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Journal of Virology, October 2002, p. 9832-9843, Vol. 76, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.19.9832-9843.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Single Mutation in the Carboxy Terminus of Reovirus Outer-Capsid Protein {sigma}3 Confers Enhanced Kinetics of {sigma}3 Proteolysis, Resistance to Inhibitors of Viral Disassembly, and Alterations in {sigma}3 Structure

Gregory J. Wilson,1,2* Emma L. Nason,3 Charles S. Hardy,1,2 Daniel H. Ebert,2,4 J. Denise Wetzel,1,2 B. V. Venkataram Prasad,3 and Terence S. Dermody1,2,4

Departments of Pediatrics,1 Microbiology and Immunology,4 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,2 Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 770303

Received 16 October 2001/ Accepted 3 June 2002

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein {sigma}3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in {sigma}3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the {sigma}3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant {sigma}3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced {sigma}3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in {sigma}3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant {sigma}3 proteins revealed structural alterations in {sigma}3 that correlate with the Y354H mutation. These results indicate that a single mutation in {sigma}3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of {sigma}3 cleavage during reovirus disassembly.

* Corresponding author. Mailing address: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-5731. Fax: (615) 343-9723. E-mail: greg.wilson{at}mcmail.vanderbilt.edu.

Journal of Virology, October 2002, p. 9832-9843, Vol. 76, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.19.9832-9843.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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