Mammalian Reovirus Nonstructural Protein mu NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein mu 2 in Transfected Cells

• Published in: Journal of virology 2002-08, Vol.76 (16), p.8285-8297
• Main Authors: Broering, T J, Parker, JSL, Joyce, P L, Kim, J, Nibert, M L
• Format: Article
• Language: English
• ISSN: 0022-538X
• DOI: 10.1128/JVI.76.16.8285-8297.2002

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 mu NS 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 super(N)). In the transfected cells, the formation of mu NS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller mu NS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of mu NS and reovirus protein mu 2 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 mu NS and mu 2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the mu 2 protein from T3D super(N) also colocalized with mu NS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing mu NS and mu 2 protein from T3D super(N) and the filamentous structures containing mu NS and mu 2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the mu 2-encoding M1 genome segment. We found that the first 40 amino acids of mu NS are required for colocalization with mu 2 but not for inclusion formation. Similarly, a fusion of mu NS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the mu 2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between mu NS and mu NSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of mu NS. The capacity of mu NS to form inclusions and to colocalize with mu 2 in transfected cells suggests a key role for mu NS in forming viral factories in reovir