title: Reovirus variants selected for resistance to ammonium chloride have mutations in viral outer-capsid protein sigma3.
abstract: Mammalian reoviruses 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 sigma3 protein and proteolytic cleavage of the mu1/mu1C 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 sigma3-encoding S4 gene. The deduced sigma3 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 sigma3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant sigma3 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 sigma3 demonstrated native capsid protein organization and minimal alterations in sigma3 structure. These results suggest that mutations in sigma3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.
authors.affiliation: Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37241, USA.
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Clark
authors.forename: J Denise
authors.initials: JD
authors.lastname: Wetzel
authors.forename: Yingqi
authors.initials: Y
authors.lastname: Gu
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Ebert
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: McAbee
authors.forename: Emily K
authors.initials: EK
authors.lastname: Stoneman
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Baer
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Zhu
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Wilson
authors.forename: B V V
authors.initials: BV
authors.lastname: Prasad
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
mesh_terms: D000222:Adaptation, Physiological; D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000235:genetics; D007739:L Cells / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D012087:Reoviridae / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D012692:Serial Passage; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D015853:Cysteine Proteinase Inhibitors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C031657:sigma NS protein, Reovirus; D000643:Ammonium Chloride; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse