title: Channel-Inactivating Mutations and Their Revertant Mutants in the Envelope Protein of Infectious Bronchitis Virus.
abstract: It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led toin vivoattenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein.IMPORTANCEThe ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Janet
authors.initials: J
authors.lastname: To
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Wahyu
authors.initials: W
authors.lastname: Surya
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: To Sing
authors.initials: TS
authors.lastname: Fung
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castelló, Spain.
authors.forename: Carmina
authors.initials: C
authors.lastname: Verdià-Bàguena
authors.affiliation: Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castelló, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Queralt-Martin
authors.affiliation: Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castelló, Spain.
authors.forename: Vicente M
authors.initials: VM
authors.lastname: Aguilella
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore dxliu0001@163.com jtorres@ntu.edu.sg.
authors.forename: Ding Xiang
authors.initials: DX
authors.lastname: Liu
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore dxliu0001@163.com jtorres@ntu.edu.sg.
authors.forename: Jaume
authors.initials: J
authors.lastname: Torres
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D001351:Infectious bronchitis virus / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008564:Membrane Potentials; D009154:Mutation; D055503:Protein Multimerization; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D057074:Virus Release
chemical_list: D007473:Ion Channels; D014759:Viral Envelope Proteins
keywords: NMR structure; channel activity; coronavirus; envelope protein; infectious bronchitis virus; small membrane protein