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Journal of Virology, May 2003, p. 5948-5963, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5948-5963.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Reovirus {sigma}NS and µNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection

Michelle M. Becker,1,2 Timothy R. Peters,2,3 and Terence S. Dermody1,2,3*

Departments of Microbiology and Immunology,1 Pediatrics,2 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 372323

Received 19 August 2002/ Accepted 24 February 2003

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, {sigma}NS and µNS, and structural protein {sigma}3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed {sigma}NS, µNS, and {sigma}3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, {sigma}NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed {sigma}NS is functional as it complements the defect in temperature-sensitive, {sigma}NS-mutant virus tsE320. In both transfected and infected cells, µNS is found in punctate cytoplasmic structures and {sigma}3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of µNS and {sigma}3 is not altered when the proteins are expressed together or with {sigma}NS. However, when expressed with µNS, {sigma}NS colocalizes with µNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both {sigma}NS and µNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, {sigma}NS interacts with µNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that {sigma}NS and µNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

* 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}vanderbilt.edu.

Journal of Virology, May 2003, p. 5948-5963, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.5948-5963.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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