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Journal of Virology, April 2001, p. 3197-3206, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3197-3206.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Adaptation of Reovirus to Growth in the Presence of Protease Inhibitor E64 Segregates with a Mutation in the Carboxy Terminus of Viral Outer-Capsid Protein sigma 3

Daniel H. Ebert,1,2 J. Denise Wetzel,2,3 David E. Brumbaugh,2,3 Stacey R. Chance,2,3 Laura E. Stobie,2,3 Geoffrey S. Baer,1,2 and Terence S. Dermody1,2,3,*

Departments of Microbiology and Immunology1 and Pediatrics3 and Elizabeth B. Lamb Center for Pediatric Research,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 27 June 2000/Accepted 4 January 2001

Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of sigma 3 protein and proteolytic cleavage of µ1/µ1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 µM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein sigma 3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of sigma 3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of sigma 3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in sigma 3 protein containing amino acid 354 influences susceptibility of sigma 3 to proteolysis during reovirus disassembly.

* Corresponding author. Mailing address: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723. E-mail: terry.dermody{at}mcmail.vanderbilt.edu.

Journal of Virology, April 2001, p. 3197-3206, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3197-3206.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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