CHARACTERIZATION OF THE DOUBLE-STRANDED-RNA GENOME SEGMENT S3 OF AVIAN REOVIRUS

The double-stranded RNA genome segment S3 of avian reovirus (ARV) S113 3 was cloned following polyadenylation of both strands and cDNA synthe sis of S3 RNA. The complete segment S3 nucleotide sequence was determi ned. 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 si ze as the known S3 gene product, protein sigma B, one of the major out er capsid proteins of avian reovirus (M-r 41471). Protein sigma B was subsequently expressed in Escherichia coli. The expressed protein sigm a B was indistinguishable from Virion protein sigma B as judged by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot ass ay, and N-terminal amino acid sequencing of several peptides generated by Staphylococcus aureus Vg protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus stra nd. The pentanacleotide sequence is common to the other genome segment SI 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 sigma B does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino ac id sequencing suggest that the most susceptible cleavage sites of sigm a B to V8 protease are located in a hydrophilic area between amino aci ds 95 and 140. (C) 1997 Elsevier Science B.V.