Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection

Abstract

ABSTRACT Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, σNS and μNS, and structural protein σ3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed σNS, μNS, and σ3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, σNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed σNS is functional as it complements the defect in temperature-sensitive, σNS-mutant virus ts E320. In both transfected and infected cells, μNS is found in punctate cytoplasmic structures and σ3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of μNS and σ3 is not altered when the proteins are expressed together or with σNS. However, when expressed with μNS, σNS colocalizes with μNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both σNS and μNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, σNS interacts with μNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that σNS and μNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.