Reovirus σNS and μNS proteins form cytoplasmic inclusion structures in the absence of viral infection

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusionsi Two viral nonstructural proteins, sigmaNS and muNS, and structural protein sigma3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed sigmaNS, muNS, and sigma3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, sigmaNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed sigmaNS is functional as it complements the defect in temperature-sensitive, sigmaNS-mutant virus tsE320. In both transfected and infected cells, muNS is found in punctate cytoplasmic structures and sigma3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of muNS and sigma3 is not altered when the proteins are expressed together or with sigmaNS. However, when expressed with sigmaNS, sigmaNS colocalizes with sigmaNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both sigmaNS and muNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, sigmaNS interacts with muNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that sigmaNS and muNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.