Structure, stability and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry

Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Journal of Structural Biology (Impact Factor: 3.23). 02/2012; 177(2):273-82. DOI: 10.1016/j.jsb.2012.01.005
Source: PubMed


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.

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    • "Based on the results of various analyses (PAGE, gel filtration chromatography, dynamic light scattering and electron microscopy), P domain proteins were observed to auto-assemble dynamically into P particles of diverse sizes, with diameters ranging between 14–25 nm (Fig. 2e). A similar assembly profile was described in a previous report on P particles of the VA98387/1998 strain as a mixture of 12-, 18-, 24-and 36-mers (Bereszczak et al., 2012). Prior studies demonstrated that HBGAs, encoded by the ABO, non-secretor and Lewis gene families, serve as NoV ligands (Huang et al., 2003, Choi et al., 2008de Rougemont et al., 2011). "
    W Su · J Gao · Y Zang · H Wu · L Wang · H Hu · X Yu · W Kong · C Jiang
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    ABSTRACT: Norovirus (NoV) is the main cause of nonbacterial infectious gastroenteritis. Due to the difficulty of culturing the virus, research on vaccine against NoV is focused on virus-like particles (VLPs). On the other hand, the P particles assembled from the P domains of NoV capsid protein become a promising vaccine candidate. GII.4 is the most prevalent genotype of NoV. While the immunogenicity of P particles derived from the GII.4 1996 variant has been investigated, the research on P particles of more recently prevalent variants is lacking. In this study, the P domain of the capsid protein of GII.4 genotype 2004 variant was expressed in Escherichia coli, purified and auto-assembled into P particles of 14-25 nm. Immunization with P particles induced specific serum antibodies with titers of 245,600 and 145,700 in mice and rabbits, respectively. The GII.4 NoV 2004 variant bound to type A, B and O secretor-positive saliva and immune sera blocked this binding, suggesting induction of neutralizing activity in such sera. Thus, this study demonstrated the immunogenicity of NoV P particles generated from E. coli and provided evidence supporting the development of this approach.
    Full-text · Article · Mar 2015 · Acta virologica
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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). "
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    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.
    Full-text · Article · Jan 2015 · Virology
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    • "In contrast, production of the P domain alone assembles different P domain complexes, including the P dimer [5], [11]–[14], the 12mer small P particle [15], the 24mer P particle [9], [16], [17] (Figure 1). These P domain complexes are interchangeable under certain conditions [18] and all P domain complexes retain binding function to HBGAs [9], [16], [19], indicating that the P domain is the carbohydrate binding domain [11]–[14]. A truncated P domain protein without the C-terminal arginine-cluster, named P polypeptide, was found in large amount in stools of NoV infected patients [20]–[22], though its biological significance remains unknown. "
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    ABSTRACT: Norovirus (NoV) P domain complexes, the 24 mer P particles and the P dimers, induced effective humoral immunity, but their role in the cellular immune responses remained unclear. We reported here a study on cellular immune responses of the two P domain complexes in comparison with the virus-like particle (VLP) of a GII.4 NoV (VA387) in mice. The P domain complexes induced significant central memory CD4(+) T cell phenotypes (CD4(+) CD44(+) CD62L(+) CCR7(+)) and activated polyclonal CD4(+) T cells as shown by production of Interleukin (IL)-2, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α. Most importantly, VA387-specific CD4(+) T cell epitope induced a production of IFN-γ, indicating an antigen-specific CD4(+) T cell response in P domain complex-immunized mice. Furthermore, P domain complexes efficiently induced bone marrow-derived dendritic cell (BMDC) maturation, evidenced by up-regulation of co-stimulatory and MHC class II molecules, as well as production of IL-12 and IL-1β. Finally, P domain complex-induced mature dendritic cells (DCs) elicited proliferation of specific CD4(+) T cells targeting VA387 P domain. Overall, we conclude that the NoV P domain complexes are efficiently presented by DCs to elicit not only humoral but also cellular immune responses against NoVs. Since the P particle is highly effective for both humoral and cellular immune responses and easily produced in Escherichia coli (E. coli), it is a good choice of vaccine against NoVs and a vaccine platform against other diseases.
    Full-text · Article · Apr 2013 · PLoS ONE
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