Characterization of the double-stranded RNA genome segment S3 of avian reovirus

The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein sigmaB, one of the major outer capsid proteins of avian reovirus (Mr 41471). Protein sigmaB was subsequently expressed in Escherichia coli. The expressed protein sigmaB was indistinguishable from virion protein sigmaB as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphyloccus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3'-terminus of their plus strands. Amino acid sequence analysis revealed that ARV sigmaB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of sigmaB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.