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Journal of Virology, September 2006, p. 9053-9063, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00276-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Gene-Specific Inhibition of Reovirus Replication by RNA Interference

Takeshi Kobayashi,1,2 James D. Chappell,1,2,3 Pranav Danthi,1,2 and Terence S. Dermody1,2,4*

Departments of Pediatrics,1 Pathology,3 Microbiology and Immunology,4 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 372322

Received 7 February 2006/ Accepted 3 July 2006

Mammalian reoviruses contain a genome of 10 segments of double-stranded RNA (dsRNA). Reovirus replication and assembly occur within distinct structures called viral inclusions, which form in the cytoplasm of infected cells. Viral nonstructural proteins µNS and {sigma}NS and core protein µ2 play key roles in forming viral inclusions and recruiting other viral proteins and RNA to these structures for replication and assembly. However, the precise functions of these proteins in viral replication are poorly defined. Therefore, to better understand the functions of reovirus proteins associated with formation of viral inclusions, we used plasmid-based vectors to establish 293T cell lines stably expressing small interfering RNAs (siRNAs) specific for transcripts encoding the µ2, µNS, and {sigma}NS proteins of strain type 3 Dearing (T3D). Infectivity assays revealed that yields of T3D, but not those of strain type 1 Lang, were significantly decreased in 293T cells stably expressing µ2, µNS, or {sigma}NS siRNA. Stable expression of siRNAs specific for any one of these proteins substantially diminished viral dsRNA, protein synthesis, and inclusion formation, indicating that each is a critical component of the viral replication machinery. Using cell lines stably expressing µNS siRNA, we developed a complementation system to rescue viral replication by transient transfection with recombinant T3D µNS in which silent mutations were introduced into the sequence targeted by the µNS siRNA. Furthermore, we demonstrated that µNSC, which lacks the first 40 amino residues of µNS, is incapable of restoring reovirus growth in the complementation system. These results reveal interdependent functions for viral inclusion proteins and indicate that cell lines stably expressing reovirus siRNAs are useful tools for the study of viral protein structure-function relationships.

* 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, September 2006, p. 9053-9063, Vol. 80, No. 18
0022-538X/06/$08.00+0     doi:10.1128/JVI.00276-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kobayashi, T., Ooms, L. S., Chappell, J. D., Dermody, T. S. (2009). Identification of Functional Domains in Reovirus Replication Proteins {micro}NS and {micro}2. J. Virol. 83: 2892-2906 [Abstract] [Full Text]  


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