ABSTRACT
The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.
Subject(s)
Adenosine Triphosphatases/genetics , Capsid/genetics , Minute Virus of Mice/genetics , Transfection , Viral Core Proteins/genetics , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Baculoviridae/genetics , Capsid/isolation & purification , Capsid/metabolism , Cell Line , Chromatography, Affinity , Cloning, Molecular , Deoxyadenine Nucleotides/metabolism , Electrophoresis, Polyacrylamide Gel , Genes, Viral , Insecta , Minute Virus of Mice/enzymology , Molecular Sequence Data , Molecular Weight , Multienzyme Complexes/genetics , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Peptides/chemical synthesis , Peptides/immunology , Plasmids , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Viral Core Proteins/isolation & purification , Viral Core Proteins/metabolism , Viral Nonstructural ProteinsABSTRACT
Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.
