tetano
Editor, Senior Moderator
J Virol. 2015 Apr 8. pii: JVI.00589-15. [Epub ahead of print]
[h=1]Temperature-sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and hence result in viral attenuation.[/h] Da Costa B[SUP]1[/SUP], Sausset A[SUP]1[/SUP], Munier S[SUP]2[/SUP], Ghounaris A[SUP]1[/SUP], Naffakh N[SUP]2[/SUP], Le Goffic R[SUP]1[/SUP], Delmas B[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The influenza virus RNA-dependent RNA polymerase catalyses 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 virus cycle. We investigated the structure and function of the PA linker (residues 197-256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichro?sm 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 vs. 37?C/33?C), suggesting an alteration of folding kinetic parameters. The ts-phenotype was associated to a reduced efficiency of replication/transcription of a pseudo-viral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts- and lethal PA mutants do 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 modulates 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 identified a PA domain as being critical of PB1-PA nuclear import and as a "hot spot" to engineer ts-mutants that could be used to design novel attenuated vaccines.
[h=4]IMPORTANCE:[/h] 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. That is the first time that a large number of ts-mutations were engineered in such a short domain, demonstrating that rationale design of ts-mutants can be achieved. We were able to associate this phenotype to a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restore the ability of the complex to move to the nucleus. Two of these ts-mutants were shown attenuated and able to produce antibodies in mice. These results are of high interest to design novel attenuated vaccines and to elaborate antiviral drugs.
Copyright ? 2015, American Society for Microbiology. All Rights Reserved.
PMID: 25855727 [PubMed - as supplied by publisher]
[h=1]Temperature-sensitive mutants in the influenza A virus RNA polymerase: alterations in the PA linker reduce nuclear targeting of the PB1-PA dimer and hence result in viral attenuation.[/h] Da Costa B[SUP]1[/SUP], Sausset A[SUP]1[/SUP], Munier S[SUP]2[/SUP], Ghounaris A[SUP]1[/SUP], Naffakh N[SUP]2[/SUP], Le Goffic R[SUP]1[/SUP], Delmas B[SUP]3[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The influenza virus RNA-dependent RNA polymerase catalyses 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 virus cycle. We investigated the structure and function of the PA linker (residues 197-256), located between its N-terminal endonuclease domain and its C-terminal structured domain that binds PB1, the polymerase core. Circular dichro?sm 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 vs. 37?C/33?C), suggesting an alteration of folding kinetic parameters. The ts-phenotype was associated to a reduced efficiency of replication/transcription of a pseudo-viral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that ts- and lethal PA mutants do 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 modulates 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 identified a PA domain as being critical of PB1-PA nuclear import and as a "hot spot" to engineer ts-mutants that could be used to design novel attenuated vaccines.
[h=4]IMPORTANCE:[/h] 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. That is the first time that a large number of ts-mutations were engineered in such a short domain, demonstrating that rationale design of ts-mutants can be achieved. We were able to associate this phenotype to a defect of transport of the PA-PB1 complex into the nucleus. Reversion substitutions restore the ability of the complex to move to the nucleus. Two of these ts-mutants were shown attenuated and able to produce antibodies in mice. These results are of high interest to design novel attenuated vaccines and to elaborate antiviral drugs.
Copyright ? 2015, American Society for Microbiology. All Rights Reserved.
PMID: 25855727 [PubMed - as supplied by publisher]