Amino terminus of reovirus nonstructural protein sigma NS is important for ssRNA binding and nucleoprotein complex formation.
Reovirus nonstructural protein sigma NS exhibits a ssRNA-binding activity thought to be involved in assembling the reovirus mRNAs for genome replication and virion morphogenesis. To extend analysis of this activity, recombinant sigma NS (r sigma NS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, r sigma NS was found in large complexes (> or = 30 S) that were disassembled into smaller, 13-19 S complexes upon treatment with RNase A. R sigma NS also bound to poly(A)-Sepharose beads both before and after purification. Treatment with high salt during purification caused r sigma NS to sediment in even smaller, 7-9 S complexes, consistent with more complete loss of RNA. To localize the RNA-binding site, limited proteolysis was used to fragment the r sigma NS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of r sigma NS, and the resulting protein no longer bound to poly(A). In addition, when r sigma NS in cell extracts was treated with thermolysin to generate the amino-terminally truncated from, it sedimented at 7-9 S, also consistent with the loss of RNA-binding capacity. To confirm these findings, a deletion mutant lacking amino acids 2-11 was constructed and expressed in insect cells from a recombinant baculovirus. The mutant protein in cell extracts showed greatly reduced poly(A)-binding activity and sedimented as 7-9 S complexes. These data suggest that the first 11 amino acids of sigma NS, which are predicted to form an amphipathic alpha-helix, are important for both ssRNA binding and formation of complexes larger than 7-9 S.
Gillian
A L
AL
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison 53706, USA.
Nibert
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites; D002460:Cell Line; D007739:L Cells; D012089:Mammalian orthoreovirus 3 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000378:metabolism*; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012087:Reoviridae / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D009698:Nucleoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C031657:sigma NS protein, Reovirus