Reovirus sigma NS protein localizes to inclusions through an association requiring the mu NS amino terminus.

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 muNS 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, sigmaNS. Although sigmaNS colocalized with muNS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of muNS. When coexpressed with muNS, sigmaNS was redistributed and colocalized with muNS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of sigmaNS to viral factories in infected cells. We have previously shown that muNS residues 1 to 40 or 41 are both necessary and sufficient for muNS association with the viral microtubule-associated protein mu2. In the present study we found that this same region of muNS is required for its association with sigmaNS. We further dissected this region, identifying residues 1 to 13 of muNS as necessary for association with sigmaNS, but not with mu2. Deletion of sigmaNS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of sigmaNS with muNS. Furthermore, when treated with RNase, a large portion of muNS was released from muNS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that muNS plays a key role in forming the reovirus factories and recruiting other components to them.