title: Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses.
abstract: The influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four viable mutants with single-codon deletions were generated; all of them exhibited a ts phenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serial in vitro passages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts mutants that could be used to design novel attenuated vaccines.
  
  
  OBJECTIVE
  In order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA-PB1 dimer to the nucleus, where viral replication occurs. These ts deletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
authors.forename: Léa
authors.initials: L
authors.lastname: Meyer
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
authors.forename: Alix
authors.initials: A
authors.lastname: Sausset
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Sedano
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Da Costa
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
authors.forename: Ronan
authors.identifier: http://orcid.org/0000-0002-2012-0064
authors.initials: R
authors.lastname: Le Goffic
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, INRA, Université Paris-Saclay, Jouy-en-Josas, France bernard.delmas@jouy.inra.fr.
authors.forename: Bernard
authors.identifier: http://orcid.org/0000-0003-2783-1093
authors.initials: B
authors.lastname: Delmas
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D003062:Codon; D004195:Disease Models, Animal; D005260:Female; D042822:Genomic Instability; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009976:Orthomyxoviridae Infections / Q000821:virology; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013696:Temperature; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D003062:Codon; D050505:Mutant Proteins; C061290:PA protein, influenza viruses; D014613:Vaccines, Attenuated; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase