Mammalian reovirus nonstructural protein RNS forms large inclusions and colocalizes with reovirus microtubule-associated protein μ2 in transfected cells

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 muNS 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 muNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller muNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of muNS and reovirus protein mu2 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 muNS and mu2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the mu2 protein from T3D(N) also colocalized with muNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing muNS and mu2 protein from T3D(N) and the filamentous structures containing muNS and mu2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the mu2-encoding M1 genome segment. We found that the first 40 amino acids of muNS are required for colocalization with mu2 but not for inclusion formation. Similarly, a fusion of muNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the mu2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between muNS and muNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of muNS. The capacity of muNS to form inclusions and to colocalize with mu2 in transfected cells suggests a key role for muNS in forming viral factories in reovirus-infected cells.