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Journal of Virology, October 2004, p. 10291-10302, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10291-10302.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Increased Ubiquitination and Other Covariant Phenotypes Attributed to a Strain- and Temperature-Dependent Defect of Reovirus Core Protein µ2

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston,¹ Harvard College Research Program,³ Ph.D. Program in Virology, Division of Medical Sciences, Harvard University, Cambridge, Massachusetts,⁴ James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York²

Received 1 March 2004/ Accepted 18 May 2004

Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the µ2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the µ2-encoding M1 genome segment, although contributions by the 3- and 2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated µ2 (Ub-µ2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by µ2 from globular strains but not with microtubules coated by µ2 from filamentous strains, and immunoprecipitations revealed that µ2 from globular strains displayed higher levels of Ub-µ2. Allelic changes at µ2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their µ2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that µ2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-µ2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.

Supplemental material for this article may be found at http://jvi.asm.org/.

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