Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N)-methanocarbathymidine.
BACKGROUND
The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear.


RESULTS
The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase.


CONCLUSIONS
We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect metabolic differences in the mutant virus.
Prichard
Mark N
MN
Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL 35233, USA. mprichard@peds.uab.edu
Kern
Earl R
ER
Quenelle
Debra C
DC
Keith
Kathy A
KA
Moyer
Richard W
RW
Turner
Peter C
PC
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011743:Pyrimidines / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014615:Vaccinia / Q000821:virology*; D014616:Vaccinia virus / Q000187:drug effects / Q000201:enzymology / Q000472:pathogenicity / Q000502:physiology*; D010948:Viral Plaque Assay; D014774:Virulence; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; C030986:pyrimidine; C469995:(north)-methanocarbathymidine; D013936:Thymidine