Project description:Noroviruses are single-stranded RNA viruses with high genomic variability. They have emerged in the last decade as a major cause of acute gastroenteritis. It remains so far unclear whether norovirus evolution is driven by sequence mutation and/or recombination. In this study, we have assessed the occurrence of recombination in the norovirus capsid gene. For this purpose, 69 complete capsid sequences of norovirus strains accessible in GenBank as well as 25 complete capsid sequences generated from norovirus-positive clinical samples were examined. Unreported recombination was detected in about 8% of norovirus strains belonging to genetic clusters I/1 (n = 1), II/1 (n = 1), II/3 (n = 1), II/4 (n = 3), and II/5 (n = 1). Recombination breakpoints were mainly located at the interface of the putative P1-1 and P2 domains of the capsid protein and/or within the P2 domain. The recombination region displayed features such as length, sequence composition (upstream and downstream GC- and AU-rich sequences, respectively), and predicted RNA secondary structure that are characteristic of homologous recombination activators. Our results suggest that recombination in the norovirus capsid gene may naturally occur, involving capsid domains presumably exposed to immunological pressure.
Project description:Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses.
Project description:Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10(-3) substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum?>?0.39). The effective population sizes of the present GII strains were large (>10(2)) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.
Project description:Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.
Project description:A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied cofactor known for human noroviruses, and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsids interact with bile acids. We found that bile acids had low-micromolar affinities for GII.1, GII.10, and GII.19 capsids but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. Amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA nonbinder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggest that bile acid functions as a loop-stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes.IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection, our evidence that an HBGA nonbinder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provide direct evidence that, like HBGAs, bile acid interaction on the capsid is an important cofactor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and the prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid nonbinders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.
Project description:Human noroviruses are the leading causative agents of epidemic and sporadic viral gastroenteritis and childhood diarrhoea worldwide. Human histo-blood group antigens (HBGA) serve as receptors for norovirus capsid protein attachment and play a critical role in infection. This makes HBGA-norovirus binding a promising target for drug development. Recently solved crystal structures of norovirus bound to HBGA have provided a structural basis for identification of potential anti-norovirus drugs and subsequently performed in silico and in vitro drug screens have identified compounds that block norovirus binding and may thereby serve as structural templates for design of therapeutic norovirus inhibitors. This review explores norovirus therapeutic options based on the strategy of blocking norovirus-HBGA binding.
Project description:BACKGROUND: Norovirus is the major cause of nonbacterial epidemic gastroenteritis, being highly prevalent in both developing and developed countries. Despite of the available monoclonal antibodies (MAbs) for different sub-genogroups, a comprehensive epitope analysis based on various bioinformatics technology is highly desired for future potential antibody development in clinical diagonosis and treatment. METHODS: A total of 18 full-length human norovirus capsid protein sequences were downloaded from GenBank. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the protein antigen spatial epitope prediction webserver (SEPPA) for predicting the possible spatial epitopes with the default threshold. The results were processed using the Biosoftware. RESULTS: Compared with GI, we found that the GII genogroup had four deletions and two special insertions in the VP1 region. The predicted conformational epitope regions mainly concentrated on N-terminal (1~96), Middle Part (298~305, 355~375) and C-terminal (560~570). We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. CONCLUSIONS: The predicted conformational epitope regions of norovirus VP1 mainly concentrated on N-terminal, Middle Part and C-terminal. We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. The overlapping with experimental epitopes indicates the important role of latest computational technologies. With the fast development of computational immunology tools, the bioinformatics pipeline will be more and more critical to vaccine design.
Project description:Genotype II.3 (GII.3) noroviruses are a major cause of sporadic gastroenteritis, particularly in children. The greater incidence of GII.3 noroviruses in the pediatric population compared to the adult demographic suggests development of herd immunity to this genotype, possibly as a consequence of limited evolution of immune epitopes. This study aimed to identify and characterize immune epitopes on the GII.3 capsid protein and to determine the level of immune cross-reactivity within the genotype. A panel of seven GII.3 virus-like particles (VLPs), representing norovirus strains isolated during 1975 to 2008, was tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with human sera and a rabbit anti-GII.3 strain-specific polyclonal serum generated against the 2008 GII.3 VLP. Immunoprecipitation of protease-digested GII.3 VLPs and sequencing of bound peptides via mass spectrometry were used to locate epitopes on the capsid. Two epitopes were investigated further using Mimotopes technology. Serum binding studies demonstrated complete intragenotype GII.3 cross-reactivity using both human and rabbit serum. Six immunoreactive regions containing epitopes were located on the GII.3 capsid protein, two within each capsid domain. Epitopes in the S and P1 domains were highly conserved within GII.3 noroviruses. P2 domain epitopes were variable and contained evolutionarily important residues and histo-blood group antigen (HBGA) binding residues. In conclusion, anti-GII.3 antibody-binding epitopes are highly cross-reactive and mostly conserved within GII.3 strains. This may account for the limited GII.3 prevalence in adults and suggests that a GII.3 strain may be a valuable inclusion in a multivalent pediatric targeted VLP vaccine. Exploration of norovirus immune epitopes is vital for effective vaccine design. IMPORTANCE This study represents an important contribution to the understanding of norovirus immunology in a pediatric genotype. The high cross-reactivity and conservation of GII.3 epitopes suggest development of herd immunity against GII.3 and indicate that a GII.3 strain would be a valuable inclusion in a pediatric targeted multivalent vaccine. Immunological understanding of pediatric norovirus strains is important since norovirus vaccines will likely target high-risk groups such as the pediatric population.
Project description:Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.
Project description:Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods. Here, we report the characterization of a broadly cross-reactive monoclonal antibody (MAb) raised against the capsid protein of a GII.3 norovirus strain. The MAb reacted with VLPs and denatured VP1 protein from GI, GII, GIV and GV noroviruses, and mapped to a linear epitope located in the inner shell domain. An alignment of all available VP1 sequences showed that the putative epitope (residues 52-56) is highly conserved across the genus Norovirus. This broadly cross-reactive MAb thus constitutes a valuable reagent for the diagnosis and study of these diverse viruses.