title: Dextran sulfate-resistant A/Puerto Rico/8/34 influenza virus is associated with the emergence of specific mutations in the neuraminidase glycoprotein.
abstract: Dextran sulfate (DS) is a negatively charged sulfated polysaccharide that suppresses the replication of influenza A viruses. The suppression was thought to be associated with inhibition of the hemagglutinin-dependent fusion activity. However, we previously showed that suppression by DS was observed not only at the initial stage of viral infection, but also later when virus is released from infected cells due to inhibition of neuraminidase (NA) activity. In the present study, we isolated DS-resistant A/Puerto Rico/8/34 (PR8) influenza viruses and analyzed the inhibition by DS. We found six mutations in NA genes of five independent resistant PR8 viruses and each resistant NA gene had two mutations. All mutations were from basic to acidic or neutral amino acids. In addition, R430L, K432E or K435E in the 430-435 region was a common mutation in all resistant NA genes. To determine which amino acid(s) are responsible for this resistance, a panel of recombinant viruses containing a PR8 and A/WSN/33(WSN) chimeric NA gene or an NA gene with different mutation(s) was generated using reverse genetics. Using recombinant viruses containing a PR8/WSN chimeric NA, we showed that one third of the C-terminal region of PR8 NA was responsible for DS-sensitivity. Recombinant viruses with a single mutation in NA replicated better than wild-type PR8 in the presence of DS, but were still DS-sensitive. However, replication of recombinant viruses with double mutations from the resistant viruses was not affected by the presence or absence of DS. In addition, resistant recombinant viruses were found to be sensitive to the NA inhibitor, oseltamivir and the oseltamivir-resistant recombinant virus was sensitive to DS. These results suggested that DS is an NA inhibitor with a different mechanism of action from the currently used NA inhibitors and that DS could be used in combination with these inhibitors to treat influenza virus infections.
authors.affiliation: Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan. Electronic address: hiyamada@nibio.go.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamada
authors.affiliation: National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Chioko
authors.initials: C
authors.lastname: Nagao
authors.affiliation: Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Ahmad M
authors.initials: AM
authors.lastname: Haredy
authors.affiliation: Division of Clinical Virology, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Mori
authors.affiliation: National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Mizuguchi
authors.affiliation: Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Yamanishi
authors.affiliation: Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
authors.forename: Shigefumi
authors.initials: S
authors.lastname: Okamoto
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D016264:Dextran Sulfate / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D007251:Influenza, Human / Q000821:virology*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053139:Oseltamivir / Q000494:pharmacology; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins; D053139:Oseltamivir; D016264:Dextran Sulfate; C487630:NA protein, influenza A virus; D009439:Neuraminidase
keywords: Dextran sulfate; Influenza; Neuraminidase