Using mass spectrometry we have detected both assembly intermediates and the final product, the T = 3 viral capsid, during reassembly of the RNA bacteriophage MS2. Assembly is only efficient when both types of quasiequivalent coat protein dimer seen in the final capsid are present in solution. NMR experiments confirm that interconversion of these conformers is allosterically regulated by sequence-specific binding of a short RNA stem-loop. Isotope pulse-chase experiments confirm that all intermediates observed are competent for further coat protein addition, i.e., they are all on the pathway to capsid formation, and that the unit of capsid growth is a coat protein dimer. The major intermediate species are dominated by stoichiometries derived from formation of the particle threefold axis, implying that there is a defined pathway toward the T = 3 shell. These results provide the first experimental evidence for a detailed mechanistic explanation of the regulation of quasiequivalent capsid assembly. They suggest a direct role for the encapsidated RNA in assembly in vivo, which is consistent with the structure of the genomic RNA within wild-type phage.
Bibliographical noteFunding Information:
We thank Drs Hugo Lago and Andrew Smith for preliminary mass spectrometry and help with data collection, respectively, and Dr Abdul Rashid for production of some of the materials used in these experiments. We also thank Professors Sheena Radford (Leeds), Bentley Fane (Arizona), and Jon King (Cambridge, Mass.) and Dr Reidun Twarock (York) for their helpful discussions of the results and the manuscript. This work was supported in part by the Leverhulme Trust, the Wellcome Trust, and the UK BBSRC. OR is a Wellcome Trust-funded PhD student.
- mass spectrometry
- RNA-protein interaction
- virus assembly