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Biomedical subjects

G Darby

Publications and source records attributed to G Darby.

At least 37 records · Page 2Linked to original sources
PubMed1987

Related functional domains in virus DNA polymerases.

Analysis of the lesions in several drug-resistant DNA polymerase mutants of herpes simplex virus along with comparative analysis of the published polymerase sequences of other human herpesviruses has shown that most lesions (five out of six) are substitutions at amino acid residues conserved in all four polymerases. Furthermore, the majority of lesions are in regions of the polypeptide where there are marked clusterings of conserved residues. On the basis of these data we have identified several domains within the polypeptide which we believe may have important functional roles in the action of the enzyme. The apparent restriction in the potential sites of lesions conferring drug resistance may explain the difficulty in selecting such mutants using acyclovir (ACV) in culture and their failure to emerge so far during ACV therapy. Extension of the comparative analysis to the polymerases of adenovirus type 2, vaccinia virus and phage phi 29 suggests that these enzymes also possess domains homologous to those most conserved in the herpes polymerases (regions I-III) and that these domains have a similar linear spatial distribution on the polypeptides. The results are discussed in relation to the known function of the DNA polymerases.

Amino Acid Sequence↗
PubMed1987

Analysis of the role of the cysteine 171 residue in the activity of herpes simplex virus type 1 thymidine kinase by oligonucleotide-directed mutagenesis.

The thymidine kinase (TK) gene from herpes simplex virus type 1 strain SC16 was cloned into bacteriophage M13 mp8 so that functional HSV-1 TK was expressed in bacteria infected with the recombinant bacteriophage, M13/TK. Oligonucleotide site-directed mutagenesis was then employed to introduce single nucleotide changes into the TK gene in M13/TK in order to alter the codon for cysteine 171 in the wild-type enzyme to a codon specifying either serine or glycine. Analysis of the mutant enzymes in bacterial extracts showed that these substitutions had little effect on the activity of the enzyme, indicating that the side chain of this residue is not involved in nucleoside binding and is not essential for the catalytic activity of the enzyme.

Base Sequence↗
PubMed1987

Specificity of the immune response of mice to herpes simplex virus glycoproteins B and D constitutively expressed on L cell lines.

Mouse L cell lines have been developed which constitutively express glycoproteins B (gB) and D (gD) of herpes simplex virus type 1. When used to study the immune response of mice to the viral glycoproteins, it was found that both gB and gD induce a delayed type hypersensitivity response and both also induce an antibody response, but only the cell line expressing gD could stimulate the production of neutralizing antibody. Virus-specific cytotoxic T lymphocytes (CTLs) recognized gB expressed by the cell line and this line could also induce CTLs in mice. Recognition of gD by major histocompatibility complex class I restricted CTLs was never seen. Vaccination of mice with the cell lines provided protection from viral challenge and inhibited the establishment of a latent infection, although gD proved to be the better protective immunogen.

Animals↗
PubMed1986

Restoration of wild-type pathogenicity to an attenuated DNA polymerase mutant of herpes simplex virus type 1.

The drug-resistant variant, RSC-26, which was derived from the herpes simplex virus type 1 wild-type strain SC16, expresses an altered DNA polymerase and has reduced pathogenicity in animal models. To determine whether the attenuation in pathogenicity was due solely to mutation in the polymerase gene, a fragment of the wild-type gene was cloned, transferred into the genome of RSC-26 and recombinants were isolated. Three recombinants examined had similar properties to wild-type virus with respect to their sensitivity to antiviral drugs, DNA polymerase activities and their pathogenicity for mice. These results strongly suggest that expression of the altered polymerase of RSC-26 results in attenuated pathogenicity.

Animals↗
PubMed1986

Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir.

Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate-specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK-activated drugs, although two were resistant to 5-(trifluoromethyl)-2'-deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered.

Acyclovir↗
PubMed1985

Selection and characterisation of acyclovir-resistant herpes simplex virus type 1 mutants inducing altered DNA polymerase activities.

A collection of TK+, ACV-resistant mutants of herpes simplex virus type 1 (HSV-1) has been derived using a selection system based on biochemically transformed cells. Evidence is presented suggesting that most of these mutants induce resistant DNA polymerase activities and are thus likely to express variant DNA polymerases. Preliminary data on the pathogenesis of these mutants show that most are similar to wild type virus in the majority of their characteristics, although they may be reduced in their ability to kill mice.

Acyclovir↗
PubMed1984

Cooperative effects between two acyclovir resistance loci in herpes simplex virus.

The acyclovir-resistant mutant SC16 R9C2 (H.J. Field, G. Darby, and P. Wildy , J. Gen. Virol. 49:115-124, 1980) has been shown to contain two resistance loci which segregate independently on recombination with wild-type virus. One locus is in thymidine kinase, and the other is in DNA polymerase. Both induced enzymes have altered properties, thymidine kinase showing a low affinity for acyclovir and low activity, and DNA polymerase showing a low affinity for acyclovir triphosphate. Other properties of both enzymes are described which distinguish them from their wild-type counterparts. Recombinants containing either mutant thymidine kinase ( RSC -11) or mutant DNA polymerase ( RSC -26), but not both, have been used to investigate the relative contribution of each lesion to resistance and pathogenicity. Although SC16 R9C2 and both recombinants grow as well as does wild-type virus in tissue culture, they are considerably attenuated in vivo, the greatest attenuation of virulence being seen with SC16 R9C2 and RSC -26. With respect to both acyclovir resistance and in vivo growth, the lesions appear to behave synergistically. Cross resistance studies have shown the recombinant RSC -26, which contains mutant DNA polymerase but which evidently expresses wild-type thymidine kinase, to be cross resistant to both 5-iodo-2'-deoxyuridine and 5-trifluoromethyl-2'-deoxyuridine but not to (E)-5-(2-bromovinyl)-2'-deoxyuridine or 9-beta-D-arabinofuranosyladenine.

Acyclovir↗
PubMed1983

Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus. Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase.

The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.

Acyclovir↗
PubMed1983

Characterization of abnormal thymidine kinases induced by drug-resistant strains of herpes simplex virus type 1.

Two TK+ acyclovir-resistant variants of herpes simplex virus (HSV) (S1 and Tr7) and one TK+ BVdU-resistant variant (B3) induce abnormal thymidine kinases with impaired ability to phosphorylate the drugs used in their isolation. These enzymes have been purified and their properties compared with those of the wild-type (wt) parent, SC16. The enzyme induced by S1 differed markedly from the other three in both its responses to salt and to pH. B3 TK recognized the enzyme's natural substrates, thymidine, deoxycytidine, dTMP and ATP as well as the wt enzyme. In contrast, Tr7 and S1 TKs failed to bind deoxycytidine and bind thymidine less well than wt. Tr7 and S1 TKs had affinities for dTMP similar to those of B3 and the wt enzymes. ATP binding to wt, Tr7 and B3 enzymes was similar but this substrate bound only weakly to S1 TK. Each mutant displayed a characteristically distinct pattern of affinities for a range of nucleoside analogue substrates, suggesting that they will show some cross-resistance to drugs which have a similar mechanism of action to acyclovir and BVdU.

Drug Resistance, Microbial↗
PubMed1982

Isolation and characterization of acyclovir-resistant strains of herpes simplex virus.

A number of clinical studies have documented herpes simplex infections which appear to be resistant to nucleoside analogs; these include idoxuridine [1,2] and acyclovir [3]. Few, if any of the viruses isolated from such patients have yet been thoroughly characterized. We have isolated a number of acyclovir-resistant mutants by selection for resistance in tissue culture. The study of the biochemical and biological properties of these mutants has given some insight into the likely nature of resistant clinical strains. We have devised a number of simple tests to allow classification of laboratory mutants. We also draw attention to some of the difficulties the clinical virologist may encounter when analyzing putative resistant virus isolated from treated patients.

Acyclovir↗
PubMed1982

Properties of a novel thymidine kinase induced by an acyclovir-resistant herpes simplex virus type 1 mutant.

The acyclovir-resistant mutant of herpes simplex virus type 1, SC16 S1, induced reduced levels of thymidine kinase activity (ca. 25% reduction) in infected cells. The activity appeared with kinetics similar to that in wild type-infected cells, and pulse-labeling experiments showed that the thymidine kinase polypeptide was synthesized at a similar rate. We showed that the enzyme was virus specific by inactivating it with antiserum raised against herpes simplex virus-infected cell proteins. The enzyme induced by the mutant had reduced electrophoretic mobility in nondenaturing gels, decreased thermal stability, and decreased affinity for several different substrates (assessed by measurement of Km values) compared with the enzyme induced by the wild type. From the data obtained we conclude that the thymidine kinase induced by the mutant has an altered specificity, probably resulting from an amino acid substitution which affects the primary binding site for nucleosides and nucleoside analogs.

Acyclovir↗
PubMed1981

Adenovirus late sequences linked to herpes simplex virus thymidine kinase may be introduced into eukaryotic cells and transcribed.

LTK-cells have been transformed to the TK+ phenotype by treatment with size-defined concatamers of HSV-1 TK DNA and Ad2 Bam H1 C fragment (42.0 - 59.5 map units). All TK+ transformants contained Ad2 DNA as well as HSV-1 TK sequences. In most cases several inserts of virus DNA were present, many in high copy numbers. Although no Ad2 transcription promoter was present in the transforming DNA, Ad2-specific sequences were detected in polyadenylated cytoplasmic RNA species from several cell lines.

Adenoviruses, Human↗
PubMed1981

Altered substrate specificity of herpes simplex virus thymidine kinase confers acyclovir-resistance.

Acyclovir (9-[2-hydroxyethoxymethyl]guanine or ACV) is a nucleoside analogue with considerable potential for the treatment of herpes simplex virus (HSV) infections in man. Two virus-coded enzymes are important in the mechanism of action of this drug: thymidine kinase (TK) which initiates its activation by converting it to the monophosphate and DNA polymerase whose action is inhibited by ACV triphosphate. Changes in either gene may confer resistance, but all reported mutations in the TK gene have resulted in failure of the resistant virus to induce appreciable levels of the enzyme. Such TK- mutants arise readily in tissue culture systems where the enzyme is non-essential for virus replication, but in animals they show considerably reduced pathogenicity and neurovirulence. We now describe the isolation of a resistant mutant which induces a TK of altered substrate specificity and we show that this virus retains pathogenicity for mice with only a slight attenuation of neurovirulence.

Acyclovir↗
PubMed1981

The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs.

Three acyclovir (ACV)-resistant mutants derived from a strain of herpes simplex virus (HSV) type 1 were studied to determine the range of their resistance to nine drugs active against HSV. Two of the mutants were thymidine kinase-deficient (TK-) and were resistant to drugs that are usually phosphorylated by HSV TK. The other mutant induced normal levels of TK; it was of special interest since TK+ viruses appear more likely to multiply well in vivo. This mutant was inhibited by "TK-mediated" drugs: idoxuridine, which is already in use, and two drugs with promising clinical potential, 1-beta-arabinofuranosylthymine and E-5-(2-bromovinyl)-2'-deoxyuridine. All of the mutants were sensitive to trifluorothymidine and 9-beta-D-arabinofuranosyladenine. These results suggest that the study of cross-resistance of HSV strains in vitro will aid in the investigation of alternative drugs for use in effective chemotherapy.

Acyclovir↗
PubMed1980

Sensitivity of viruses to phosphorylated 9-(2-hydroxyethoxymethyl)guanine revealed in TK-transformed cells.

Vaccinia and pseudorabies viruses are resistant to ACV [Acyclovir or 9-(2-hydroxyethoxymethyl)guanine] in normal cells. However, both viruses are sensitive in thymidine kinase (TK)-transformed cells in which the resident HSV-specific TK is able to phosphorylate the drug. This demonstrates the sensitivity of these viruses to phosphorylated ACV and suggests a wider antiviral activity for the phosphorylated drug.

Acyclovir↗