Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant.
The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.
Threadgill
D S
DS
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960.
Steagall
W K
WK
Flaherty
M T
MT
Fuller
F J
FJ
Perry
S T
ST
Rushlow
K E
KE
Le Grice
S F
SF
Payne
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002415:Cats; D003588:Cytopathogenic Effect, Viral; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015684:Gene Products, pol / Q000096:biosynthesis; D006736:Horses; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011755:Pyrophosphatases / Q000172:deficiency / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014779:Virus Replication
D015684:Gene Products, pol; D011993:Recombinant Fusion Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase