Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

Bennett, Nicholas J.; Götte, Matthias

doi:10.3390/v5010054

Cited by 15 publications

(15 citation statements)

References 62 publications

(109 reference statements)

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“…The different profiles of HSV-1 and HCMV mutant susceptibility to foscarnet as well as the altered viral fitness could be related to subtle conformational differences resulting from the interaction between amino acids, specific to each enzyme, that are located at the interface between the fingers domain and the N-terminal domain. These results further demonstrate the usefulness of the bacteriophage RB69 polymerase gp43 as a surrogate enzyme for HSV-1 and HCMV in the discovery and development of new antiherpetic drugs (44).…”

Section: Discussionsupporting

confidence: 52%

Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

Piret

Goyette

Eckenroth

et al. 2015

J Virol

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DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the ␣-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC 50 s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC 50 ) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45-and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains. IMPORTANCEDNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the ␣-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.A ll antiviral agents currently approved for the treatment of herpes simplex virus (HSV) and human cytomegalovirus (HCMV) infections ul...

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Section: Discussionsupporting

confidence: 52%

Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

Piret

Goyette

Eckenroth

et al. 2015

J Virol

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“…The bacteriophage RB69 DNApol is one of the most studied at the structural and functional levels, and there are currently 122 entries in the protein data bank (http://www.rcsb.org/pdb/results/results.do?outformat=&qrid=C9789076&tabtoshow=Current) (30,36–40). Although RB69 DNApol lacks the pre-NH 2 -terminal domain, it is a good surrogate model for herpesvirus DNApol, especially regarding structural changes involved in catalysis and ligand binding (DNA, dNTPs) (36).…”

Section: Structural Features Of Dna Polymerase B Familymentioning

confidence: 99%

“…Although RB69 DNApol lacks the pre-NH 2 -terminal domain, it is a good surrogate model for herpesvirus DNApol, especially regarding structural changes involved in catalysis and ligand binding (DNA, dNTPs) (36). HSV-1 DNApol structure is also a good structural model for the other HHVs since the sequence identity is high among the members of the herpesviridae resulting in conserved protein-folding (32).…”

Section: Structural Features Of Dna Polymerase B Familymentioning

confidence: 99%

“…Their polar and/or positively charged side chains allow establishment of electrostatic or H-bond interactions with the incoming dNTPs. This domain is critical for catalysis of the polymerase reaction, and the Finger A and Finger B helices are subject to an important rotation to adopt an optimal position for substrate recognition (36). A close view of the human DNApol α active site shows the same arrangement as the RB69 and HSV-1 DNApols [pdb: 4Q5V; (42)] (Figure 5).…”

Section: Structural Features Of Dna Polymerase B Familymentioning

confidence: 99%

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Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

Topalis¹,

Gillemot²,

Snoeck³

et al. 2016

Nucleic Acids Res

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Emergence of drug-resistance to all FDA-approved antiherpesvirus agents is an increasing concern in immunocompromised patients. Herpesvirus DNA polymerase (DNApol) is currently the target of nucleos(t)ide analogue-based therapy. Mutations in DNApol that confer resistance arose in immunocompromised patients infected with herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV), and to lesser extent in herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV) and human herpesvirus 6 (HHV-6). In this review, we present distinct drug-resistant mutational profiles of herpesvirus DNApol. The impact of specific DNApol amino acid changes on drug-resistance is discussed. The pattern of genetic variability related to drug-resistance differs among the herpesviruses. Two mutational profiles appeared: one favoring amino acid changes in the Palm and Finger domains of DNApol (in α-herpesviruses HSV-1, HSV-2 and VZV), and another with mutations preferentially in the 3′-5′ exonuclease domain (in β-herpesvirus HCMV and HHV-6). The mutational profile was also related to the class of compound to which drug-resistance emerged.

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“…22,23 In all enzymes, the 3′,5′-exonuclease activity had been deleted to eliminate confounding effects of editing activities in the enzyme assays. When evaluated for their anti-DNA polymerase activity, the adenine (and also thymine)-butenyl-α-CNPs were highly inhibitory against HCMV-UL54 (IC 50 : ≤0.5 μM) but completely inactive against bacteriophage WT RB69 and the hybrid RB69/ABC5 (IC 50 : > 100 μM).…”

Section: Resultsmentioning

confidence: 99%

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates

John¹,

Kim²,

Bennett

et al. 2015

J. Med. Chem.

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Alpha-carboxynucleoside phosphonates (α-CNPs) are novel viral DNA polymerase inhibitors that do not need metabolic conversion for enzyme inhibition. The prototype contains a cyclopentyl linker between nucleobase and α-carboxyphosphonate and preferentially (50- to 100-fold) inhibits HIV-1 RT compared with herpetic DNA polymerases. A synthetic methodology involving three steps has been developed for the synthesis of a series of novel α-CNPs, including a Rh(II) catalyzed O-H insertion which connects the carboxyphosphonate group to a linker moiety and attachment of a nucleobase to the other end of the linker by a Mitsunobu reaction followed by final deprotection. Replacing the cyclopentyl moiety in the prototype α-CNPs by a more flexible entity results in a selectivity shift of ~ 100-fold in favor of the herpetic DNA polymerases when compared to HIV-1 RT. The nature of the kinetic interaction of the acyclic α-CNPs against the herpetic DNA polymerases differs from the nature of the nucleobase-specific kinetic interaction of the cyclopentyl α-CNPs against HIV RT.

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Utility of the Bacteriophage RB69 Polymerase gp43 as a Surrogate Enzyme for Herpesvirus Orthologs

Cited by 15 publications

References 62 publications

Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates

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