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Molecular-cloning and characterization of deoxyuridine triphosphatase from feline immunodeficiency virus (fiv)

Academic Article
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Overview

authors

  • Wagaman, P. C.
  • Hasselkuslight, C. S.
  • Henson, M.
  • Lerner, D. L.
  • Phillips, T. R.
  • Elder, John

publication date

  • 1993

journal

  • Virology  Journal

abstract

  • The feline lentivirus, FIV, contains dUTPase (DU) as part of the enzyme cassette encoded by the viral pol gene (Elder et al., 1992, J. Virol. 66, 1791-1794). The enzyme is processed from the Pol polyprotein and is packaged into infectious virions. We report here the basic characteristics of the viral enzyme, including substrate specificity, ion requirements, and pH optimum. We also report the overexpression of DU in Escherichia coli and insertional mutagenesis of the enzyme in the context of the complete provirus or DU alone. The enzyme requires Mg2+ for full activity and competition studies employing unlabeled dNTPs indicated that DU has an absolute preference for dUTP. The pH optimum for FIV DU is pH 7.0. The limits of the protein dictate a species of M(r) 14,350, which agrees precisely with the determination by ion spray mass spectroscopy of DU isolated from virions. Cleavage sites at the junctions between DU, RT, and IN, as defined by N-terminal amino acid sequencing of each protein species, are consistent with predictions for sites of cleavage by aspartate protease. In-frame insertional mutagenesis at Tyr 75 of DU abolishes activity. Cells transfected with proviruses containing this mutation express virion-associated reverse transcriptase activity but lack DU activity. The resultant virions replicate slower than those possessing wild-type DU. Tests are currently underway to evaluate the consequences of DU mutagenesis on in vivo phenotype.

subject areas

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cats
  • Cells, Cultured
  • Cloning, Molecular
  • DNA Mutational Analysis
  • Escherichia coli
  • Hydrogen-Ion Concentration
  • Immunodeficiency Virus, Feline
  • Magnesium
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Pyrophosphatases
  • Recombinant Proteins
  • Sequence Homology, Amino Acid
  • Substrate Specificity
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Identity

International Standard Serial Number (ISSN)

  • 0042-6822

Digital Object Identifier (DOI)

  • 10.1006/viro.1993.1501

PubMed ID

  • 8396797
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Additional Document Info

start page

  • 451

end page

  • 457

volume

  • 196

issue

  • 2

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