Mutant cells selected during persistent reovirus infection do not express mature cathepsin L and do not support reovirus disassembly

Geoffrey S. Baer, Daniel Ebert, Chia J. Chung, Ann H. Erickson, Terence S. Dermody

Research output: Contribution to journalArticle

Abstract

Persistent reovirus infections of murine L929 cells select cellular mutations that inhibit viral disassembly within the endocytic pathway. Mutant cells support reovirus growth when infection is initiated with infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly formed following proteolysis of viral outer-capsid proteins. However, mutant cells do not support growth of virions, indicating that these cells have a defect in virion-to-ISVP processing. To better understand mechanisms by which viruses use the endocytic pathway to enter cells, we defined steps in reovirus replication blocked in mutant cells selected during persistent infection. Subcellular localization of reovirus after adsorption to parental and mutant cells was assessed using confocal microscopy and virions conjugated to a fluorescent probe. Parental and mutant cells did not differ in the capacity to internalize virions or distribute them to perinuclear compartments. Using pH-sensitive probes, the intravesicular pH was determined and found to be equivalent in parental and mutant cells. In both cell types, virions localized to acidified intracellular organelles. The capacity of parental and mutant cells to support proteolysis of reovirus virions was assessed by monitoring the appearance of disassembly intermediates following adsorption of radiolabeled viral particles. Within 2 h after adsorption to parental cells, proteolysis of viral outer-capsid proteins was observed, consistent with formation of ISVPs. However, in mutant cells, no proteolysis of viral proteins was detected up to 8 h postadsorption. Since treatment of cells with E64, an inhibitor of cysteine-containing proteases, blocks reovirus disassembly, we used immunoblot analysis to assess the expression of cathepsin L, a lysosomal cysteine protease. In contrast to parental cells, mutant cells did not express the mature, proteolytically active form of the enzyme. The defect in cathepsin L maturation was not associated with mutations in procathepsin L mRNA, was not complemented by procathepsin L overexpression, and did not affect the maturation of cathepsin B, another lysosomal cysteine protease. These findings indicate that persistent reovirus infections select cellular mutations that affect the maturation of cathepsin L and suggest that alterations in the expression of lysosomal proteases can modulate viral cytopathicity.

Original languageEnglish (US)
Pages (from-to)9532-9543
Number of pages12
JournalJournal of Virology
Volume73
Issue number11
StatePublished - 1999
Externally publishedYes

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Reoviridae Infections
cathepsin L
Cathepsin L
Reoviridae
mutants
infection
virion
Virion
cells
proteolysis
Proteolysis
Adsorption
adsorption
Cysteine Proteases
cysteine proteinases
Capsid Proteins
coat proteins
mutation
Mutation
proteinases

ASJC Scopus subject areas

  • Immunology

Cite this

Mutant cells selected during persistent reovirus infection do not express mature cathepsin L and do not support reovirus disassembly. / Baer, Geoffrey S.; Ebert, Daniel; Chung, Chia J.; Erickson, Ann H.; Dermody, Terence S.

In: Journal of Virology, Vol. 73, No. 11, 1999, p. 9532-9543.

Research output: Contribution to journalArticle

Baer, Geoffrey S. ; Ebert, Daniel ; Chung, Chia J. ; Erickson, Ann H. ; Dermody, Terence S. / Mutant cells selected during persistent reovirus infection do not express mature cathepsin L and do not support reovirus disassembly. In: Journal of Virology. 1999 ; Vol. 73, No. 11. pp. 9532-9543.
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AU - Dermody, Terence S.

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