Norwalk Virus Assembly and Stability Monitored by Mass Spectrometry

Abstract

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 VP1 60 (3.36-MDa) and VP1 80 (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 (VP1 180 ). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T ‫؍‬ 3 VP1 180 particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1 60 and VP1 80 species. Molecular & Cellular Proteomics 9: 1742-1751, 2010.

Qualitative and Quantitative Analysis of the Binding of GII.4 Norovirus Variants onto Human Blood Group Antigens

“…1D) (37). For the Bristol isolate, the VLPs were smaller, with diameters averaging 21.3 Ϯ 4.7 nm, which suggests that they might have been subunit particles and might have been composed of 60 monomers (also called VP1 60 ) (3,37). The observation by electron microscopy of the cesium chloride-purified VLPs from the Hunter variant showed that the preparation was essentially composed of full and empty particles of 35 nm in diameter (Fig.…”

“…The majority of the VLP preparations presented globular particles with an average diameter ranging between 30 and 38 nm (US95/96, 37.2 Ϯ 3.7 nm; Hunter, 29.6 Ϯ 3.2 nm; Den Haag, 37.6 Ϯ 4.8 nm; Osaka, 36.8 Ϯ 6.2 nm) and corresponded to complete VLPs (also called VP1 180 ) as described previously (Fig. 1D) (37). For the Bristol isolate, the VLPs were smaller, with diameters averaging 21.3 Ϯ 4.7 nm, which suggests that they might have been subunit particles and might have been composed of 60 monomers (also called VP1 60 ) (3,37).…”

Norwalk Virus Minor Capsid Protein VP2 Associates within the VP1 Shell Domain

“…Based on structural and mass spectroscopy studies, it is proposed that capsid assembly in NV and likely in other noroviruses proceeds by the formation of pentamers of VP1 dimers (41,42). Although we note that it is not necessary for VP2 to interact with all of the dimers in the capsid, VP2 may bind to Ile-52, provide stability to VP1 dimers, and promote nucleation whereby assembly proceeds in a cascade until a fully closed capsid is formed.…”

Sampling Protein Form and Function with the Atomic Force Microscope

“…The images were recorded using jumping mode AFM (see Ref. 86 for full experimental details with the sole difference that these images were taken in sodium phosphate buffer).…”