Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus.
We 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.
Department of Veterinary Microbiology, Royal Veterinary and Agricultural University of Copenhagen, Denmark.
J
J
Christensen
M
M
Pedersen
B
B
Aasted
S
S
Alexandersen
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000454:Aleutian Mink Disease Virus / Q000201:enzymology* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000302:isolation & purification*; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism; D005814:Genes, Viral; D009036:Moths; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000276:immunology / Q000302:isolation & purification*; D015678:Viral Structural Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D002413:Cations, Divalent; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases