Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

Rebecca M Dubois,Stephen W White,Charles J Russell,Richard J Heath,Hassan Zaraket,Muralidhar Reddivari,Félix A Rey

doi:10.1371/journal.ppat.1002398

Rebecca M Dubois, Stephen W White + Show 5 more

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https://doi.org/10.1371/journal.ppat.1002398

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Abstract

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

Highlights

Pathogenic avian influenza (HPAI) viruses kill up to 100% of infected poultry flocks and may cause high mortality rates when transmitted to humans [1,2]
The acid stability of the HA protein is identified as a novel virulence factor for emerging H5N1 influenza viruses
moderately pathogenic (MP) and highly pathogenic (HP) HA proteins have different acid stabilities To determine how the HA proteins from the two isolates differ in their biochemical properties, the proteins were expressed in cell culture and compared for expression, cleavage, receptor binding, and activation pH (Figure 1, S1)

Summary

Introduction

Pathogenic avian influenza (HPAI) viruses kill up to 100% of infected poultry flocks and may cause high mortality rates when transmitted to humans [1,2]. The continued circulation of H5N1 and potential emergence of an H5N1 human pandemic virus remain everpresent threats. The hemagglutinin (HA) surface glycoprotein promotes viral entry through its receptor binding and membrane fusion functions [5], and mutations in HA have been shown to modulate the pathogenicity, host range specificity, transmissibility, and pandemic potential of influenza viruses [1,6,7]. HA is synthesized as a trimeric HA0 protein that must be activated for membrane fusion by post-translational cleavage into a high-energy HA1/HA2 complex. The multi-basic HA0 cleavage sites of H5 and H7

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