Synthesis in Escherichia coli of avian reovirus core protein sigma A and its dsRNA-binding activity

The genome segment S2 of avian reovirus (ARV) S1133 was cloned and sequence d. The entire S2 nucleotide sequence is 1325 bp long with one long open rea ding frame that encodes a protein of 415 amino acids, corresponding to sigm a A, 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 sigma A contains a carboxy-terminal region (one-fou rth of the protein) mat is formed from alpha-helices and beta-turns, and th e remainder (three-fourths of the protein) is formed predominantly from bet a-strands and beta-turns. Analysis of binding activity to poly(rl)-poly(rC) -agarose suggested that ARV protein A present in total virus-infected chick en embryo fibroblasts (CEF) had dsRNA-binding activity. To further characte rize the binding activity, protein sigma A was subsequently expressed in Es cherichia coli BL21(DE3) cells as a fusion protein and isolated by metal ch ef ate affinity chromatography. The expressed protein e sigma A was further purified through a Superdex 75 HR 10/30 column after digestion of me purif ied fusion peptide with enterokinase. The expressed protein e sigma A has t he same molecular weight as virion protein sigma A purified from ARV-infect ed CEF and is indistinguishable from virion protein sigma A by immunoblot a nalysis. The e sigma A binds cooperatively alpha P-32-labeled dsRNA probe p roduced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reacti on is blocked by homologous ARV dsRNA or heterologous infectious bursal dis ease virus dsRNA and poly(rl)-poly(rC), but not by salmon sperm DNA. The re sults indicate that the expressed protein e sigma A has dsRNA-binding activ ity similar to that of sigma A obtained from infected cells, and its bindin g is sequence-independent. (C) 2000 Academic Press.