title: Synthesis in Escherichia coli of avian reovirus core protein varsigmaA and its dsRNA-binding activity.
abstract: The genome segment S2 of p6ian reovirus (ARV) S1133 was cloned and sequenced. The entire S2 nucleotide sequence is 1325 bp long with one long open reading frame that encodes a protein of 415 amino acids, corresponding to varsigmaA, a major core protein of ARV. S2 possesses a pentanucleotide, TCATC, at the 3'-terminus of its plus strand, common to other known genome segments of ARV and to 10 genome segments of mammalian reovirus. Amino acid sequence analysis revealed that varsigmaA contains a carboxy-terminal region (one-fourth of the protein) that is formed from alpha-helices and beta-turns, and the remainder (three-fourths of the protein) is formed predominantly from beta-strands and beta-turns. Analysis of binding activity to poly(rI)-poly(rC)-agarose suggested that ARV protein A present in total virus-infected chicken embryo fibroblasts (CEF) had dsRNA-binding activity. To further characterize the binding activity, protein varsigmaA was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chelate affinity chromatography. The expressed protein evarsigmaA was further purified through a Superdex 75 HR 10/30 column after digestion of the purified fusion peptide with enterokinase. The expressed protein evarsigmaA has the same molecular weight as virion protein varsigmaA purified from ARV-infected CEF and is indistinguishable from virion protein varsigmaA by immunoblot analysis. The evarsigmaA binds cooperatively alpha (32)P-labeled dsRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reaction is blocked by homologous ARV dsRNA or heterologous infectious bursal disease virus dsRNA and poly(rI)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein evarsigmaA has dsRNA-binding activity similar to that of varsigmaA obtained from infected cells, and its binding is sequence-independent.
authors.affiliation: Department of Veterinary Medicine, National Chung Hsing University, Taichung, 403, Taiwan.
authors.forename: H S
authors.initials: HS
authors.lastname: Yin
authors.forename: J H
authors.initials: JH
authors.lastname: Shien
authors.forename: L H
authors.initials: LH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018165:Orthoreovirus / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C403292:sigmaA protein, avian reovirus; D012965:Sodium Chloride