title: An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.
abstract: Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.
  
  
  OBJECTIVE
  Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Zanin
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Susu
authors.initials: S
authors.lastname: Duan
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Sook-San
authors.initials: SS
authors.lastname: Wong
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Gyanendra
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Pradyumna
authors.initials: P
authors.lastname: Baviskar
authors.affiliation: Ceva Animal Health, Lenexa, Kansas, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Collin
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Russell
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Subrata
authors.initials: S
authors.lastname: Barman
authors.affiliation: Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hause
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA richard.webby@stjude.org.
authors.forename: Richard
authors.initials: R
authors.lastname: Webby
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D059645:Mutation Rate; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D000072417:Protein Domains / Q000235:genetics*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D000596:Amino Acids; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase
keywords: influenza; neuraminidase; stalk