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Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus
journal contribution
posted on 1995-03-01, 00:00 authored by J Christensen, M Pedersen, B Aasted, Soren AlexandersenSoren AlexandersenWe have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.
History
Journal
Journal of virologyVolume
69Issue
3Pagination
1802 - 1809Publisher
American Society for MicrobiologyLocation
Washington, D.C.ISSN
0022-538XeISSN
1098-5514Language
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
1995, American Society for MicrobiologyUsage metrics
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No categories selectedKeywords
Adenosine TriphosphatasesAleutian Mink Disease VirusAnimalsAntibodies, MonoclonalAntigens, ViralCations, DivalentCloning, MolecularDNA HelicasesGenes, ViralMothsMutagenesis, Site-DirectedNucleopolyhedrovirusRecombinant ProteinsSolubilityStructure-Activity RelationshipViral Nonstructural ProteinsViral Structural ProteinsScience & TechnologyLife Sciences & BiomedicineVirologyACUTE INTERSTITIAL PNEUMONIAMINUTE VIRUSAUTONOMOUS PARVOVIRUSTRANS-ACTIVATIONBACULOVIRUS VECTORHELICASE ACTIVITYINSECT CELLSP38 PROMOTERT-ANTIGENMICE DNA
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