Figure 1.
Characterization of the HP and MP HA proteins.
(A) Western blot of HA expression. Values below the HA2 bands are the cleavage ratio, calculated by dividing HA2 by total HA (i.e., HA0 + HA2). The values listed are the mean of 3 independent experiments. (B) HA protein expression. Total expression (solid bars) was measured by Western blot and surface expression (open bars) was measured by flow cytometry. Values were normalized to 100 % for HP HA (+ HP NA). (C) Hemadsorption of chicken and turkey erythrocytes to cell surface-expressed HA proteins. Bound erythrocytes were lysed, and released hemoglobin was measured at 415 nm. Values were normalized to 100% for HP HA (+ HP NA). (D and E) pH of HA protein conformational changes. HP and MP HA proteins were co-expressed with their homologous NA proteins. Activation pH values for HA proteins were measured by flow cytometry using conformation specific monoclonal antibodies Vn04-09 and Vn04-16, that preferentially recognize prefusion and acid-activated forms of the H5N1 HA protein, respectively. (F) Activation pH of the HA protein, expressed as the midpoint of the pH of conformational changes (closed bars) and the highest pH at which syncytia formation occurs (open bars). Error bars in panels B, C, and F represent the standard deviation of triplicate experiments.
Figure 2.
Enzymatic activities of the NA proteins from MP and HP influenza viruses.
NA enzymatic activity was determined by using a fluorescence-based assay for transiently expressed NA protein (A) and for prestandardized virus (B).
Figure 3.
Biochemical properties of wild-type and mutant HA proteins.
(A) The pH of HA activation was determined as the average of the highest pH at which syncytia formation occurs and the midpoint of the pH of conformational changes. (B) HA protein expression. Closed bars represent total expression as determined by using Western blot analysis, and open bars represent cell-surface expression analyzed by flow cytometry. (C) HA protein cleavage ratio, measured as described in Figure 1A. (D) Hemadsorption of chicken and turkey erythrocytes to cell surface-expressed HA protein. Wild-type and mutant HP HAs were co-expressed in presence HP NA in all experiments. Values depict the average ± standard deviation of at least 3 independent experiments (for total expression and cleavage) or triplicate experiments for surface expression and hemadsorption. HP, highly pathogenic. MP, moderately pathogenic. rHA1, HP HA possessing D104N, I115T, E131D, and L142H mutations.
Figure 4.
HA activation pH and pathogenicity in chickens.
The activation pH values are averages of the midpoint pH of conformational changes and the highest pH at which syncytia form. Pathogenicity is expressed as the reciprocal of the LD50 values in chickens.
Figure 5.
Crystal structures of the MP and HP HA proteins.
(A) Schematic of the HA protein after proteolytic cleavage into HA1 and HA2 subunits. The receptor-binding domain (RBD) consists of the receptor-binding sub-domain (green) and the vestigial esterase sub-domain (yellow). The F' fusion sub-domain in HA1 is colored blue and the HA2 stalk domain is colored red. The locations of the 7 amino acids that differ between the MP and HP HA proteins are highlighted in magenta. (B) Crystal structure of MP HA trimer determined at 2.50Å. One protomer is colored as in panel A (with HA1 in blue, HA2 in red, and 6 amino acids that differ in HP HA shown in magenta spheres). The seventh amino acid (337) that differs in HP HA is removed after cleavage at the polybasic cleavage site between HA1 and HA2. Glycosylation carbohydrates observed in the electron-density maps at HA1 residues Asn34 and Asn169 are shown as a ball-and-stick model. The remaining 2 HA protomers are colored grey. (C) Crystal structure of one protomer of MP HA. The domains of the protein are identified with color: receptor-binding sub-domain (green), vestigial esterase sub-domain (yellow), F' fusion sub-domain (blue), and HA2 stalk domain (red). Important structural features include the 110-helix in the vestigial esterase sub-domain and helix A, B loop, and helix C in the HA2 stalk domain. (D) Superposition of 1 protomer from the crystal structure of MP HA (blue) and 2 crystal structures of HP HA (yellow). HP HA crystallized in 2 forms; their structures were determined at 3.10Å (Crystal form 1) and 2.95Å (Crystal form 2). The coordinates for all 3 crystal structures have been deposited at the Protein Data Bank (PDB entry 3S11 [MP HA], PBD entry 3S12 [HP HA Crystal form 1], and PDB entry 3S13 [HP HA Crystal form 2]). All residues are labeled using H3 numbering.
Table 1.
Crystallographic data.
Figure 6.
Structures at sites of sequence variation between the MP and HP HA proteins.
(A) Zoomed-in stereo view of residues 104 and 115 at the N- and C-terminal ends of the 110-helix of HA1 in MP HA (blue) and HP HA (yellow). The corresponding HA2 backbone is colored red and HA2 sidechains of the other two protomers are colored white. Residues from HA2 are denoted with a “2” subscript. Dotted lines represent hydrogen bonds and are colored to match the corresponding HA protein with MP HA in blue and HP HA in yellow. Note the interaction between the 110-helix with the B loops from 2 HA2 protomers. (B) Zoomed-in stereo view of residues 216 and 221 in MP HA (blue) and HP HA (yellow) and their location at the trimer interface. The adjacent protomers across the RBD-RBD interface for MP and HP HA are all shown in gray. In both A and B, the left and middle panels represent the divergent pair of stereoimages while the middle and right panels represent the convergent pair of stereoimages. All residues are labeled using H3 numbering.
Table 2.
Prevalance of amino-acid residues at positions 104 and 115 of the HA protein.