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 DELTADUpol Escherichia coli expression cassette and a DELTADU proviral clone. Expression of the recombinant DELTADUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of DELTADU 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 DELTADU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of DELTADU 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.