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 activ ity thought to be involved in assembling the reovirus mRNAs for genome replication and Virion morphogenesis. To extend analysis of this acti vity, 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 (greater than or equal to 30 S) that wer e disassembled into smaller, 13-19 S complexes upon treatment with RNa se A. R sigma NS also bound to poly(A)Sepharose beads both before and after purification. Treatment with high salt during purification cause d 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, limi ted proteolysis was used to fragment the r sigma NS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amin o 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 wi th thermolysin to generate the amino-terminally truncated form, it sed imented at 7-9 S, also consistent with the loss of RNA-binding capacit y. To confirm these findings, a deletion mutant lacking amino acids 2- 11 was constructed and expressed in insect cells from a recombinant ba culovirus. 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 predicte d to form an amphipathic alpha-helix, are important for both ssRNA bin ding and formation of complexes larger than 7-9 S. (C) 1998 Academic P ress.