Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

• Published in: Molecular & cellular proteomics Vol. 9; no. 8; pp. 1742 - 1751
• Main Authors: Shoemaker, Glen K., van Duijn, Esther, Crawford, Sue E., Uetrecht, Charlotte, Baclayon, Marian, Roos, Wouter H., Wuite, Gijs J.L., Estes, Mary K., Prasad, B. V. Venkataram, Heck, Albert J.R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2010; The American Society for Biochemistry and Molecular Biology
• Subjects: Capsid - chemistry; Capsid - ultrastructure; Microscopy, Atomic Force; Norwalk virus - physiology; Norwalk virus - ultrastructure; Osmolar Concentration; Particle Size; Spectrometry, Mass, Electrospray Ionization - methods; Virus Assembly - physiology
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.M900620-MCP200

Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP160 (3.36-MDa) and VP180 (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1180). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP160 and VP180 species.