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Journal of Virology, May 2005, p. 6194-6206, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6194-6206.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Carboxyl-Proximal Regions of Reovirus Nonstructural Protein µNS Necessary and Sufficient for Forming Factory-Like Inclusions

Teresa J. Broering ,^1,2,, Michelle M. Arnold,^1,3, Cathy L. Miller,¹ Jessica A. Hurt,⁴ Patricia L. Joyce,^2, and Max L. Nibert^1,3,4*

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,¹ Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706,² Ph.D. Programs in Virology,³ Biological and Biomedical Sciences, Division of Medical Sciences, Harvard University, Cambridge, Massachusetts 02138⁴

Received 1 September 2004/ Accepted 14 January 2005

Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein µNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of µNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue µNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of µNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of µNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in µNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across µNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of µNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.

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* Corresponding author. Mailing address: 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: mnibert{at}hms.harvard.edu.

T.J.B. and M.M.A. contributed equally to this study.

Present address: Massachusetts Biologic Laboratories, Jamaica Plain, MA 02130.

Present address: Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

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Journal of Virology, May 2005, p. 6194-6206, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6194-6206.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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