Characterization and mutational studies of equine infectious anemia virus dUTPase

Hai Shao, Michael D. Robek, Deborah S. Threadgill, Lori S. Mankowski, Craig E. Cameron, Frederick J. Fuller, Susan L. Payne

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 103 to 5 x 104 units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a K(m) in the range of 3 to 8 μM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PP(i). The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is giycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-Ioops also abolish EIAV dUTPase activity.

Original languageEnglish (US)
Pages (from-to)181-191
Number of pages11
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number2
StatePublished - May 23 1997

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'Characterization and mutational studies of equine infectious anemia virus dUTPase'. Together they form a unique fingerprint.

Cite this