摘要
The genome segment S2 of avian 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 σA, 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 σA contains a carboxy-terminal region (one-fourth of the protein) that is formed from α-helices and β-turns, and the remainder (three-fourths of the protein) is formed predominantly from β-strands and β-turns. Analysis of binding activity to poly(rl)-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 σA was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chelate affinity chromatography. The expressed protein eσA was further purified through a Superdex 75 HR 10/30 column after digestion of the purified fusion peptide with enterokinase. The expressed protein eσA has the same molecular weight as virion protein σA purified from ARV-infected CEF and is indistinguishable from virion protein σA by immunoblot analysis. The eσA binds cooperatively α 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(rl)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein eσA has dsRNA-binding activity similar to that of σA obtained from infected cells, and its binding is sequence-independent.