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Journal of Virology, August 2002, p. 8285-8297, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8285-8297.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Teresa J. Broering,1,2 John S. L. Parker,1 Patricia L. Joyce,2,{dagger} Jonghwa Kim,1,2 and Max L. Nibert1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 537062

Received 28 February 2002/ Accepted 9 May 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 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 (T3DN). 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 T3DN 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 T3DN 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.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-4828. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu.

{dagger} Present address: Department of Radiation Oncology, University of North Carolina—Chapel Hill, Chapel Hill, NC 27599.

Journal of Virology, August 2002, p. 8285-8297, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8285-8297.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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