1. Amsacta moorei entomopoxvirus expresses an active superoxide dismutase.
- Author
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Becker MN, Greenleaf WB, Ostrov DA, and Moyer RW
- Subjects
- Amino Acid Sequence, Animals, Blotting, Northern, Blotting, Western, Cell Line, Cloning, Molecular, Copper analysis, Dimerization, Entomopoxvirinae growth & development, Escherichia coli genetics, Escherichia coli metabolism, Gene Deletion, Gene Expression Regulation, Viral, Genes, Viral, Models, Molecular, Molecular Sequence Data, Molecular Weight, RNA, Messenger analysis, RNA, Viral analysis, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Alignment, Superoxide Dismutase chemistry, Superoxide Dismutase isolation & purification, Viral Proteins metabolism, Zinc analysis, Entomopoxvirinae enzymology, Entomopoxvirinae genetics, Lepidoptera virology, Superoxide Dismutase genetics, Superoxide Dismutase metabolism
- Abstract
The entomopoxvirus from Amsacta moorei serves as the prototype of the group B entomopoxviruses. One of the interesting genes found in Amsacta moorei entomopoxvirus (AmEPV) is a superoxide dismutase (sod) (open reading frame AMV255). Superoxide dismutases (SODs) catalyze the conversion of superoxide radicals to hydrogen peroxide and oxygen. Many vertebrate poxviruses contain a sod gene, but to date, none have been demonstrated to be active. There are three families of SODs, characterized by their metal ion-binding partners, Fe, Mn, or Cu and Zn. Poxvirus enzymes belong to the Cu-Zn SOD family. Unlike inactive vertebrate poxvirus SODs, AMVSOD contains all the amino acids necessary for function. We expressed and purified a 6X-His-tagged version of the AMVSOD in Escherichia coli. The recombinant AMVSOD demonstrates superoxide dismutase activity both in an in situ gel assay and by stopped flow spectrophotometry. The k(cat)/K(m) for AMVSOD is 4 x 10(7) M(-1)s(-1). In infected cells, the AMVSOD protein behaves as a dimer and is catalytically active; however, disruption of the gene in AMEPV has little or no effect on growth of the virus in cell culture. An analysis of mRNA expression indicates that AMVsod is expressed late during infection of Lymantria dispar (Ld652) cells and produces a discrete nonpolydisperse transcript. Characterization of protein expression with a monoclonal antibody generated against AMVSOD confirms that the AMVSOD protein can be classified as a late, postreplicative gene. Therefore, AMVSOD is the first example of an active poxvirus SOD.
- Published
- 2004
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