Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein μNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication.