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.
Highlights
Spherical viral capsids assemble by two distinct mechanisms [1]
The genomic RNA footprint on the capsid proteins within the virion was determined by an RNA crosslinking and peptide mapping assay (RCAP), used previously to map the interaction between the capsid and the nucleic acid of brome mosaic virus [63]
RNA and RNA–peptide complexes were precipitated with lithium chloride, the cross-links were reversed and the released peptides were identified by MALDI-ToF mass spectrometry (Fig. 2a)
Summary
Spherical viral capsids assemble by two distinct mechanisms [1]. Many double-stranded DNA (http://creativecommons.org/licenses/by/4.0/).J Mol Biol (2016) 428, 431–448Packaging Signal-Mediated Bacteriophage MS2 Assembly phages and Herpesviruses form a protein-only shell of coat and scaffold proteins, a pro-capsid, into which the genome is actively pumped via specialzsed energy-requiring enzymes as the scaffold proteins are removed. The free energy for this process has been thought to come mostly from favorable electrostatic interactions between the negatively charged RNA and positively charged residues and domains in the CPs [3,4,5,6,7,8,9,10,11,12] Adding to this view are the observations from in vitro reassembly experiments showing that many viral CPs can assemble into virus-like particles in the absence of RNA, in the presence of non-cognate RNAs, or even anionic polymers. Virions from natural infections predominantly encapsidate their cognate genomes, with only minor misincorporation of cellular or degraded RNAs [18,19] These outcomes hint at more complex regulation of the assembly process
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