Amino terminus of reovirus nonstructural protein σNS is important for ssRNA binding and nucleoprotein complex formation

Abstract

Reovirus nonstructural protein σNS 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 σNS (rσNS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, rσNS was found in large complexes (≤30 S) that were disassembled into smaller, 13-19 S complexes upon treatment with RNase A. RσNS also bound to poly(A)-Sepharose beads both before and after purification. Treatment with high salt during purification caused rσNS 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 rσNS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of rσNS, and the resulting protein no longer bound to poly(A). In addition, when rσNS in cell extracts was treated with thermolysin to generate the amino- terminally truncated form, 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 σNS, which are predicted to form an amphipathic α-helix, are important for both ssRNA binding and formation of complexes larger than 7-9 S.