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Journal of Virology, February 2001, p. 1459-1475, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1459-1475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reovirus sigma NS Protein Is Required for Nucleation of Viral Assembly Complexes and Formation of Viral Inclusions

Michelle M. Becker,1,2 Mehmet I. Goral,1,2,3 Paul R. Hazelton,4 Geoffrey S. Baer,1,2 Steven E. Rodgers,1,2 Earl G. Brown,5 Kevin M. Coombs,4 and Terence S. Dermody1,2,3,*

Departments of Microbiology and Immunology1 and Pediatrics3 and Elizabeth B. Lamb Center for Pediatric Research,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba,4 and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario,5 Canada

Received 12 June 2000/Accepted 26 October 2000

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigma NS and core protein µ2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigma NS and µ2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigma NS and µ2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigma NS-mutant virus tsE320, sigma NS is distributed diffusely in the cytoplasm and µ2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with µ2-mutant virus tsH11.2, µ2 is distributed diffusely in the cytoplasm and the nucleus. However, sigma NS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigma NS precedes µ2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigma NS nucleate sites of viral replication to which other viral proteins, including µ2, are recruited to commence dsRNA synthesis.

* Corresponding author. Mailing address: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723. E-mail: terry.dermody{at}mcmail.vanderbilt.edu.

Journal of Virology, February 2001, p. 1459-1475, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1459-1475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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