tetano
Editor, Senior Moderator
J Virol. 2016 Jan 20. pii: JVI.03101-15. [Epub ahead of print]
[h=1]Codon deletions in the influenza A virus PA gene generate temperature-sensitive viruses.[/h] Meyer L[SUP]1[/SUP], Sausset A[SUP]1[/SUP], Sedano L[SUP]1[/SUP], Da Costa B[SUP]1[/SUP], Le Goffic R[SUP]1[/SUP], Delmas B[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The influenza virus RNA-dependent RNA polymerase which is composed of three subunits, PB1, PB2, and PA, catalyses genome replication and transcription within the cell nucleus. The PA linker (residues 197-256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts) and attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at 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 single-codon deletion viable mutants were generated; all of them exhibited a ts-phenotype that was associated to a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at restrictive temperature (39.5?C). Mice infections showed that the four mutants were attenuated and induced antibodies able to protect from a lethal homologous wild-type virus challenge. Serial in vitro passages of two deletion mutants at 39.5?C and 37?C did not allowed to restore a wt-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.
[h=4]IMPORTANCE:[/h] In order to generate genetically stable live influenza A vaccines, we constructed viruses with single-codon deletion in a discrete domain of the RNA-polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found associated to a defect of transport of the PA-PB1 dimer to the nucleus where viral replication occurs. These deletion ts-mutants were shown to be attenuated, able to produce antibodies in mice and protect from a lethal challenge. Assays to select revertants able to grow efficiently at 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 influenza genetically stable live vaccines.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 26792748 [PubMed - as supplied by publisher]
[h=1]Codon deletions in the influenza A virus PA gene generate temperature-sensitive viruses.[/h] Meyer L[SUP]1[/SUP], Sausset A[SUP]1[/SUP], Sedano L[SUP]1[/SUP], Da Costa B[SUP]1[/SUP], Le Goffic R[SUP]1[/SUP], Delmas B[SUP]2[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] The influenza virus RNA-dependent RNA polymerase which is composed of three subunits, PB1, PB2, and PA, catalyses genome replication and transcription within the cell nucleus. The PA linker (residues 197-256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts) and attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at 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 single-codon deletion viable mutants were generated; all of them exhibited a ts-phenotype that was associated to a reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using a fluorescent-tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at restrictive temperature (39.5?C). Mice infections showed that the four mutants were attenuated and induced antibodies able to protect from a lethal homologous wild-type virus challenge. Serial in vitro passages of two deletion mutants at 39.5?C and 37?C did not allowed to restore a wt-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.
[h=4]IMPORTANCE:[/h] In order to generate genetically stable live influenza A vaccines, we constructed viruses with single-codon deletion in a discrete domain of the RNA-polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found associated to a defect of transport of the PA-PB1 dimer to the nucleus where viral replication occurs. These deletion ts-mutants were shown to be attenuated, able to produce antibodies in mice and protect from a lethal challenge. Assays to select revertants able to grow efficiently at 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 influenza genetically stable live vaccines.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 26792748 [PubMed - as supplied by publisher]