title: A single amino acid at the hemagglutinin cleavage site contributes to the pathogenicity and neurovirulence of H5N1 influenza virus in mice.
abstract: H5 influenza viruses containing a motif of multiple basic amino acids at the hemagglutinin (HA) cleavage site (HACS) are highly pathogenic in chicken but display different virulence phenotypes in mammals. Previous studies have shown that multiple basic amino acids of H5N1 influenza virus are a prerequisite for lethality in mice. However, it remains unclear which specific residue at the cleavage site affects the pathogenicity of H5N1 in mammals. A comprehensive genetic analysis of the H5N1 HACS showed that residues at P6 (position 325, by H3 numbering) were the most polymorphic, including serine (S), arginine (R), deletion (*), glycine (G), and isoleucine (I). To determine whether a single residue at P6 could affect virulence, we introduced different mutations at P6 of an avirulent clade 7 H5N1 strain, rg325G, by reverse genetics. Among the recombinant viruses, the rg325S virus showed the highest cleavage efficiency in vitro. All these viruses were highly pathogenic in chicken but exhibited different virulences in mice. The rg325S virus exhibited the highest pathogenicity in terms of unrestricted organ tropism and neurovirulence. Remarkably, the HA-325S substitution dramatically increased the pathogenicity of H5N1 viruses of other clades, including clades 2.2, 2.3.2, and 2.3.4, indicating that this residue impacts genetically divergent H5N1 viruses. An analysis of predicted structures containing these mutations showed that the cleavage site loop with 325S was the most exposed, which might be responsible for the efficient cleavage and high virulence. Our results demonstrate that an amino acid substitution at the P6 cleavage site alone could modulate the virulence of H5N1 in mice.
authors.affiliation: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yipeng
authors.initials: Y
authors.lastname: Sun
authors.forename: Honglei
authors.initials: H
authors.lastname: Sun
authors.forename: Juan
authors.initials: J
authors.lastname: Pu
authors.forename: Yuhai
authors.initials: Y
authors.lastname: Bi
authors.forename: Yi
authors.initials: Y
authors.lastname: Shi
authors.forename: Xishan
authors.initials: X
authors.lastname: Lu
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Zhu
authors.forename: George F
authors.initials: GF
authors.lastname: Gao
authors.forename: Hanchun
authors.initials: H
authors.lastname: Yang
authors.forename: Jinhua
authors.initials: J
authors.lastname: Liu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D005585:Influenza in Birds / Q000821:virology*; D007251:Influenza, Human / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D014774:Virulence
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus