Aphidicolin resistance in Herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site

Hall, Jennifer; Wang, Yisheng; Pierpont, John W.; Berlin, Mark S.; Rundlett, Stephen E.; Woodward, S.

doi:10.1093/nar/17.22.9231

Cited by 43 publications

(50 citation statements)

References 55 publications

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“…4), but PAA'5 is hypersensitive to GCV, while AraA r7 is sensitive . This kind of difference in phenotype conferred by mutations at adjacent residues has been previously observed in HSV and human cytomegalovirus (Gibbs et al, 1988;Hall et al, 1989;Sullivan et sl., 1993). An interesting property of some of the mutants studied here is resistance to PCV and either hypersensitivity (PAA r5) or sensitivity to the closely related drug GCV ( Fig.…”

Section: Implications For Polymerase Substrate Recognitionsupporting

confidence: 64%

“…In particular, seven such mutations have now been identified that affect conserved region III (Larder et al, 1987;Gibbs et al, 1988;Hall et al, 1989;this report). This supports the suggestion that region III may "be involved directly in drug and substrate binding (Larder and Darby, 1987;Gibbs et al, 1988).…”

Section: Implications For Polymerase Substrate Recognitionmentioning

confidence: 99%

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Penciclovir-Resistance Mutations in the Herpes Simplex Virus DNA Polymerase Gene

Chiou

Kumura

et al. 1995

Antivir Chem Chemother

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SummaryPenciclovir is the active form of the orally available prodrug famciclovir, which is entering clinical use for herpesvirus infections. Like aciclovir, penciclovir is an acyclic guanosine analogue that is phosphorylated by viral thymidine kinase and whose triphosphate can inhibit viral DNA polymerase. We tested several well-characterized herpes simplex virus mutants with aciclovir-resistance mutations in the viral DNA polymerase gene for altered sensitivity to penciclovir. The mutants varied in their susceptibilities to penciclovir with one exhibiting 2-fold hypersensitivity, one marginal resistance and three about 3-fold resistance. Marker rescue and DNA sequencing analyses mapped the penciclovir-resistance mutation of one mutant, AraA r7, to a single base change that alters a glycine to a cysteine at residue 841 within conserved region III of a-like DNA polymerases. The results have implications for the mechanism of selective action of penciclovir, for the potential for development of resistance in the clinic, and for the substrate recognition properties of herpes simplex virus DNA polymerase.

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Section: Implications For Polymerase Substrate Recognitionsupporting

confidence: 64%

Section: Implications For Polymerase Substrate Recognitionmentioning

confidence: 99%

Penciclovir-Resistance Mutations in the Herpes Simplex Virus DNA Polymerase Gene

Chiou

Kumura

et al. 1995

Antivir Chem Chemother

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“…A competitive inhibition mechanism was proposed with dCTP and perhaps dTTP and non-competitive ones with the other dNTP substrates [17 and references therein]. Studies have been attempted [18] to identify the protein residues which participate in aphidicolin interactions by characterizing mutations within the aphidicolin-sensitive DNA pol of herpes simplex virus type, which alter the substrate specificity of this enzyme. The interaction model which emerged from these studies suggested that the binding sites for aphidicolin and dNTPs are in close proximity and overlap substantially.…”

Section: Buadatpmentioning

confidence: 99%

“…Thus, although Hall et al [18] as well as Copeland and Wang [19] have derived from their experiments a binding position for aphidicolin close to the nucleotide binding site, it is possible that other domains of pol a may be required for the inhibitory effects of that molecule. Despite its four OH-groups, aphidicolin is mainly of hydrophobic nature.…”

Section: Buadatpmentioning

confidence: 99%

Targeting human DNA polymerase α for the inhibition of keratinocyte proliferation. Part 1. Homology model, active site architecture and ligand binding

Richartz

Höltje

Brandt

et al. 2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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In order to understand the binding modes of human DNA polymerase a (pol a) inhibitors on a molecular level, a 3D homology model of the active site of the enzyme was proposed based on the application of molecular modelling methods and molecular dynamic simulations using available crystal coordinates of pol a relatives. Docking results for a series of known nucleotide analogue inhibitors were consistent with reported experimental binding data and offered the possibility to elucidate structure-activity relationships via investigations of active site-inhibitor interactions. Furthermore, the study could explain, at least partially, the inhibitory effect of aphidicolin on pol a. In molecular dynamics simulations, aphidicolin occupied the catalytic centre, but acted in a not truly competitive manner with respect to nucleotides. It destabilized the replicating "closed" form of the pol a and transferred the enzyme into the inactive "open" conformation. This result is consistent with recent experiments on the binding mode of aphidicolin.

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“…PCNA orthologues are not found in most baculovirus genomes, but a homologue of the pcna gene in the AcMNPV genome has been reported (6, 33). The AcMNPV DNApol consists of several conserved domains, designated regions I through VII, which are in the order region IV-region II-region VI-region IIIregion I-region VII-region V and are shared among ␣-like DNApols of eukaryotes and various DNA viruses (5,17,23,46). These conserved regions were postulated to be functional domains involved in substrate recognition (23).…”

mentioning

confidence: 99%

Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

Feng

Thumbi

Jong

et al. 2012

J Virol

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c Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.T he molecular mechanisms of baculovirus DNA replication have been studied in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the genus Alphabaculovirus of the family Baculoviridae. Baculoviruses are distinctive in that they have a biphasic replication cycle producing budded virions (BVs) early in infection, and later in infection, single or multiple nucleocapsids are enveloped in the ring zone to form occlusion-derived virus (ODV), which becomes embedded within polyhedra (14). AcMNPV replicates its large (134-kb), circular, double-stranded DNA genome (1) in the nucleus of infected cells following a molecular interaction between virus-encoded transacting factors and cis-acting DNA sequences. To date, the functional roles of only some trans-acting factors involved in DNA replication have been elucidated (24,31).The AcMNPV DNA polymerase (DNApol) plays an essential role in AcMNPV DNA replication (27,48). The 3-kb DNApol open reading frame (ORF) encodes a polypeptide of 984 amino acids (aa) with a predicted molecular mass of 114.31 kDa (46). Previous biochemical studies suggested that baculovirus-encoded DNApol is most closely related to ␣-and ␦-like DNApols of various DNA viruses, such as herpes simplex viruses (HSVs), adenoviruses, and poxviruses (18,30). Moreover, the AcMNPV DNApol is sensitive to aphidicolin, a feature common to ␣-like DNApols (18,37,43). The ␦-like (replicative-form) DNApol requires proliferating cell nuclear antigen (PCNA) (a proc...

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Aphidicolin resistance in Herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site

Cited by 43 publications

References 55 publications

Penciclovir-Resistance Mutations in the Herpes Simplex Virus DNA Polymerase Gene

Penciclovir-Resistance Mutations in the Herpes Simplex Virus DNA Polymerase Gene

Targeting human DNA polymerase α for the inhibition of keratinocyte proliferation. Part 1. Homology model, active site architecture and ligand binding

Selection and Characterization of Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase Mutations

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