Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2

Óttar Rolfsson*, Stefani Middleton, Iain W. Manfield, Simon J. White, Baochang Fan, Robert Vaughan, Neil A. Ranson, Eric Dykeman, Reidun Twarock, James Ford, C. Cheng Kao, Peter G. Stockley

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)431-448
Number of pages18
JournalJournal of Molecular Biology
Volume428
Issue number2
DOIs
Publication statusPublished - 29 Jan 2016

Bibliographical note

Publisher Copyright:
© 2015 The Authors.

Other keywords

  • assembly mechanism
  • packaging signals
  • RNA virus
  • RNA-protein interaction

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