title: Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation.
abstract: The influenza virus RNA-dependent RNA polymerase catalyzes genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits PB1, PB2, and PA are critical steps in the virus life cycle. We investigated the structure and function of the PA linker (residues 197 to 256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichroism experiments revealed that the PA linker by itself is structurally disordered. A large series of PA linker mutants exhibited a temperature-sensitive (ts) phenotype (reduced viral growth at 39.5°C versus 37°C/33°C), suggesting an alteration of folding kinetic parameters. The ts phenotype was associated with a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts and lethal PA mutants did not efficiently recruit PB1 to reach the nucleus at 39.5°C. A protein complementation assay using PA mutants, PB1, and β-importin IPO5 tagged with fragments of the Gaussia princeps luciferase showed that increasing the temperature negatively modulated the PA-PB1 and the PA-PB1-IPO5 interactions or complex stability. The selection of revertant viruses allowed the identification of different types of compensatory mutations located in one or the other of the three polymerase subunits. Two ts mutants were shown to be attenuated and able to induce antibodies in mice. Taken together, our results identify a PA domain critical for PB1-PA nuclear import and that is a "hot spot" to engineer ts mutants that could be used to design novel attenuated vaccines.
  
  
  OBJECTIVE
  By targeting a discrete domain of the PA polymerase subunit of influenza virus, we were able to identify a series of 9 amino acid positions that are appropriate to engineer temperature-sensitive (ts) mutants. This is the first time that a large number of ts mutations were engineered in such a short domain, demonstrating that rational design of ts mutants can be achieved. We were able to associate this phenotype with a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restored the ability of the complex to move to the nucleus. Two of these ts mutants were shown to be attenuated and able to produce antibodies in mice. These results are of high interest for the design of novel attenuated vaccines and to develop new antiviral drugs.
authors: INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
authors: Bruno
authors: B
authors: Da Costa
authors: INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
authors: Alix
authors: A
authors: Sausset
authors: Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, Paris, France CNRS, URA3015, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, Paris, France.
authors: Sandie
authors: S
authors: Munier
authors: INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
authors: Alexandre
authors: A
authors: Ghounaris
authors: Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, Département de Virologie, Paris, France CNRS, URA3015, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Unité de Génétique Moléculaire des Virus à ARN, Paris, France.
authors: Nadia
authors: N
authors: Naffakh
authors: INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
authors: Ronan
authors: http://orcid.org/0000-0002-2012-0064
authors: R
authors: Le Goffic
authors: INRA, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France bernard.delmas@jouy.inra.fr.
authors: Bernard
authors: http://orcid.org/0000-0003-2783-1093
authors: B
authors: Delmas
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002942:Circular Dichroism; D005260:Female; D005816:Genetic Complementation Test; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000502:physiology* / Q000528:radiation effects*; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding / Q000528:radiation effects; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication / Q000528:radiation effects*
chemical_list: D050505:Mutant Proteins; C061290:PA protein, influenza viruses; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase