Cargando…

Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation

Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. How...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Jessica W., Yang, Lijuan, Santos, Celia, Hassan, Sergio A., Collins, Peter L., Buchholz, Ursula J., Le Nouën, Cyril
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8751989/
https://www.ncbi.nlm.nih.gov/pubmed/34965283
http://dx.doi.org/10.1371/journal.ppat.1010191
_version_ 1784631798200795136
author Chen, Jessica W.
Yang, Lijuan
Santos, Celia
Hassan, Sergio A.
Collins, Peter L.
Buchholz, Ursula J.
Le Nouën, Cyril
author_facet Chen, Jessica W.
Yang, Lijuan
Santos, Celia
Hassan, Sergio A.
Collins, Peter L.
Buchholz, Ursula J.
Le Nouën, Cyril
author_sort Chen, Jessica W.
collection PubMed
description Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.
format Online
Article
Text
id pubmed-8751989
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-87519892022-01-12 Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation Chen, Jessica W. Yang, Lijuan Santos, Celia Hassan, Sergio A. Collins, Peter L. Buchholz, Ursula J. Le Nouën, Cyril PLoS Pathog Research Article Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate. Public Library of Science 2021-12-29 /pmc/articles/PMC8751989/ /pubmed/34965283 http://dx.doi.org/10.1371/journal.ppat.1010191 Text en https://creativecommons.org/publicdomain/zero/1.0/This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Chen, Jessica W.
Yang, Lijuan
Santos, Celia
Hassan, Sergio A.
Collins, Peter L.
Buchholz, Ursula J.
Le Nouën, Cyril
Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title_full Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title_fullStr Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title_full_unstemmed Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title_short Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
title_sort reversion mutations in phosphoprotein p of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8751989/
https://www.ncbi.nlm.nih.gov/pubmed/34965283
http://dx.doi.org/10.1371/journal.ppat.1010191
work_keys_str_mv AT chenjessicaw reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT yanglijuan reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT santoscelia reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT hassansergioa reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT collinspeterl reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT buchholzursulaj reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation
AT lenouencyril reversionmutationsinphosphoproteinpofacodonpairdeoptimizedhumanrespiratorysyncytialvirusconferincreasedtranscriptionimmunogenicityandgeneticstabilitywithoutlossofattenuation