Evidence for a Herpesvirus Saimiri-specified DNA Polymerase Activity which is Aphidicolin-resistant and Phosphonoacetate-sensitive

O’Hare, Peter; Honess, R. W.

doi:10.1099/0022-1317-64-5-1013

Cited by 10 publications

(5 citation statements)

References 33 publications

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“…Bodemer & B. Fleckenstein, personal communication). In common with the DNA polymerase activities of a number of other herpesviruses, the activity specified by HVS is normally inhibited by phosphonoacetic acid (PAA) (O'Hare & Honess, 1983b). Doses of PAA sufficient to inhibit virus DNA replication inhibit the synthesis of the majority of virus-induced polypeptides [e.g.…”

Section: Introductionmentioning

confidence: 99%

DNA-binding Proteins Specified by Herpesvirus Saimiri

Blair¹,

Honess²

1983

Journal of General Virology

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SUMMARYHerpesvirus saimiri-specific proteins from the nuclear fractions of productively infected owl monkey kidney cells were dissociated from virus and host DNA by treatment with 2 M-NaCI or separation on Urografin density gradients. Empty virus capsids remained intact and could be separated from major non-structural proteins (110K, 51K and 48K) and from a subset of structural proteins (130K, 29K and 12K), either by Urografin gradient sedimentation or differential centrifugation. The DNA in such soluble extracts of nuclear proteins was efficiently removed by spermine precipitation, together with the host cell histones and large fractions of the 130K and 12K structural proteins. Proteins in the spermine-soluble fraction were analysed by affinity chromatography on columns of single-stranded calf thymus DNA coupled to cellulose. Two major structural proteins (130K and 12K), whose synthesis was sensitive to phosphonoacetic acid (PAA), and one minor PAA-resistant structural protein (29K) bound to DNA-cellulose. The major PAA-resistant 11 OK non-structural protein and the PAA-resistant non-structural 51K and 48K phosphoproteins were efficiently released into the spermine-soluble fraction and also bound to DNA-cellulose as did the 76K protein and minor species of 42K, 39K, 34K, 25K and 21K. Virus-specific proteins were eluted from such columns by buffers containing 0.4 M-NaC1 or by heparin in lowsalt buffers. Polypeptides from virus particles, infected cell extracts, or samples of eluates from DNA-cellulose chromatography, were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose filters and probed for their ability to bind labelled polynucleotides. The non-structural 51K phosphoprotein, the 12K and 29K structural proteins and a 100K virion polypeptide all bound labelled DNA. However, the binding activities of the 130K protein from virions or purified by affinity chromatography and of the 11 OK polypeptide could not be demonstrated reproducibly after transfer from SDS gels to nitrocellulose. Comparisons of the present results on the properties of the herpesvirus saimiri-specified DNA-binding proteins with published accounts of the DNA-binding proteins of other herpesviruses, suggest some striking similarities with the DNA-binding proteins of the Epstein-Barr virus.

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Section: Introductionmentioning

confidence: 99%

DNA-binding Proteins Specified by Herpesvirus Saimiri

Blair¹,

Honess²

1983

Journal of General Virology

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“…The timeordered synthesis is also seen on the mRNA level. By using PAA as an inhibitor of the virus-specific DNA polymerase (23), two classes of herpesvirus saimiri gene products can be distinguished: the early mRNAs or proteins which are synthesized without prior DNA replication and the late mRNAs and proteins. Synthesis of late products is turned on when viral DNA is produced.…”

Section: Discussionmentioning

confidence: 99%

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis

Hell¹,

Modrow²,

Wolf³

1985

J Virol

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Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products.

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“…During the course of experiments comparing the action of the tetracyclic diterpenoid, aphidicolin, on the synthesis of DNA in cells infected with HSV and herpesvirus saimiri, we found that a variant of HSV selected for high level resistance to PAA had acquired an unselected hypersensitivity to aphidicolin (O'Hare & Honess, 1983). In contrast to the analysis of resistance, the hypersensitive phenotype is a very convenient property for genetic analysis.…”

Section: Hsv-i[mp17]tsh) or Aphidicolin Hypersensitivity (Eg Hsv-i[mentioning

confidence: 96%

“…However, detailed studies of the mode of action of this interesting inhibitor on cellular ~-polymerases are likely to be constrained by the difficulties of acquiring the relevant genetic information. Although the inhibitor in its present form seems unlikely to be a useful agent for the treatment of herpesvirus infections (O'Hare & Honess, 1983;present Table 1 and Fig. 1 ; at least one strain of pseudorabies virus also plaques and grows efficiently in the presence of 0-5 Ixg/ml, unpublished results) the HSV DNA polymerase provides an excellent system with which to explore the molecular genetics of aphidicolin resistance, sensitivity and hypersensitivity in a replicative DNA polymerase.…”

Section: Discussionmentioning

confidence: 99%

Single Mutations at Many Sites within the DNA Polymerase Locus of Herpes Simplex Viruses Can Confer Hypersensitivity to Aphidicolin and Resistance to Phosphonoacetic Acid

Honess

Purifoy

Young

et al. 1984

Journal of General Virology

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SUMMARYAphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-ct activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase in vitro. A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin. A number of mutants with temperature-sensitive (ts) lesions in the polymerase gene also showed increased aphidicolin sensitivity (e.g. HSV-I[mP17]tsH) or aphidicolin hypersensitivity (e.g. HSV-I[KOS]tsD9, tsC4).Resistance or hypersensitivity of virus growth and DNA synthesis in vivo were correlated with resistance or hypersensitivity of virus DNA polymerase reactions in vitro. Resistance phenotypes were closely linked to the polymerase gene during recombination with outside markers. Moreover, the selection of aphidicolin-resistant mutants from hypersensitive variants with independent PAA resistance or ts mutations in the polymerase gene could result in co-selection for PAA-sensitive and ts + phenotypes. Confirmation that multiple independent mutations could determine aphidicolin hypersensitivity was obtained by studies of recombination between independent hypersensitive variants. Aphidicolin-resistant recombinant progeny were formed with recombination frequencies (0.4 to 2.6~) compatible with intragenic events. With parental hypersensitive variants which were products of limited PAA selection, or with the ts polymerase mutations, aphidicolin-resistant recombinants were PAA-sensitive and/or ts +. The segregation of other markers (ts, plaque morphology) amongst recombinant progeny permitted the orientation of multiple determinants of PAA resistance and aphidicolin hypersensitivity with respect to other markers in the polymerase gene and in other genes. The nature of residues determined at any one of a constellation of separate sites within the polymerase locus can determine resistance or sensitivity to antiviral drugs and aphidicolin hypersensitivity associated with changes at the polymerase locus facilitates high resolution genetic analysis of this locus. INTRODUCTION Herpesviruses characteristically specify the synthesis of a novel DNA polymerase activity which is essential for the replication of virus DNA in productively infected cells. For herpes simplex viruses of type l and type 2 (HSV-1, HSV-2) there is now good evidence that t Present address:

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Evidence for a Herpesvirus Saimiri-specified DNA Polymerase Activity which is Aphidicolin-resistant and Phosphonoacetate-sensitive

Cited by 10 publications

References 33 publications

DNA-binding Proteins Specified by Herpesvirus Saimiri

DNA-binding Proteins Specified by Herpesvirus Saimiri

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis

Single Mutations at Many Sites within the DNA Polymerase Locus of Herpes Simplex Viruses Can Confer Hypersensitivity to Aphidicolin and Resistance to Phosphonoacetic Acid

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