title: Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.
abstract: The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC), Madrid, Spain; Ciber de Enfermedades Respiratorias (ISCIII), Madrid, Spain.
authors.forename: Maite
authors.initials: M
authors.lastname: Pérez-Cidoncha
authors.affiliation: School of Biology, Centre for Biomolecular Sciences, University of St Andrews, St Andrews, United Kingdom.
authors.forename: Marian J
authors.initials: MJ
authors.lastname: Killip
authors.affiliation: Fundació d'Investigació Sanitària de les Illes Balears (FISIB), Bunyola, Mallorca, Spain.
authors.forename: Víctor J
authors.initials: VJ
authors.lastname: Asensio
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC), Madrid, Spain; Ciber de Enfermedades Respiratorias (ISCIII), Madrid, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Fernández
authors.affiliation: Laboratory Microbial Pathogenesis, Fundació d'Investigació Sanitària de les Illes Balears (FISIB), Bunyola, Mallorca, Spain; Ciber de Enfermedades Respiratorias (ISCIII), Madrid, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Bengoechea
authors.affiliation: School of Biology, Centre for Biomolecular Sciences, University of St Andrews, St Andrews, United Kingdom.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Randall
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC), Madrid, Spain; Ciber de Enfermedades Respiratorias (ISCIII), Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Ortín
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007113:Immunity, Innate; D009980:Influenza A virus / Q000235:genetics / Q000502:physiology*; D007372:Interferons / Q000096:biosynthesis* / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D017931:DNA Primers; C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins; D007372:Interferons