title: Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2.
abstract: Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)-RNA and maturation protein (MP)-RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Óttar
authors.initials: Ó
authors.lastname: Rolfsson
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA; The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Stefani
authors.initials: S
authors.lastname: Middleton
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Iain W
authors.initials: IW
authors.lastname: Manfield
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: White
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Baochang
authors.initials: B
authors.lastname: Fan
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Vaughan
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Neil A
authors.initials: NA
authors.lastname: Ranson
authors.affiliation: Department of Biology and Mathematics and York Centre for Complex Systems Analysis, University of York, York YO10 5DD, United Kingdom.
authors.forename: Eric
authors.initials: E
authors.lastname: Dykeman
authors.affiliation: Department of Biology and Mathematics and York Centre for Complex Systems Analysis, University of York, York YO10 5DD, United Kingdom.
authors.forename: Reidun
authors.initials: R
authors.lastname: Twarock
authors.affiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.
authors.forename: James
authors.initials: J
authors.lastname: Ford
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: C Cheng
authors.initials: CC
authors.lastname: Kao
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Stockley
mesh_terms: D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D017909:Levivirus / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; C096923:maturation protein, Enterobacterio phage MS2
keywords: RNA virus; RNA–protein interaction; assembly mechanism; packaging signals