title: Analysis of influenza virus hemagglutinin receptor binding mutants with limited receptor recognition properties and conditional replication characteristics.
abstract: To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, O. Wayne Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322, USA.
authors.forename: Konrad C
authors.initials: KC
authors.lastname: Bradley
authors.forename: Summer E
authors.initials: SE
authors.lastname: Galloway
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.forename: Xuezheng
authors.initials: X
authors.lastname: Song
authors.forename: Jamie
authors.initials: J
authors.lastname: Heimburg-Molinaro
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Ganesh R
authors.initials: GR
authors.lastname: Talekar
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.forename: David A
authors.initials: DA
authors.lastname: Steinhauer
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D004285:Dogs; D004912:Erythrocytes / Q000378:metabolism / Q000821:virology; D005822:Genetic Vectors; D006168:Guinea Pigs; D006386:Hemagglutination Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000378:metabolism*; D006736:Horses; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000821:virology; D051379:Mice; D046228:Microarray Analysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009975:Orthomyxoviridae / Q000472:pathogenicity; D009976:Orthomyxoviridae Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011991:Receptors, Virus / Q000378:metabolism*; D012756:Sheep; D014421:Turkey; D014779:Virus Replication
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011134:Polysaccharides; D011991:Receptors, Virus