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  • 1
    Keywords: RECEPTOR ; RISK ; POLYMORPHISMS ; SUSCEPTIBILITY LOCUS ; VARIANTS ; IDENTIFICATION ; GENOME-WIDE ASSOCIATION ; PROGESTERONE ; GENOTYPE IMPUTATION ; GRANULOSA-CELL TUMOR
    Abstract: Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains. IMPORTANCE: We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.
    Type of Publication: Journal article published
    PubMed ID: 25505081
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  • 2
    Keywords: INFECTION ; SUSCEPTIBILITY ; RECOGNITION ; PATTERNS ; RECEPTORS ; STRUCTURAL BASIS ; EPOCHAL EVOLUTION ; NORWALK VIRUS ; BINDING SPECIFICITY ; P2 DOMAIN
    Abstract: Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly repositioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications. IMPORTANCE: Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can accommodate numerous HBGA types.
    Type of Publication: Journal article published
    PubMed ID: 25428879
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  • 3
    Keywords: COMPLEX ; STRUCTURAL BASIS ; BLOOD GROUP ANTIGENS ; NORWALK VIRUS ; MACROMOLECULAR CRYSTALLOGRAPHY
    Abstract: Human noroviruses bind histo-blood group antigens (HBGAs) and this interaction is thought to be important for an infection. We identified two additional fucose-binding pockets (termed fucose-3/4 sites) on a genogroup II human (GII.10) norovirus-protruding (P) dimer using X-ray crystallography. Fucose-3/4 sites were located between two previously determined HBGA binding pockets (termed fucose-1/2 sites). We found that four fucose molecules were capable of binding altogether at fucose-1/2/3/4 sites on the P dimer, though the fucose molecules bound in a dose-dependent and step-wise manner. We also showed that HBGA B-trisaccharide molecules bound in a similar way at the fucose-1/2 sites. Interestingly, we discovered that the monomers of the P dimer were asymmetrical in an unliganded state and when a single B-trisaccharide molecule bound, but were symmetrical when two B-trisaccharide molecules bound. We postulate that the symmetrical dimers might favor HBGA binding interactions at fucose-1/2 sites.
    Type of Publication: Journal article published
    PubMed ID: 25980740
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  • 4
    Abstract: The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions. The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.
    Type of Publication: Journal article published
    PubMed ID: 27167457
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  • 5
    Abstract: Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. The genogroup II genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Most human noroviruses bind HBGAs, while some strains were found to have minimal or no HBGA interactions. Here, we explain some possible structural constraints for several noroviruses that were found to bind poorly to HBGAs by using X-ray crystallography. We showed that one aspartic acid was flexible or positioned away from the fucose moiety of the HBGAs and this likely hindered binding, although other fucose-interacting residues were perfectly oriented. Interestingly, a neighboring loop also appeared to influence the loop hosting the aspartic acid. These new findings might explain why some human noroviruses bound HBGAs poorly, although further studies are required.
    Type of Publication: Journal article published
    PubMed ID: 27303720
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