title: Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein.
abstract: Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals.
  
  
  OBJECTIVE
  We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Hanson
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Masaki
authors.initials: M
authors.lastname: Imai
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Masato
authors.initials: M
authors.lastname: Hatta
authors.affiliation: Departments of Cell and Molecular Biology and Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: McBride
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Imai
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Taft
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Gongxun
authors.initials: G
authors.lastname: Zhong
authors.affiliation: ERATO Infection-Induced Host Responses Project, Saitama, Japan.
authors.forename: Tokiko
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: College of Life and Health Sciences, Chubu University, Aichi, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Neumann
authors.affiliation: Departments of Cell and Molecular Biology and Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Paulson
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA ERATO Infection-Induced Host Responses Project, Saitama, Japan Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan kawaokay@svm.vetmed.wisc.edu.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawaoka
mesh_terms: D000220:Adaptation, Biological; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000502:physiology*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D020125:Mutation, Missense; D055550:Protein Stability; D011991:Receptors, Virus / Q000378:metabolism; D013696:Temperature; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D050505:Mutant Proteins; D011991:Receptors, Virus; C109963:hemagglutinin, avian influenza A virus