Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutations to the DNA polymerase locus.

Seven herpes simplex virus mutants which have been previously shown to be resistant to arabinosyladenine were examined for their sensitivities to four types of antiviral drugs. These drugs were a pyrophosphate analog, four nucleoside analogs altered in their sugar moieties, two nucleoside analogs altered in their base moieties, and one altered in both. The seven mutants exhibited five distinct phenotypes based on their sensitivities to the drugs relative to wild-type strain KOS. All mutants exhibited resistance to acyclovir and arabinosylthymine, as well as marginal resistance to iododeoxyuridine, whereas all but one exhibited resistance to phosphonoformic acid. The mutants exhibited either sensitivity or hypersensitivity to other drugs tested--2'-nor-deoxyguanosine, 5-methyl-2'-fluoroarauracil, 5-iodo-2'-fluoroarauracil, and bromovinyldeoxyuridine--some of which differed only slightly from drugs to which the mutants were resistant. These results suggest ways to detect and treat arabinosyladenine-resistant isolates in the clinic. Antiviral hypersensitivity was a common phenotype. Mutations conferring hypersensitivity to 2'-nor-deoxyguanosine in mutant PAAr5 and to bromovinyldeoxyridine in mutant tsD9 were mapped to nonoverlapping regions of 1.1 and 0.8 kilobase pairs, respectively, within the herpes simplex virus DNA polymerase locus. Thus, viral DNA polymerase mediates sensitivity to these two drugs. However, we could not confirm reports of mutations in the DNA polymerase locus conferring resistance to these two drugs. All of the mutants exhibited altered sensitivity to two or more types of drugs, suggesting that single mutations affect recognition of the base, sugar, and triphosphate moieties of nucleoside triphosphates by viral polymerase.     

Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus.

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.     

Acyclovir-resistant mutants of herpes simplex virus type 1 express altered DNA polymerase or reduced acyclovir phosphorylating activities.

The biochemical properties of four acyclovir-resistant mutants are described. Two of these mutants, PAAr5 and BWr, specified nucleotidyl transferase (DNA polymerase) activities which were less sensitive to inhibition by acyclovir triphosphate than their wild-type counterparts. Another mutant, IUdRr, exhibited reduced ability to phosphorylate acyclovir. The fourth mutant, ACGr4, both induced an altered DNA polymerase and failed to phosphorylate appreciable amounts of acyclovir. BWr, a new acyclovir-resistant mutant derived from the Patton strain of herpes simplex virus type 1, induced a DNA polymerase resistant to inhibition by acyclovir triphosphate, but, unlike the polymerases induced by PAAr5 and ACGr4, still sensitive to phosphonoacetic acid. Resistance of BWr to acyclovir mapped close to the PAAr locus and was separable from mutations in the herpes simplex virus thymidine kinase gene by recombination analysis.     

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.     

Molecular genetics of herpes simplex virus. III. Fine mapping of a genetic locus determining resistance to phosphonoacetate by two methods of marker transfer.

We have transferred a genetic locus determining resistance to phosphonoacetic acid (PAAr) from one herpes simplex viral genome to another by two methods of marker transfer. One method requires recombination between an intact DNA molecule and a restriction endonuclease DNA fragment, whereas the other requires repair of a partial heteroduplex formed between the two DNA molecules. These two methods mapped the PAAr locus between positions 0.45 and 0.53 map units on the physical map of the viral DNA. Fine mapping of the PAAr locus showed that it maps at or near an EcoRI restriction endonuclease site at either 0.46 or 0.49 map units. We also describe and compare the two methods of marker transfer.     

Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin.

Fourteen mutants known or likely to contain mutations in the herpes simplex virus DNA polymerase gene were examined for their sensitivity to aphidicolin in plaque reduction assays. Eleven of these exhibited some degree of hypersensitivity to the drug; altered aphidicolin-sensitivity correlated with altered sensitivity to the pyrophosphate analog, phosphonoacetic acid. The DNA polymerase specified by one of these mutants, PAAr5, required roughly seven-fold less aphidicolin to inhibit its activity by 50% than did polymerase specified by its parental strain. Mutations responsible for the aphidicolin-hypersensitivity phenotype of PAAr5 were mapped to an 0.8 kbp region in the herpes simplex virus DNA polymerase locus. These data taken together indicate that 1) mutations in the herpes simplex virus DNA polymerase gene can confer altered sensitivity to aphidicolin, 2) that the HSV polymerase is sensitive to aphidicolin in vivo, and 3) that amino acid alterations which affect aphidicolin binding may affect the pyrophosphate exchange-release site as well, suggesting that aphidicolin binds in close proximity to this site.     

Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain.

In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.     

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.     

Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants.

Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.     

A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin.

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.     

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.     

Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity.

We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.     

Inhibition of the reproduction of a herpes simplex I virus carrying mutations in the thymidine kinase and DNA polymerase genes

Sensitivity of Herpes Simplex Virus type I (HSV-I) mutants carrying genetic defect in the DNA polymerase and thymidine kinase genes to the action of some drugs was studied. TK- mutant of HSV-I was resistant to Ara-T and ACG and sensitive to PAA, Ara-A as well as to ribavirin and ADEA. PAAr mutant of HSV-I was resistant to PAA, Ara-A, ACG and sensitive to Ara-T, ribavirin and ADEA. A double mutant of HSV-I-TK-, PAAr was resistant to all drugs, except for ribavirin and ADEA. To inhibit reproduction of HSV with genetic defect, it is important using drugs of independent mode of action on the function of defective viral gene.     

A single amino acid substitution in the ICP27 protein of herpes simplex virus type 1 is responsible for its resistance to leptomycin B

Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.     

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.     

Characterization of herpes simplex virus 2 temperature-sensitive mutants whose lesions map in or near the coding sequences for the major DNA-binding protein.

By marker rescue with cloned herpes simplex virus 2 DNA fragments, we have mapped the temperature-sensitive mutations of a series of herpes simplex virus 2 mutants to a region of the herpes simplex virus 2 genome that lies within or near the coding sequences for the major DNA-binding protein, ICP8. In cells infected with certain of these mutants at the nonpermissive temperature, the association of the major DNA-binding protein with the cell nucleus was defective. In these cells, the DNA-binding protein accumulated in the cytoplasmic and the crude nuclear detergent wash fractions. At the permissive temperature, the maturation of the mutant ICP8 was similar to that of the wild-type viral protein. With the remainder of the mutants, the nuclear maturation of ICP8 was similar to that encoded by the wild-type virus at the nonpermissive and permissive temperatures as assayed by cell fractionation.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.