Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region.
The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity.
Miller
J E
JE
Department of Biological Sciences, University of California, Santa Barbara 93106.
Samuel
C E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D036022:Capsid Proteins; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012087:Reoviridae / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014771:Virion / Q000737:chemistry / Q000378:metabolism
D036022:Capsid Proteins; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C053179:sigma protein 3, Reovirus; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase