10 results on '"Fyfe JA"'
Search Results
2. Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine.
- Author
-
Fyfe JA, McKee SA, and Keller PM
- Subjects
- Bromodeoxyuridine pharmacology, Drug Resistance, Microbial, Genetic Code, Phosphorylation, Simplexvirus drug effects, Substrate Specificity, Acyclovir pharmacology, Antiviral Agents pharmacology, Bromodeoxyuridine analogs & derivatives, Nucleoside-Phosphate Kinase isolation & purification, Phosphotransferases isolation & purification, Simplexvirus enzymology, Thymidine Kinase isolation & purification
- Abstract
Virus-coded thymidine (dThd) kinases were purified by affinity chromatography from a parental strain (SC16) and two strains (SC16 B3 and SC16 S1) of herpes simplex virus, Type 1, with altered drug sensitivities. These latter two strains were less sensitive, respectively, to E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and to both BrVdUrd and 9-(2-hydroxyethoxymethyl)guanine (acyclovir). The enzymes were characterized with respect to physical and catalytic properties. The enzyme from SC16 B3 was very similar to the parental enzyme except in its substrate specificity and kinetic constants. It catalyzed the phosphorylation of BrVdUrd at a relative rate that was 110% of the rate with dThd versus a relative rate of 140% with the parental enzyme. The apparent Km value for BrVdUrd was 6 microM versus 0.1 microM for the parental enzyme. The reaction kinetics with acyclovir were similar for the two enzymes. The SC16 B3 enzyme catalyzed the phosphorylation of dTMP, but at only 2% the efficiency of the parental enzyme; phosphorylation of the monophosphate of BrVdUrd (BrVdUMP) was not detected with the SC16 B3 enzyme. The enzyme from the SC16 S1 variant had a much narrower phosphate acceptor specificity than the enzyme from the parental virus. BrVdUrd was a substrate but with a relative rate of 30% and an apparent Km value of 4 microM; acyclovir was neither detectably phosphorylated nor a good inhibitor. BrVdUMP was not detectably phosphorylated. The relative efficiencies of the two variant enzymes for acyclovir phosphorylation correlated well with the sensitivities of the viruses to this compound. In contrast, the relative efficiencies of the second phosphorylation step (BrVdUMP to BrVdUDP) were most consistent with the sensitivities of the viruses to BrVdUrd.
- Published
- 1983
3. Differential phosphorylation of (E)-5-(2-bromovinyl)-2'-deoxyuridine monophosphate by thymidylate kinases from herpes simplex viruses types 1 and 2 and varicella zoster virus.
- Author
-
Fyfe JA
- Subjects
- Bromodeoxyuridine metabolism, Cytosol metabolism, Kinetics, Phosphorylation, Thymidine metabolism, Bromodeoxyuridine analogs & derivatives, Herpesvirus 3, Human enzymology, Simplexvirus enzymology, Thymidine Kinase metabolism
- Abstract
5-(2-Bromovinyl)-2'-deoxyuridine (BrVdUrd) is a potent and selective inhibitor of herpes simplex virus Type I (HS-I) and varicella zoster (VZ) virus replication but is much less potent against herpes simplex virus Type II (HS-II) replication. A possible enzymatic basis for this difference is reported here to involve the virus-coded dThd-dTMP kinases (EC 2.7.1.75) from the three virus strains. The thymidine kinases from the three virus strains were purified by affinity chromatography. In addition to catalyzing the phosphorylation of nucleosides, each of the three purified enzymes catalyzed the phosphorylation of thymidylate to its diphosphate but at strikingly different rates. The relative amounts of extractable virus-coded thymidylate kinases were estimated to be 100/2/40 for cells infected with HS-I, HS-II, and VZ viruses, respectively. Extracts of cells infected with HS-I virus catalyzed the phosphorylation of the monophosphate of BrVdUrd to its diphosphate. In contrast, the product was not detected with extracts from cells infected with HS-II virus. THe ratios of rates with 0.5 mM BrVdUrd monophosphate versus 0.1 mM dTMP as substrates for each of the purified dThd-dTMP kinases from HS-I, HS-II, and VZ viruses and the dTMP kinase from host cells were, respectively, 0.09, less than 0.002, 0.03, and less than 0.0002. These observations correlate with the relative sensitivities of these viruses to BrVdUrd in cell culture and suggest that, if BrVdUrd exerts its effect as a triphosphate, the inefficient phosphorylation of the monophosphate contributes to the insensitivity of HS-II virus to this agent.
- Published
- 1982
4. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine.
- Author
-
Fyfe JA, Keller PM, Furman PA, Miller RL, and Elion GB
- Subjects
- Guanine pharmacology, Kinetics, Structure-Activity Relationship, Substrate Specificity, Antiviral Agents pharmacology, Guanine analogs & derivatives, Simplexvirus enzymology, Thymidine Kinase metabolism
- Published
- 1978
5. Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity.
- Author
-
Ellis MN, Keller PM, Fyfe JA, Martin JL, Rooney JF, Straus SE, Lehrman SN, and Barry DW
- Subjects
- Acyclovir metabolism, Acyclovir pharmacology, Adult, Animals, Antiviral Agents metabolism, Antiviral Agents pharmacology, Arabinonucleosides metabolism, Arabinonucleosides pharmacology, Cloning, Molecular, DNA Restriction Enzymes, DNA, Viral analysis, Drug Resistance, Microbial, Enzyme Induction, Female, Herpes Simplex microbiology, Humans, Kinetics, Male, Mice, Mice, Inbred BALB C, Phosphorylation, Recurrence, Simplexvirus drug effects, Simplexvirus genetics, Substrate Specificity, Thymidine analogs & derivatives, Thymidine metabolism, Thymidine pharmacology, Thymidine Kinase metabolism, Vero Cells, Simplexvirus enzymology, Thymidine Kinase biosynthesis
- Abstract
In vitro and in vivo studies were done on a herpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied 125I-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-beta-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these clones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Km') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]guaine (BW B759U) were 2- to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight- and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.
- Published
- 1987
- Full Text
- View/download PDF
6. Enzymatic phosphorylation of acyclic nucleoside analogs and correlations with antiherpetic activities.
- Author
-
Keller PM, Fyfe JA, Beauchamp L, Lubbers CM, Furman PA, Schaeffer HJ, and Elion GB
- Subjects
- Animals, Chlorocebus aethiops, Kinetics, Nucleosides pharmacology, Phosphorylation, Simplexvirus drug effects, Structure-Activity Relationship, Substrate Specificity, Thymidine Kinase analysis, Virus Replication drug effects, Antiviral Agents metabolism, Nucleosides metabolism, Simplexvirus metabolism
- Published
- 1981
- Full Text
- View/download PDF
7. Selectivity of antiviral effectiveness derived from differences of herpes simplex virus-coded thymidine kinases.
- Author
-
Fyfe JA
- Subjects
- Animals, Bromodeoxyuridine toxicity, Cell Line, Chlorocebus aethiops, Kidney, Simplexvirus drug effects, Thymidine Kinase isolation & purification, Acyclovir toxicity, Antiviral Agents toxicity, Bromodeoxyuridine analogs & derivatives, Simplexvirus genetics, Thymidine Kinase genetics
- Published
- 1984
- Full Text
- View/download PDF
8. Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine.
- Author
-
Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, and Schaeffer HJ
- Subjects
- Cell Line, DNA-Directed DNA Polymerase metabolism, Guanine metabolism, Guanine Nucleotides metabolism, Kinetics, Phosphotransferases metabolism, Simplexvirus enzymology, Thymidine Kinase metabolism, Antiviral Agents, Guanine analogs & derivatives, Simplexvirus drug effects
- Abstract
A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).
- Published
- 1977
- Full Text
- View/download PDF
9. Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir.
- Author
-
Kit S, Sheppard M, Ichimura H, Nusinoff-Lehrman S, Ellis MN, Fyfe JA, and Otsuka H
- Subjects
- Autoradiography, Base Sequence, Cells, Cultured, Cloning, Molecular, Humans, Simplexvirus genetics, Acyclovir pharmacology, Genes, Viral, Simplexvirus enzymology, Thymidine Kinase genetics
- Abstract
To identify the nucleotide changes that occur in drug-induced thymidine kinase (TK) mutants of herpes simplex virus type 2 (HSV-2), we compared the nucleotide sequences of the tk genes of two mutant HSV-2 clones isolated from a patient who had been treated with acyclovir [9-(2-hydroxyethoxymethyl)guanine; ACV] with the nucleotide sequence of the parental TK+ HSV-2(8703) strain isolated from the same patient. One of the mutants, TK-altered (TKA) HSV-2(9637), was ACV resistant but induced the incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. The nucleotide sequence of the tk gene of mutant TKA HSV-2(9637) had a single change (G to A) at nucleotide 668, which would cause an arginine-to-histidine substitution at amino acid residue 223 of the TK polypeptide. The second ACV-resistant mutant, TK- HSV-2(8710), did not induce detectable incorporation of [14C]thymidine into the DNA of infected rabbit skin cells. This mutant exhibited a deletion of a single base at nucleotide 217 of its nucleotide sequence. This deletion would cause a frameshift mutation at amino acid residue 73 and chain termination at amino acid residue 86 of the TK polypeptide. The nucleotide sequence of TK+ HSV-2(8703) was the same as that of the laboratory strain, TK+ HSV-2(333). The nucleotide sequence of a bromodeoxyuridine-resistant TK- HSV-2(333) mutant of TK+ HSV-2(333) also exhibited a single-base deletion, but at nucleotide 439. This deletion would cause a frameshift mutation at amino acid residue 147 and chain termination at amino acid residue 182. The frameshift mutations of TK- HSV(8710) and TK- HSV-2(333), respectively, occurred in sequences in which C was repeated three times and G was repeated seven times. The results raise the possibility that TK- frameshift mutations of HSV-2 may be common.
- Published
- 1987
- Full Text
- View/download PDF
10. Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.
- Author
-
Furman PA, St Clair MH, Fyfe JA, Rideout JL, Keller PM, and Elion GB
- Subjects
- Animals, Cell Line, DNA biosynthesis, DNA-Directed DNA Polymerase metabolism, Guanosine metabolism, Guanosine pharmacology, Haplorhini, Kidney, Simplexvirus metabolism, Templates, Genetic, DNA Replication drug effects, DNA, Viral biosynthesis, Guanine Nucleotides pharmacology, Guanosine analogs & derivatives, Nucleic Acid Synthesis Inhibitors, Simplexvirus drug effects
- Abstract
The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.
- Published
- 1979
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.