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Journal of Virology, February 2004, p. 1882-1892, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.1882-1892.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Reovirus Nonstructural Protein µNS Recruits Viral Core Surface Proteins and Entering Core Particles to Factory-Like Inclusions

Teresa J. Broering,1 Jonghwa Kim,1,2 Cathy L. Miller,1 Caroline D. S. Piggott,1,3 Jason B. Dinoso,1 Max L. Nibert,1* and John S. L. Parker1*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706,2 Harvard College Research Program, Harvard University, Cambridge, Massachusetts 021383

Received 30 July 2003/ Accepted 28 October 2003

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein µNS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with µNS. We found that all three of these proteins—{lambda}1, {lambda}2, and {sigma}2—localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with µNS, on the other hand, each core surface protein colocalized with µNS in globular inclusions, supporting the initial hypothesis. We also found that {lambda}1, {lambda}2, and {sigma}2 each localized to filamentous inclusions formed upon the coexpression of µNS and µ2, a structurally minor core protein that associates with microtubules. The first 40 residues of µNS, which are required for association with µ2 and the RNA-binding nonstructural protein {sigma}NS, were not required for association with any of the three core surface proteins. When coexpressed with µ2 in the absence of µNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with µ2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of µNS that had been preformed in those cells, providing evidence that µNS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins µNS and µ2 in this process.

* Corresponding author. Mailing address for Max L. Nibert: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 645-3680. Fax: (617) 738-7664. E-mail: max_nibert{at}hms.harvard.edu. Present address for John S. L. Parker: James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853. Phone: (607) 256-5626. Fax: (607) 256-5608. E-mail: jsp7{at}cornell.edu.

Journal of Virology, February 2004, p. 1882-1892, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.1882-1892.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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