IMPORTANCEOur 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. Human noroviruses are responsible for most epidemic outbreaks of gastroenteritis. There are still no antivirals or vaccines approved, despite the discovery of these viruses over 4 decades ago (1). Noroviruses are genetically and antigenically diverse (2), yet a single genetic cluster (genogroup II, genotype 4 [GII.4]) has dominated over the past decade (3). The GII.4 noroviruses evolve ϳ5% every year and are believed to have a mechanism that allows them to evade the immune system or alter receptor binding profiles (4-6). However, immunity to noroviruses is still poorly understood (7).Human noroviruses interact with histo-blood group antigens (HBGAs), and this is thought to be important for viral infections (8-11). A recent report showed for the first time that human noroviruses infect B cells and that HBGAs (synthetic or from HBGAexpressing enteric bacteria) can enhance the infection (12). HBGAs are also found as soluble antigens in saliva and are expressed on epithelial cells. Genetic polymorphisms in genes that control their synthesis are known to provide intraspecies diversity (13). To date, based on the ABH and Lewis HBGA types, at least nine different HBGAs have been found to interact with human noroviruses. Individuals expressing the O type are thought to have a significantly higher infection rate than those for individuals with other blood types (11). The GII noroviruses are thought to have preferences for HBGAs in a strain-dependent manner (14)(15)(16)(17)(18)(19).Expression of the norovirus capsid protein in insect cells results in the formation of virus-like particles (VLPs) that are antigenically similar to native virions. The X-ray crystal structure of prototype (GI.1) norovirus VLPs identified two domains: the shell (S) and protruding (P) domains (20). The S domain forms a scaffold surrounding the viral RNA, whereas the P domain is thought to contain the determinants for cell attachment and strain diversity. The P domain can be further subdivided into P1 and P2 subdomains, and each subdomain likely has unique functions. In this study, we determined the X-ray crystal structures of P domains from three epidemic GII.4 variants, from 2004GII.4 variants, from , 2006GII.4 variants, from , and 2012, in complex with a panel of HBGAs in order to elucidate HBGA binding mechanisms. Our data showed that the GII.4 noroviruses bound numerous HBGA types and that bindi...

dHisto-blood group antigens (HBGAs) are important binding factors for norovirus infections. We show that two human milk oligosaccharides, 2=-fucosyllactose (2=FL) and 3-fucosyllactose (3FL), could block norovirus from binding to surrogate HBGA samples. We found that 2=FL and 3FL bound at the equivalent HBGA pockets on the norovirus capsid using X-ray crystallography. Our data revealed that 2=FL and 3FL structurally mimic HBGAs. These results suggest that 2=FL and 3FL might act as naturally occurring decoys in humans. Mothers' milk has long been seen as a great source of infant nutrition and protection against a large number of pathogens. Human milk oligosaccharides (HMOs), the third-mostabundant (10 to 15 g/liter) components of human milk, are thought to be in part accountable for these health benefits (1). HMOs are unconjugated complex glycans, and more than 200 isomers are known. HMOs consist of combinations of different monosaccharide building blocks, including fucose, glucose, galactose, N-acetylglucosamine, and the sialic acid derivative N-acetylneuraminic acid. HMOs have been demonstrated to protect against human noroviruses, rotavirus, and certain bacteria (reviewed in reference 2).Human noroviruses are also known to interact with histoblood group antigens (HBGAs), and the interaction is thought to be important for infection (3-6). HBGAs can be found as soluble antigens in saliva and are expressed on epithelial cells. HBGAs consist of monosaccharide building blocks similar to those of HMOs, and at least nine different HBGA types have been found to interact with human norovirus (7-12). HMOs are thought to act as a "receptor decoy" for certain pathogens, since HMOs and HBGAs mimic each other structurally. However, little is known about how HMOs block norovirus infections. One study found that human milk was able to block genogroup I genotype 1 (GI.1) and GII.4 norovirus strains from binding to saliva samples (13). A follow-up study suggested that certain HMOs might compete with the HBGA binding sites on the GI.1 and GII.4 norovirus capsid (14). Despite the fact that human noroviruses are the dominant cause of acute gastroenteritis, there are still no suitable antivirals or vaccines commercially available.In this study, we analyzed the ability of two HMOs, i.e., 2=-fucosyllactose (2=FL) and 3-fucosyllactose (3FL), to block GII.10 norovirus virus-like particles (VLPs) from binding to HBGAs (Fig. 1A). A slightly modified blocking enzyme-linked immunosorbent assay (ELISA) was developed using both porcine gastric mucin type III (PGM) and human saliva (A and B types) (3, 15). The PGM sample was confirmed to contain a mixture of A and H types using specific anti-HBGA monoclonal antibodies (data not shown).The GII.10 VLPs were expressed and purified as previously described (16). The untreated VLPs were first examined for binding to PGM and saliva samples using a direct ELISA. Maxisorp 96-well plates were coated with 100 l per well of 10 g/ml PGM for 4 h at room temperature. The saliva samples were processed in a si...

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.

Recent reports suggest that human genogroup II genotype 17 (GII.17) noroviruses are increasing in prevalence. We analyzed the evolutionary changes of three GII.17 capsid protruding (P) domains. We found that the GII.17 P domains had little cross-reactivity with antisera raised against the dominant GII.4 strains. X-ray structural analysis of GII.17 P domains from 2002 to 2014 and 2015 suggested that surface-exposed substitutions on the uppermost part of the P domain might have generated a novel 2014-2015 GII.17 variant. Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. In the past decade, genogroup II genotype 4 (GII.4) norovirus strains were those mostly responsible for epidemic outbreaks (1-3). However, a GII.17 variant norovirus was found recently to cause an alarming number of outbreaks in certain parts of Asia in 2014 to 2015 (4-8). Before this time, the GII.17 norovirus was only a minor cause of infections, although it was first described in 1978 (9). Researchers are now reporting that the GII.17 variant is emerging in other parts of the world, and molecular epidemiologists have warned that the GII.17 norovirus might replace the predominant GII.4 norovirus (10).Noroviruses have a single-stranded, positive-sense RNA genome of 7.5 to 7.7 kb. The genome contains three open reading frames (ORFs). The first ORF (ORF1) encodes nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp), ORF2 encodes capsid protein (VP1), and ORF3 encodes a minor capsid protein (VP2) (11). The X-ray crystal structure of the prototype (GI.1) virus-like particles (VLPs) identified two domains, the shell (S) domain and the protruding (P) domain, which can be further subdivided into P1 and P2 subdomains (12). The S domain surrounds the viral RNA, whereas the P domain contains the determinants for cell attachment and antigenicity. Human noroviruses are known to bind histo-blood group antigens (HBGAs), and the interaction is thought to be important for infection (13)(14)(15)(16). Two recent reports indicated that, similarly to other GII noroviruses, the recent GII.17 strains bind a panel of different HBGA types (4,8).Human noroviruses are believed to evolve in a manner similar to that seen with influenza viruses, where new norovirus genotype variants emerge every other year. Evolving strains with an ϳ5% amino acid change can reinfect the same individual (17). Data on short-and long-term immunity to human norovirus are still unclear, although vaccines are currently been tested in clinical trials (18,19). Unfortunately, the vaccines, which can include VLPs or P domains (20, 21), may not protect from antigenically divergent strains (18-21). Here, we report the first X-ray crystal structure of GII.17 norovirus P domains and describe the cross-reactivities with antibodies (Abs) raised against GII.4 strains, which are targeted by the current vaccines in clinical trials.Three different GII.17 norovirus strains were selected for antibody binding and structural analysis: a nonprevalent 2002 strain (Sai...

CitationCrystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite. Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.