The present study is the first report on the functional activity of a parapoxvirus-encoded dUTPase. The dUTPase gene of the attenuated orf virus (ORFV), strain D1701, was expressed as a bacterial thioredoxin fusion protein. In vitro assays showed that ORFV dUTPase was highly specific for dUTP as substrate. The enzyme was active over a broad pH range (pH 6·0–9·0), with maximal enzymatic activity at pH 7·0 in the presence of Mg2+ cations. Kinetic studies of the recombinant ORFV dUTPase revealed an apparent Km of 4·0 μM, which is more similar to that of the mammalian or African swine fever virus enzyme than to the Km of vaccinia virus dUTPase. Enzyme activity was also found with purified ORFV particles, indicating its virion association.
BeardsleyG. P.,
AbelsonH. T.1980; A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides. Analytical Biochemistry 105:311–318
BergmanA. C.,
NymanP. O.,
LarssonG.1998; Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1. FEBS Letters 441:327–330
BertaniL. E.,
HäggmarkA.,
ReichardP.1963; Enzymatic synthesis of deoxyribonucleotides. II. Formation and interconversion of deoxyuridine phosphates. Journal of Biological Chemistry 238:3407–3413
ClimieS.,
LutzT.,
RadulJ.,
Sumner-SmithM.,
VandenbergE.,
McIntoshE.1994; Expression of trimeric human dUTP pyrophosphatase in Escherichia coli and purification of the enzyme. Protein Expression and Purification 5:252–258
CottoneR.,
BüttnerM.,
BauerB.,
HenkelM.,
HettichE.,
RzihaH.-J.1998; Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701. Virus Research 56:53–67
DixonL. K.,
TwiggS. R. F.,
BaylisS. A.,
VydelingumS.,
BristowC.,
HammondJ. M.,
SmithG. L.1994; Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1). Journal of General Virology 75:1655–1684
DukerN. J.,
GrantC. L.1980; Alterations in the levels of deoxyuridine triphosphatase, uracil-DNA glycosylase and AP endonuclease during the cell cycle. Experimental Cell Research 125:493–497
FisherF. B.,
PrestonV. G.1986; Isolation and characterization of herpes simplex virus type 1 mutants which fail to induce dUTPase activity. Virology 148:190–197
FlemingS. B.,
BlokJ.,
FraserK. M.,
MercerA. A.,
RobinsonA. J.1993; Conservation of gene structure and arrangement between vaccinia virus and orf virus. Virology 195:175–184
FlemingS. B.,
LyttleD. J.,
SullivanJ. T.,
MercerA. A.,
RobinsonA. J.1995; Genomic analysis of a transposition-deletion variant of orf virus reveals a 3·3 kbp region of non-essential DNA. Journal of General Virology 76:2969–2978
FlemingS. B.,
McCaughanC. A.,
AndrewsA. E.,
NashA. D.,
MercerA. A.1997; A homolog of interleukin-10 is encoded by the poxvirus orf virus. Journal of Virology 71:4857–4861
HokariS.,
SakagishiY.1987; Purification and characterization of deoxyuridine triphosphate nucleotidohydrolase from anemic rat spleen: a trimer composition of the enzyme protein. Archives of Biochemistry and Biophysics 253:350–356
JönsA.,
GranzowH.,
KuchlingR.,
MettenleiterT. C.1996; The UL49.5 gene of pseudorabies virus codes for an O -glycosylated structural protein of the viral envelope. Journal of Virology 70:1237–1241
KöppeB.,
Menendez-AriasL.,
OroszlanS.1994; Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity. Journal of Virology 68:2313–2319
KremmerE.,
SommerP.,
HolzerD.,
GaletskyS. A.,
MolochkovV. A.,
GurtsevitchV.,
WinkelmannC.,
LisnerR.,
NiedobitekG.,
GrässerF. A.1999; Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8) ORF54 encodes a functional dUTPase expressed in the lytic replication cycle. Journal of General Virology 80:1305–1310
LiangX.,
TangM.,
MannsB.,
BabiukL. A.,
ZambT. J.1993; Identification and deletion mutagenesis of the bovine herpesvirus 1 dUTPase gene and a gene homologous to herpes simplex virus UL49.5. Virology 195:42–50
LiangX.,
ChowB.,
BabiukL. A.1997; Study of immunogenicity and virulence of bovine herpesvirus 1 mutants deficient in the UL49 homolog, UL49.5 homolog and dUTPase genes in cattle. Vaccine 15:1057–1064
LichtensteinD. L.,
RushlowK. E.,
CookR. F.,
RaabeM. L.,
SwardsonC. J.,
KocibaG. J.,
IsselC. J.,
MontelaroR. C.1995; Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity. Journal of Virology 69:2881–2888
LyttleD. J.,
FraserK. M.,
FlemingS. B.,
MercerA. A.,
RobinsonA. J.1994; Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus. Journal of Virology 68:84–92
McGeochD. J.1990; Protein sequence comparisons show that the ‘pseudoproteases’ encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family. Nucleic Acids Research 18:4105–4110
McInnesC. J.,
WoodA. R.,
NettletonP. F.,
GilrayJ.2001; Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is non-essential for replication. Virus Genes 22:141–150
MassungR. F.,
JayaramaV.,
MoyerR. W.1993; DNA sequence analysis of conserved and unique regions of swinepox virus: identification of genetic elements supporting phenotypic observations including a novel G protein-coupled receptor homologue. Virology 197:511–528
MayrA.,
HerlynM.,
MahnelH.,
DancoA.,
ZachA.,
BostedtH.1981; Control of ecthyma contagiosum (pustular dermatitis) of sheep with a new parenteral cell culture live vaccine. Zentralblatt für Veterinarmedizin. Reihe B 28:535–552 (in German
MercerA. A.,
FraserK. M.,
StockwellP. A.,
RobinsonA. J.1989; A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus. Virology 172:665–668
OliverosM.,
Garcia-EscuderoR.,
AlejoA.,
VinuelaE.,
SalasM. L.,
SalasJ.1999; African swine fever virus dUTPase is a highly specific enzyme required for efficient replication in swine macrophages. Journal of Virology 73:8934–8943
PardoE. G.,
GutierrezC.1990; Cell cycle- and differentiation stage-dependent variation of dUTPase activity in higher plant cells. Experimental Cell Research 186:90–98
PylesR. B.,
SawtellN. M.,
ThompsonR. L.1992; Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency. Journal of Virology 66:6706–6713
RobinsonA. J.,
LyttleD. J.1992; Parapoxviruses. Their biology and potential as recombinant vaccines. In Recombinant Poxviruses pp 285–327 Edited by
BinnsM. M.,
SmithG. L.
Boca Raton, FL: CRC Press;
RossJ.,
WilliamsM.,
CohenJ. I.1997; Disruption of the varicella-zoster virus dUTPase and the adjacent ORF9A gene results in impaired growth and reduced syncytia formation in vitro . Virology 234:186–195
RzihaH.-J.,
HenkelM.,
CottoneR.,
MeyerM.,
DehioC.,
BüttnerM.1999; Parapoxviruses: potential alternative vectors for directing the immune response in permissive and non-permissive hosts. Journal of Biotechnology 73:235–242
SavoryL. J.,
StackerS. A.,
FlemingS. B.,
NivenB. E.,
MercerA. A.2000; Viral vascular endothelial growth factor plays a critical role in orf virus infection. Journal of Virology 74:10699–10706
SlabaughM. B.,
RosemanN. A.1989; Retroviral protease-like gene in the vaccinia virus genome. Proceedings of the National Academy of Sciences, USA 86:4152–4155
SommerP.,
KremmerE.,
BierS.,
KönigS.,
ZaludP.,
ZeppezauerM.,
JonesF. J.,
Mueller-LantzschN.,
GrässerF. A.1996; Cloning and expression of the Epstein–Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme. Journal of General Virology 77:2795–2805
ThreadgillD. S.,
SteagallW. K.,
FlahertyM. T.,
FullerF. J.,
PerryS. T.,
RushlowK. E.,
Le GriceS. F. J.,
PayneS. L.1993; Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. Journal of Virology 67:2592–2600
TyeB. K.,
NymanP. O.,
LehmanI. R.,
HochhauserS.,
WeissB.1977; Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA. Proceedings of the National Academy of Sciences, USA 74:154–157
WilliamsM. V.1984; Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type 1. Purification and characterization of induced enzyme. Journal of Biological Chemistry 259:10080–10084
WilliamsM. V.,
ChengY.1979; Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia. Journal of Biological Chemistry 254:2897–2901