Reovirus σNS Protein Localizes to Inclusions through an Association Requiring the μNS Amino Terminus

Abstract

ABSTRACT Cells infected with mammalian reoviruses contain phase-dense inclusions, called viral factories, in which viral replication and assembly are thought to occur. The major reovirus nonstructural protein μNS forms morphologically similar phase-dense inclusions when expressed in the absence of other viral proteins, suggesting it is a primary determinant of factory formation. In this study we examined the localization of the other major reovirus nonstructural protein, σNS. Although σNS colocalized with μNS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of μNS. When coexpressed with μNS, σNS was redistributed and colocalized with μNS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of σNS to viral factories in infected cells. We have previously shown that μNS residues 1 to 40 or 41 are both necessary and sufficient for μNS association with the viral microtubule-associated protein μ2. In the present study we found that this same region of μNS is required for its association with σNS. We further dissected this region, identifying residues 1 to 13 of μNS as necessary for association with σNS, but not with μ2. Deletion of σNS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of σNS with μNS. Furthermore, when treated with RNase, a large portion of σNS was released from μNS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that μNS plays a key role in forming the reovirus factories and recruiting other components to them.