Synthesis in Escherichia coli of avian reovirus core protein varsigmaA and its dsRNA-binding activity.
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.
Yin
H S
HS
Department of Veterinary Medicine, National Chung Hsing University, Taichung, 403, Taiwan.
Shien
J H
JH
Lee
L H
LH
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*
D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C403292:sigmaA protein, avian reovirus; D012965:Sodium Chloride