Structures (A) and the structure-based sequence alignment (B) of the P domains of OIF virus.

<p>(A), Overall structure (ribbon representation, green) of OIF P protein monomer (left) and its dimeric form (ribbon representation, green and pink) in complex with Le^a trisaccharide (stick representation, indicated by arrows) (right). Secondary structural elements are labeled in the P monomer (left). (B), the structure-based sequence alignment of the P domains of GII.21 OIF, GII.4 VA387, GII.9 VA207, GII.10 VN026 and GII.12 Hiro. Regions spanning the P1 and P2 subdomains are indicated by arrows. Identical residues are highlighted with blue background, while similar residues are shown in red characters. Residues forming the HBGA binding interface of the GII.21 OIF are indicated in red frames, while the conserved amino acid residues forming the conventional GII HBGA binding interface are indicated by blue stars. The seven surface loops that constitute the two types of HBGA binding interfaces are indicated by black frames. The letters in blue backgrounds indicate the identical amino acid sequences, while the red letters indicate the similar amino acids among the five NoVs. Blue box fames both identical and similar residues.</p

Summary of the mutagenesis study of the three sites of the HBGA-binding interface of MOH (GII-5) and VA207 (GII-9) predicted by sequence alignment with that of Norwalk virus (GI) and VA387 (GII).

1<p>The number of “+” indicated the relative binding affinity of the wild type (WT) and mutant P particles at 16.7 ng/µl to HBGAs: “+++” indicates an OD₄₅₀ >2.0; “++” between 1.0 and 2.0; “+” between 0.3 and 1.0; while “−” indicates an OD₄₅₀ <0.3, suggesting a complete loss of binding. Saliva samples of type A, B, AB, and O were from secretors containing A, B, A/B and H antigen, respectively.</p

Statistics of structure refinement.

<p>a. R_work = Σ||F_obs|-|F_cal|||/Σ|Fobs|, R_free = ΣT||F_obs|-|F_cal|||/ΣT|F_obs|, where F_obs and F_cal are observed and calculated structure factors, respectively.</p><p>b. R_free, T is a randomly selected test data set (5%) of total reflections and was set aside before structure refinement.</p><p>Statistics of structure refinement.</p

Statistics of data-collection.

<p>a. Values in parentheses correspond to the shell of highest resolution.</p><p>b. R_merge = Σi|Ii–<i>|/ΣiIi, where Ii and <i> are the observed and mean intensity of related reflections with common indices h,k, and l.</i></i></p><p><i><i>Statistics of data-collection.</i></i></p

Primers used for cloning of P domain and site-directed mutagenesis to generate single mutation in the HBGA-binding interface and around regions.

<p>Primers used for cloning of P domain and site-directed mutagenesis to generate single mutation in the HBGA-binding interface and around regions.</p

Phylogenetic and sequence analysis of genogroup (G) II noroviruses (NoVs).

<p>(A), Phylogenetic tree of GII NoVs that is re-drawn according to [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005025#ppat.1005025.ref052" target="_blank">52</a>]. The unique genetic branch consisting of GII.13 and GII.21 genotypes is indicated by a green frame. (B), P domain sequence alignment of NoVs representing each of the 20 GII genotypes with a focus on the residues at the HBGA binding interface. The conserved amino acids constituting the GII conventional HBGA binding interface are indicated with colored letter in frames. The GenBank accession numbers of the P domain sequences are: AY038600.3 (VA387), U07611 (Hawaii), AY134748 (SMV), U22498 (Mexico), X86557 (Lordsdale), AF397156 (MOH), AF414407 (Florida269), AJ277608 (Leeds), AF195848 (Amsterdam), AAK84676 (VA207), AF427118 (Erfurt), AB074893 (SW918), AJ277618 (Wortley), AY113106 (Fayettevil), AY130761 (M7), AY130762 (J23), AY502010 (Tiffin), AY502009 (CS-E1), AY823304 (SW101), AY823306 (SW170), EU373815 (Lucken), AY675554 (OIF), and AB083780.1 (YURI).</p

Identification of the HBGA-binding interface of OIF virus and its interaction with the Le^a trisaccharide.

<p>(A), (Fo-Fc) omit electron density map of Le^a trisaccharide. The omit map was created using the final structure of OIF P domain without Le^a trisaccharide, and the mesh map was contoured at 2.0 sigma (blue) around the selection site, with a coverage of 1.6Å radius. Carbon, oxygen and nitrogen atoms are indicated in green, red and blue, respectively. (B), Surface representation of the P dimer of OIF virus in complex with Le^a trisaccharide (stick representation). (C and D), Close-ups of top (C) and side (D) views of the HBGA binding interface (surface representations) with indications of the residues that form the HBGA binding interface (colored in green). The Le^a trisaccharide (stick representation) is also indicated. The individual saccharides of the Le^a-trisaccharide are colored differently: β-Galactose, yellow; α-Fucose, purple; and N-acetyl glucosamine, grey. (E and F), the interacting networks between the side/main chains of the amino acids at the binding interface with the β-Galactose (Gal) and α-Fucose (Fuc) (Le-Fuc). The hydrogen bonds are indicated by dashed lines. The water molecule is presented as a red ball (E) or a circled W (F).</p

Sequence alignments of the HBGA-binding interfaces of various GI and GII noroviruses.

<p>Sequence of the three major components (red letters) of the HBGA-binding interfaces of 10 genogroup I (GI) (A) and 17 genogroup II (GII) (B) noroviruses, representing each of the 8 GI and 17 GII genetic types, respectively, are aligned based on the two known binding interfaces of Norwalk virus (GI) and VA387 (GII). Star symbols label the residues that have been experimentally shown to be required for binding to HBGAs. The two strains that have no detectable binding to examined HBGAs are underlined. The accession numbers of the sequence are: M87661 (Norwalk virus), L23828 (KY 89), L07418 (SOV), AF414403 (HLL), U04469 (DSV), AY038598 (VA115), AB042808 (Chiba), AJ277614 (Musgrove), AY502008 (Wiscon), AJ277609 (Winchester), AF538679 (Boxer), AY038600 (VA387), U07611 (Hawaii), AY134748 (SMV), U22498 (Mexico), X86557 (Lordsdale), AF397156 (MOH), AF414407 (Florida269), AJ277608 (Leeds), AF195848 (Amsterdam), AAK84676 (VA207), AF427118 (Erfurt), AB074893 (SW918), AJ277618 (Wortley), AY113106 (Fayettevil), AY130761 (M7), AY130762 (J23), AY502010 (Tiffin), AY502009 (CS-E1).</p

A schematic relationship of the known carbohydrate-binding phenotypes of caliciviruses.

<p>Six calicivirus genera may be evolved from a common calicivirus ancestor and at least one strain from four genera (orange) has been shown to bind to carbohydrates. Similarly, five norovirus genogroups (G) may be evolved from a common norovirus ancestor and two of the three human norovirus genogroups have been demonstrated to recognize HBGAs (purple). GI and GII noroviruses share conserved genogroup-specific HBGA-binding interfaces and both genogroups contain strains binding to either A/B/H antigens (A/B binding groups, yellow) or Lewis antigens (Lewis binding group, green). GII-13 (Blue) is a unique genotype that does not share conserved binding sites with other GII genotypes and thus may represent a sublineage parallel to other GII genotypes, in which a strain (OIF) has been shown to bind to Lewis antigens (green). Arrows indicate the direction of evolution. Solid line shows the evolutionary lineages with defined binding to HBGAs, while the dashed line shows the lineages with unknown interaction with carbohydrates. RHDV, rabbit hemorrhagic disease virus; TV, Tulane virus; FCV, feline calicivirus; OIF, norovirus strain that was isolated from troops deployed to the Operation of Iraqi Freedom.</p

The crystal structures of the HBGA-binding interfaces of Norwalk virus (GI-1) and VA387 (GII-4).

<p>The surface models of the P dimers (top views) with indications of the HBGA-binding interfaces (colored regions) are shown in (A) and (B) with one monomer being shown in darker gray than another. Enlargements of the HBGA-binding interfaces are shown in (C) and (D) correspondingly with labels of individual amino acids, in which the prime symbol indicates a residue of another protomer. The three major components of the binding interfaces are colored in green (site I), red (site II), and orange (site III), respectively, while the trisaccharides binding to the interface are in yellow in (A) and (B) or in variable colors (C-cyans, O-red, and N-blue) in (C) and (D). The amino acids around the interface that affect the binding specificity are in light blue. (E) and (F) are schematic diagrams of hydrogen bonding network (dash lines) between the amino acids of the P dimers of Norwalk virus (E), or VA387 (F) and the A- or B- type trisaccharides. The water-bridged hydrogen bonds are indicated by W. (A) to (D) were prepared by software PyMOL version 1.0 (Delano Scientific), while (E) and (F) by software ChemDraw Pro version 11.0 (Adept Scientific). (E) is adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005058#pone.0005058-Bu1" target="_blank">[13]</a> with permission. The original data were published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005058#pone.0005058-Bu1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005058#pone.0005058-Cao1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005058#pone.0005058-Choi1" target="_blank">[15]</a>.</p