Reovirus variants selected for resistance to ammonium chloride have mutations in viral outer-capsid protein σ3

Kimberly M. Clark, J. Denise Wetzel, Yingqi Gu, Daniel Ebert, Stephanie A. McAbee, Emily K. Stoneman, Geoffrey S. Baer, Yuwei Zhu, Gregory J. Wilson, B. V V Prasad, Terence S. Dermody

Research output: Contribution to journalArticle

Abstract

Mammalian reovirases are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the σ3 protein and proteolytic cleavage of the μ1/μ1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the σ3-encoding S4 gene. The deduced σ3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of σ3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant σ3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of σ3 demonstrated native capsid protein organization and minimal alterations in σ3 structure. These results suggest that mutations in σ3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.

Original languageEnglish (US)
Pages (from-to)671-681
Number of pages11
JournalJournal of Virology
Volume80
Issue number2
DOIs
Publication statusPublished - Jan 2006
Externally publishedYes

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ASJC Scopus subject areas

  • Immunology

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