Reovirus Nonstructural Protein Sigma Ns Recruits Viral Rna To Replication Organelles

The function of the mammalian orthoreovirus (reovirus) sigma NS nonstructural protein is enigmatic. sigma NS 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 sigma NS-RNA binding in reovirus replication, we engineered sigma NS 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 sigma NS, we established a complementation system in which wild-type or mutant forms of sigma NS could be tested for the capacity to overcome inhibition of sigma NS expression. Mutations in sigma NS that disrupt RNA binding also diminish viral replication and sigma NS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein mu NS) when sigma NS is present and capable of binding RNA. Collectively, these findings indicate that sigma NS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for sigma NS in reovirus replication.IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus mu NS proteins assemble into exclusive factory scaffolds that require reovirus sigma NS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for sigma NS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.