and 1039 may play a role in binding the phosphate backbone of the duplex DNA product.
The different PMEAr, PMEDAPr and PFAr clones were not significantly impaired in their ability to kill mice after intracerebral inoculation. This finding is in agreement with previous findings indicating that different drug-resistant DNA polymerase mutants may vary in their neurovirulence capacity (Field & Darby, 1980 ; Field & Coen, 1986
; Sacks et al., 1989
; Snoeck et al., 1994
; Coen, 1996a
; Pelosi et al., 1998a
). In contrast, the HPMPCr clones were much less neurovirulent for mice than the wild-type KOS strain, suggesting that the molecular basis for drug-resistance at the level of the DNA polymerase may affect the neurovirulence of the mutants. In the case of the HPMPAr isolates, the clones B5 and A3, with the Ile-1028 to Thr mutation, showed reduced neurovirulence compared with the wild-type KOS strain. However, the HPMPAr clone D1, with the Leu-1007 to Met mutation and the HPMPAr clone F1, which did not present mutations at position 1007 or 1028, had neurovirulence for mice similar to that of the parental KOS strain.
HSV mutants that are resistant to antiviral drugs due to mutations in the TK and/or DNA polymerase genes are an increasingly serious problem for immunocompromised patients (Safrin, 1996 ). The vast majority of HSV mutants that are attenuated in neurovirulence exhibit defects in replication in brain and/or in the peripheral nervous system (PNS) (Chou et al., 1990
; Pyles et al., 1992
). However, one DNA polymerase mutant, which is ACVr due to an Arg-842 to Ser mutation in the conserved region III of the DNA polymerase, has recently been reported to be attenuated in neurovirulence, although it is able to replicate in the PNS and in the brain similarly to wild-type virus (Pelosi et al., 1998b
). Understanding how the different DNA polymerase mutations attenuate neurovirulence specifically may help to reveal the potential mechanism(s) of HSV pathogenesis.
The fact that selection of HSV-1 strains resistant to PME and HPMP derivatives is associated with different mutations at the level of the viral DNA polymerase has important consequences for the treatment of drug-resistant mutants that could arise during therapy with ANP analogues or with other drugs (e.g. ACV and PFA), emphasizing the importance of monitoring the drug susceptibility profile of isolates from patients with clinical drug resistance. Although selection of HSV-1 strains resistant to HPMPC has not been described in the clinic, it is important to know their pattern of sensitivity and their neurovirulence. The long-lasting antiviral effect of ANP derivatives, a remarkable feature of this class of compounds, allows infrequent administration of HPMPC in the treatment of HSV infections, thus reducing the probability of selection of HPMPCr strains (Hitchcock et al., 1996 ; Naesens et al., 1997
). We have demonstrated the usefulness of HPMPC in the treatment of ACVr/PFAr HSV infections, as well as the feasibility of alternating ACV and HPMPC therapy for the treatment of alternating ACVs (ACV-sensitive) and ACVr HSV infections in immunocompromised patients, since, as a rule, recurrences following HPMPC therapy show a reversion of the ACVr to the ACVs phenotype (Snoeck et al., 1994
).
An HSV-2 isolate with reduced susceptibility to HPMPC selected in vitro has been reported by Mendel et al. (1997) . This resistant virus had a reduced susceptibility to HPMPC but no change in its susceptibility to ACV and PFA. Notably, this strain proved to be severely compromised in its virulence for mice. Genotypic analysis of this HPMPCr HSV-2 identified a single mutation (Gly-506 to Ser) located between region IV and region A within the proposed catalytic domain of the DNA polymerase.
It should be noted that multi-step drug selection of HSV-1 with PFA, PMEA and PMEDAP resulted in a homogeneous virus population, since all the different clones presented the same phenotypic and genotypic profile. In contrast, selection with HPMPC or HPMPA resulted in a heterogeneous virus population, since different amino acid changes that conferred resistance to HPMPC and HPMPA were identified, indicating that different regions of the HSV DNA polymerase may be involved in the interaction with these compounds. However, mutations in the DNA polymerase gene occur with low frequency due to the fact that there exists only a restricted number of sites in the DNA polymerase gene at which changes may occur that result in drug resistance while maintaining a functional DNA polymerase. The cloning and sequencing of the DNA polymerase genes of the HPMPCr and HPMPAr clones that were negative for the mutations identified here is currently in progress. Marker transfer experiments to confirm that the polymerase mutations described in the present study confer the drug-resistance phenotype are also in progress. In addition, the possibility that mutations in other HSV DNA replication genes may play a role should be investigated further. This may be relevant to the HPMPCr isolates (clones C3 and A4) that showed hypersensitivity to the pyrophosphate analogues, since mutations in the viral major DNA-binding protein (ICP8) are known to cause PAA hypersensitivity (Chiou et al., 1985 ). This could also be relevant to the PMEAr and PMEDAPr isolates listed in Table 1
, which have identical pol genes but a 10-fold different sensitivity to PMEDAP.
In conclusion, HSV-1 strains resistant to HPMP derivatives are distinct from those that are resistant to PME derivatives. Furthermore, in contrast to the ACVr, PFAr and PMEr mutations, the mutations arising under selective pressure of HPMPC and HPMPA are unique and occur in non-conserved positions of the viral DNA polymerase. This has important consequences for antiviral chemotherapy, as it may form a scientific basis for the use of HPMPC in the treatment of ACVr and/or PFAr HSV infections, as well as for the avoidance of selection of multi-drug-resistant virus in vivo. Since the (neuro)virulence of the virus strains seems to be associated with the type of drug resistance, mutation monitoring seems to be even more advisable.
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Acknowledgments |
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References |
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Received 14 July 1999;
accepted 12 November 1999.