Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

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.

H5N1亚型高致病性禽流感病毒仍持续威胁农业生产与人类健康。本研究借助生物化学与X射线晶体学技术，揭示了血凝素（hemagglutinin, HA）蛋白的氨基酸变异如何影响H5N1禽流感病毒在鸡体内的致病性。研究发现，高致病性（HP）A/chicken/Hong Kong/YU562/2001与中度致病性（MP）A/goose/Hong Kong/437-10/1999两株H5N1分离株的HA蛋白表达量与切割效率相近，且二者的受体结合特性无显著差异；然而，HA1受体结合结构域（receptor-binding domain, RBD）的酯酶样亚结构域（vestigial esterase sub-domain）内104位与115位的氨基酸变异，可调控HA蛋白的激活pH阈值：HP型与MP型HA蛋白分别在pH 5.7与pH 5.3的环境下激活以完成膜融合。总体而言，在观测到的pH 5.2至6.0范围内，H5N1在鸡体内的致病性提升与HA激活pH的升高呈正相关，该规律适用于突变型与嵌合型HA蛋白。本研究解析了MP型HA蛋白的晶体结构，分辨率达2.50埃，同时解析了两株HP型HA蛋白的晶体结构，分辨率分别为2.95埃与3.10埃；调控HA蛋白酸稳定性的104与115位氨基酸，位于酯酶样亚结构域内110螺旋的N端与C端，该螺旋可与HA2茎结构域的B环发生相互作用。110螺旋与茎结构域之间的相互作用似乎在调控HA蛋白酸稳定性的过程中发挥关键作用，进而影响流感病毒的复制与致病机制。总体而言，H5N1禽流感病毒要在禽类体内呈现高致病性，其HA蛋白需具备适宜的激活pH阈值。