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J Mol Biol. 2016 Jan 29;428(2 Pt B):431-48. doi: 10.1016/j.jmb.2015.11.014. Epub 2015 Dec 1.

Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2.

Author information

1
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
2
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA; The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.
3
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
4
Department of Biology and Mathematics and York Centre for Complex Systems Analysis, University of York, York YO10 5DD, United Kingdom.
5
The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.

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.

KEYWORDS:

RNA virus; RNA–protein interaction; assembly mechanism; packaging signals

PMID:
26608810
PMCID:
PMC4751978
DOI:
10.1016/j.jmb.2015.11.014
[Indexed for MEDLINE]
Free PMC Article

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