Substrate specificity of homogeneous monkeypox virus uracil-DNA glycosylase.
Weak or nonexistent smallpox immunity in today's human population raises concerns about the possibility of natural or provoked genetic modifications leading to re-emergence of variola virus and other poxviruses. Thus, the development of new antiviral strategies aimed at poxvirus infections in humans is a high priority. The DNA repair protein uracil-DNA glycosylase (UNG) is one of the viral enzymes important for poxvirus pathogenesis. Consequently, the inhibition of UNG is a rational therapeutic strategy for infections with poxviruses. Monkeypox virus, which occurs naturally in Africa, can cause a smallpox-like disease in humans. Here, the monkeypox virus UNG (mpUNG) is characterized and compared to vaccinia virus UNG (vUNG) and human UNG (hUNG). The mpUNG protein excises uracil preferentially from single-stranded DNA. Furthermore, mpUNG prefers the U.G pair over the U.A pair and does not excise oxidized bases. Both mpUNG and vUNG viral proteins are strongly inhibited by physiological concentrations of NaCl and MgCl2. Although the two viral DNA repair enzymes have similar substrate specificities, the kcat/KM values of mpUNG are higher than those of vUNG. The mpUNG protein was strongly inhibited by 5-azauracil and to a lesser extent by 4(6)-aminouracil and 5-halogenated uracil analogues, whereas uracil had no effect. To develop antiviral drugs toward mpUNG, we also validated a repair assay using the molecular beacons containing multiple uracil residues. Potential targets and strategies for combating pathogenic orthopoxviruses, including smallpox, are discussed.
Duraffour
Sophie
S
Laboratoire de virologie, Centre de Recherche du Service de Santé des Armées, Grenoble, France.
Ishchenko
Alexander A
AA
Saparbaev
Murat
M
Crance
Jean-Marc
JM
Garin
Daniel
D
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008993:Monkeypox virus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry*
D004277:DNA, Single-Stranded; D007527:Isoenzymes; D011994:Recombinant Proteins; D014764:Viral Proteins; D015636:Magnesium Chloride; D012965:Sodium Chloride; D006639:Histidine; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase; C039856:5-azauracil