Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2

Area of Science:

• Molecular Virology
• Structural Biology
• Biochemistry

Background:

• Bacteriophage MS2 is a model system for studying viral assembly.
• Understanding protein-RNA interactions is crucial for viral particle formation.
• Previous studies predicted RNA packaging signals (PSs) but lacked direct interface data.

Purpose of the Study:

• To determine the precise peptide and oligonucleotide sequences at the capsid protein (CP)-RNA and maturation protein (MP)-RNA interfaces in bacteriophage MS2.
• To elucidate the mechanism of MS2 virion assembly, specifically the role of packaging signals (PSs).
• To compare RNA secondary structures in protein-free states versus within the virion.

Main Methods:

• Cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS).
• Cross-linking immunoprecipitation sequencing (CLIP-Seq).
• Chemical RNA footprinting.

Main Results:

• Identified conserved CP-RNA and MP-RNA interfaces within every MS2 viral particle.
• Observed that CP-binding sites span the viral genome, consistent with predicted packaging signals (PSs).
• Found that some PSs require RNA refolding from their solution structures to form virion contacts; MP RNA-binding sites are in the N-terminal half, with conserved C-termini involved in pilin binding.

Conclusions:

• MS2 virion assembly is mediated by a packaging signal (PS)-dependent mechanism.
• Viral protein interactions with RNA involve an induced-fit process.
• The study provides unambiguous support for the PS-mediated assembly model of MS2.