Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

ABSTRACT Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.

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Characterisation of the substrate specificity of homogeneous vaccinia virus uracil-DNA glycosylase

Nucleic Acids Research ◽

10.1093/nar/gkg672 ◽

2003 ◽

Vol 31 (16) ◽

pp. 4950-4957 ◽

Cited By ~ 25

Author(s):

N. Scaramozzino

Keyword(s):

Substrate Specificity ◽

Vaccinia Virus ◽

Dna Glycosylase ◽

Uracil Dna Glycosylase

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Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Journal of Biological Chemistry ◽

10.1074/jbc.m511239200 ◽

2005 ◽

Vol 281 (6) ◽

pp. 3439-3451 ◽

Cited By ~ 57

Author(s):

Eleni S. Stanitsa ◽

Lisa Arps ◽

Paula Traktman

Keyword(s):

Vaccinia Virus ◽

Dna Polymerase ◽

Dna Glycosylase ◽

Viral Dna ◽

Uracil Dna Glycosylase ◽

Processivity Factor

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A New Class of Uracil–DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme

Molecules ◽

10.3390/molecules26216668 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6668

Author(s):

Inga R. Grin ◽

Grigory V. Mechetin ◽

Rustem D. Kasymov ◽

Evgeniia A. Diatlova ◽

Anna V. Yudkina ◽

...

Keyword(s):

Vaccinia Virus ◽

Viral Infections ◽

Dna Glycosylase ◽

Dna Glycosylases ◽

Uracil Dna Glycosylase ◽

Cytosine Deamination ◽

Strand Breaks ◽

Promising Target ◽

Competitive Mechanism ◽

Wide Range

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

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