title: Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice.
abstract: In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity.
  
  
  OBJECTIVE
  Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.
authors.affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Hooper
authors.affiliation: Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Crowe
authors.affiliation: Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA jbloom@fhcrc.org.
authors.forename: Jesse D
authors.identifier: http://orcid.org/0000-0003-1267-3408
authors.initials: JD
authors.lastname: Bloom
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D004285:Dogs; D005260:Female; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology / Q000502:physiology*; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology / Q000502:physiology*; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism*; D009976:Orthomyxoviridae Infections / Q000473:pathology / Q000821:virology; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism; D059386:Reverse Genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D053585:Virus Attachment; D014776:Virus Cultivation
chemical_list: D050505:Mutant Proteins; D011991:Receptors, Virus; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase