Restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host

Abstract

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (HA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 HA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 HA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

FIG. 1.

Hemadsorption of horse or guinea pig erythrocytes by avian or human influenza A virus HA. (A) Vero cells were infected with either A/Duck/Singapore/97 (D/S) or A/Sydney/5/97 (SYD) at a multiplicity of infection of >1. Twenty-four hours postinfection, a 0.5% suspension of horse or guinea pig erythrocytes was added for 1 h at room temperature. Following washing with phosphate-buffered saline, Vero cells and bound erythrocytes were lysed. The absorbance of the clarified lysate was measured at 540 nm. (B) Vero cells were infected with FPV directing the expression of T7 polymerase and were then transfected with plasmids directing the expression of mutant H5 HA (1 μg) and HK156 M2 (0.2 μg) proteins. Forty-eight hours posttransfection, cells were treated with 5.5 mU of bacterial neuraminidase (Vibrio cholerae sialidase; Roche) per ml for 1 h. Then a 0.5% suspension of horse or guinea pig erythrocytes was added, and after 1 h of incubation at room temperature, cells were washed and lysed for absorbance measurement. Cells infected with FPV T7 but untransfected were processed in the same way, and the mean absorbance from these wells was subtracted as background. All transfections were performed in triplicate, and mean absorbance values with standard errors are shown. WT, wild type.

FIG. 2.

Modeling of point mutations into the receptor binding site of H5 HA. (A) Wild type; (B) G228S mutant; (C) Q226L mutant; (D) Q226V mutant; (E) LSS double mutant; (F) VSS double mutant. Native residues 226Q, 227S, and 228G are shown in orange, pink, and yellow, respectively. Mutations to a hydrogen bonding residue (serine) are shown in red, and mutations to a hydrophobic residue (leucine-valine) are shown in blue.

FIG. 3.

(A and B) Human and avian virus M2 protein expression. Forty-eight hours following transfection of 0.2 μg of plasmid DNA, cells were incubated with primary antibodies against M2. A mouse monoclonal antibody (14C2) was used to detect the human M2 proteins (A), and a rabbit polyclonal antiserum raised against a swine influenza virus M2, kindly provided by P. Heinen, was used to detect the avian M2 proteins (B). Fluorescein isothiocyanate-conjugated secondary antibody was used, and the mean fluorescence intensity of each sample was measured by fluorescence-activated cell sorter analysis. (C) Representation of the ability of different M2 proteins to support hemadsorption by H5 HA. Vero cells were transfected with 1 μg of plasmid directing the expression of HK156 HA and 0.2 μg of plasmid expressing different avian or human viral M2 proteins and incubated either with (grey bars) or without (black bars) 5 μM amantadine. Forty-eight hours posttransfection, cells were treated with 5.5 mU of bacterial neuraminidase (V. cholerae sialidase) per ml, and a 0.5% suspension of horse erythrocytes was added. After 1 h of incubation at room temperature, cells were washed and lysed for absorbance measurement at 540 nm. Cells infected with FPV T7 but untransfected were processed in the same way, and the mean absorbance from these wells was subtracted as background. All transfections were performed in triplicate, and mean absorbance values with standard errors are shown. D/S, A/Duck/Singapore/3/97; 156, A/HK/156/97; Pan, A/Panama/99; Syd, A/Sydney/5/97.