Mammalian Reovirus Nonstructural Protein μNS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein μ2 in Transfected Cells

Abstract

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these “viral factories” are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein μNS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3D^N). In the transfected cells, the formation of μNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller μNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of μNS and reovirus protein μ2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing μNS and μ2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the μ2 protein from T3D^N also colocalized with μNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing μNS and μ2 protein from T3D^N and the filamentous structures containing μNS and μ2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the μ2-encoding M1 genome segment. We found that the first 40 amino acids of μNS are required for colocalization with μ2 but not for inclusion formation. Similarly, a fusion of μNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the μ2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between μNS and μNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of μNS. The capacity of μNS to form inclusions and to colocalize with μ2 in transfected cells suggests a key role for μNS in forming viral factories in reovirus-infected cells.