Structure, stability and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry
ABSTRACT Expression of the protruding (P) domain of the norovirus capsid protein, in vitro, results in the formation of P dimers and larger oligomers, 12-mer and 24-mer P particles. All these P complexes retain the authentic antigenicity and carbohydrate-binding function of the norovirus capsid. They have been used as tools to study norovirus-host interactions, and the 24-mer P particle has been proposed as a vaccine and vaccine platform against norovirus and other pathogens. In view of their pharmaceutical interest it is important to characterise the structure, stability and dynamics of these protein complexes. Here we use a native mass spectrometric approach. We analyse the P particles under both non-reducing and reducing conditions, as it is known that the macromolecular assemblies are stabilised by inter-subunit disulphide bonding. A novel 18-mer complex is identified, and we show that under reducing conditions the 24-mer dissociates into P dimers that reassemble into the 12-mer small P particle and another novel 36-mer complex. The collisional cross-sections of the 12-mer and 24-mer P particles determined by ion mobility MS are in good agreement with theoretical predictions and electron microscopy data. We propose a model structure for the 18-mer based on ion mobility experiments. Our results demonstrate the interchangeable nature and dynamic relationship of all P domain complexes and confirm their binding activity to the host receptors - human histo blood group antigens (HBGAs). These findings, together with the identification of the 18-mer and 36-mer P complexes add new information to the intriguing interactions of the norovirus P domain.
- SourceAvailable from: Leyi Wang
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- "On the other hand, the P domain alone can form different complexes, including 24 mer P particles (Tan et al., 2008a; Tan et al., 2011b; Tan and Jiang, 2005b), 12 mer small P particles (Tan et al., 2011a) and P dimers (Tan et al., 2004a; Tan et al., 2008c). These P domain complexes are interchangeable in certain condition (Bereszczak et al., 2012) and they all bind to histo-blood group antigens (HBGAs) (Tan et al., 2011a; Tan et al., 2004a; Tan and Jiang, 2005b), the viral receptors or attachment factors of NoVs (Tan and Jiang, 2005a, 2007, 2010, 2011). NoV P domain complexes have been useful tools for study of NoV–HBGA interactions (Tan et al., 2004a; Tan et al., 2008b; Tan et al., 2006; Tan et al., 2008c; Tan et al., 2009). "
ABSTRACT: Noroviruses (NoVs), an important cause of gastroenteritis in humans, recognize human histo-blood group antigens (HBGAs) as receptors. The crystal structures of the protruding (P) domain of a GII.10 NoV (Vietnam 026) in complex with various HBGA oligosaccharides were elucidated. However, the HBGA binding profile of this virus remains unknown. In this study, we determined the saliva and oligosaccharide binding profiles of this virus and the roles of amino acids that are involved in HBGA binding. Our data showed that Vietnam 026 bound to all ABO secretor and non-secretor saliva with clear signals detected by monoclonal antibodies against H3, H1, Le(y), Le(a) and sialyl Le(a). Mutagenesis study confirmed the binding site determined by the crystallography study, in which single mutations wiped out the binding function. We also identified amino acids surrounding the central binding pocket that may participate in the binding by affecting the HBGA binding specificity of the GII.10 NoV. Copyright © 2014 Elsevier Inc. All rights reserved.Virology 01/2015; 476C:386-394. DOI:10.1016/j.virol.2014.12.039 · 3.28 Impact Factor
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ABSTRACT: Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987-1997), contemporary (2004-2009), and broad (1987-2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294-298 and 368-372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393-395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution.PLoS Pathogens 05/2012; 8(5):e1002705. DOI:10.1371/journal.ppat.1002705 · 8.06 Impact Factor
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ABSTRACT: Noroviruses (NoVs) are important pathogens causing epidemic acute gastroenteritis that affects millions of people worldwide. The protruding (P) domain of the NoV capsid protein, the surface antigen of NoV, forms a 24-mer subviral particle called the P particle that is an excellent candidate vaccine against NoVs. The P particles are easily produced in Escherichia coli, highly stable and highly immunogenic. Each P domain has three surface loops that can be used for foreign antigen presentation, making the P particles a useful platform for vaccine development against other infectious diseases. This article summarizes the discovery, structure, development and applications of the P particles as a vaccine against NoVs, as well as a vaccine platform against rotavirus, influenza virus and possibly other pathogens in the future.Nanomedicine 06/2012; 7(6):889-97. DOI:10.2217/nnm.12.62 · 5.82 Impact Factor