Binding to selected regions of reovirus mRNAs by a nonstructural reovirus protein

When assembled into 13--19S particles, the reovirus nonstructural protein sigma-NS selectively binds single-stranded RNAs. Sedimentation analyses combined with binding to nitrocellulose membrane filters showed that 1--2 pmol of reovirus mRNAs from the large, medium, or small size classes saturated in vitro the binding site(s) on 13--19S particles containing 100 pmol of sigma-NS. All mRNA segments in each size class bound to particles, and no mRNAs in one size class excluded the binding of mRNAs in any other class. In competition experiments, the maximal binding of all reovirus mRNAs to particles of sigma-NS was achieved when medium and small mRNAs were bound before the large mRNAs. This preferred order of addition of mRNAs to sigma-NS resulted in a marked increase in the size of some of the complexes. This finding suggests that the addition of large mRNAs last to particles promoted the formation of complexes with more than one RNA segment bound per particle. The 13--19S particles of sigma-NS protected 20- to 40-nucleotide RNA fragments from nuclease digestion. At least one of the protected fragments from mRNAs of each size class included the 3' terminus; the remaining were from internal regions of the mRNAs. The protected RNA fragments rebound to particles during a second or third cycle of binding in a configuration in which they were fully protected from nuclease digestion. We conclude that binding of particles of sigma-NS to reovirus mRNAs was not at random sites but was to specific regions unique for members of each size class.