Phylogenetic tree of 103 HAs from H5N1 strains.

<p>Phylogenetic trees are inferred from protein sequences by the Neighbor-Joining method and rooted using A/turkey/England/1991(red text). Estimates of the statistical significance of the phylogenies are calculated by performing 1,000 bootstrap replicates. The lengths of the horizontal line are proportional to the numbers of protein sequence differences, as indicated by the scale bars. Different clades classified by the WHO are shown as grayish-white bars. Virus with glycosylation site 158N deficiency are labeled in red, whereas glycosylation site 169N deficiency are labeled in green, and the dual deficiencies are labeled in purple. Several important mutation strains mentioned in the article are labeled in blue.</p

The binding energies collected from flexible docking.

<p>The binding energies are calculated from the three lowest energies provided in the largest clusters. A notable tendency is the increasing binding energy accompanying with increasing glycosylation.</p

The influence of vicinal N-glycans on the RBD of HA trimer.

<p>(A) The volumetric topologies of the N-glycans near the RBD during the 5 ns MD simulation. The complex N-glycans on the sites 158N and 169N would swing dramatically. (B) The distances from the weight center of the RBD to the three topological centers of the N-glycans are calculated at 10 ps intervals, three colors represent the corresponding distances in (A).</p

The amino-acid residues in the RBD of H5N1 HA.

<p>(A) The RBD in H5N1 HA consists of three secondary structure elements, Loop130, Loop220 and Helix190, together with four 100% conserved residues at the bottom (red). The residues with a conservation rate higher than 99% are labeled in yellow. (B) The statistics of the conserved amino-acid residues in the RBD. The predominant amino-acid residues are underlined, and the number of different types of mutations are shown as various blocks. Those with conservation rates lower than 99% are labeled. All of the residue numbers were adopted from the H3 HA numbering system.</p

Schematic diagrams for HA and SA receptors models.

<p>(A) Five initial models of HA are represented by 04VN, MG, HM, DS and FG respectively. Only one glycan is added on 158N near the RBD. (B) Four glycans on glycosylated HAs are represented by MG, HM, DS and FG respectively. (C) Eight sialoglycans and one pentaglucose are used for docking assays. The abbreviations for each glycan are as follows: lactosialyltetraoses (LSTa/LSTc), disialyllacto-N-tetraose (3DSLNT/6DSLNT) and bisialyantennas based on two monosaccharides (bisialyantennary mannose, BM3/BM6 and bisialyantennary GlcNAc, BG3/BG6). The sequence of monosaccharides and glycosidic bonds are illustrated using Consortium for Functional Glycomics nomenclature.</p

Sequence alignment of A/Hong Kong/486/97, A/Viet Nam/1203/2004(3GBM), A/Hong Kong/213/2003, A/Cambodia/S1211394/2008, A/Egypt/2321-NAMRU3/2007, A/Anhui/1/2005 and A/chicken/Shanxi/10/2006.

<p>The RBDs are shown in blue lines with the identical amino acids marked with asterisks. The glycosylation site 158N are boxed in red, whereas the four most conserved residues at the bottom of the RBD are boxed in green.</p

The N-J trees of two glycoproteins in IVs with the corresponding distribution chart of glycosites.

<p>The phylogenetic trees of HAs and NAs were constructed using three to ten representative amino acid sequences in each subtype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049224#pone.0049224.s003" target="_blank">File S1</a>). The distribution charts of glycosites, colored according to the statistics of conservation in each HA or NA subtype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049224#pone.0049224.s004" target="_blank">File S2</a>), are shown in various strips. The red, green and blue color represent the levels of conservation of “>95%”, “5%∼95%” and “<5%”, respectively. The conserved cysteines are shown in yellow strips. (A) The N-J tree of HA subtypes with the corresponding distribution chart of glycosites. (B) The N-J tree of NA subtypes with the corresponding distribution chart of glycosites.</p

The conservative rate of glycosites in the HA of H5N1.

<p>The conservative rate of glycosites in the HA of H5N1.</p

The full NAs and HAs structure and the distribution of glycosites.

<p>Those most conserved glycosites (>95%) are shown in red spheres, while the remainder are shown in cyan spheres. (A) The bottom view of the global domain of the NA monomer. (B) The side view of a NA monomer, the deletion of the stem domain would decrease four Glycosites. (C) The top view of the NA tetramer. (D) The side view of the global domain in the HA monomer. (E) The side view of the stem domain in the HA monomer, the 559NGS glycosite in the fusion peptide domain cannot be seen due to limited data. (F) The side view of the HA trimer.</p

Structural overviews of the glycans attached to glycosites 104 (A), 286 (B) and 71 (C) and their shielding regions on HA of human seasonal influenza H1N1 viruses.

<p>The glycosites are numbered in white.</p