Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment

ABSTRACT We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venu...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Yuri Furusawa, Shinya Yamada, Tiago Jose da Silva Lopes, Jayeeta Dutta, Zenab Khan, Divya Kriti, Harm van Bakel, Yoshihiro Kawaoka
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	foreign gene insertion genetic stability influenza recombinant virus virus polymerase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/89b8272724554fa485028d1ea5512c61
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:89b8272724554fa485028d1ea5512c61
record_format	dspace
spelling	oai:doaj.org-article:89b8272724554fa485028d1ea5512c612021-11-15T15:59:40ZInfluenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment10.1128/mBio.01794-192150-7511https://doaj.org/article/89b8272724554fa485028d1ea5512c612019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01794-19https://doaj.org/toc/2150-7511ABSTRACT We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venus-PR8, we discovered that the PB2-E712D mutation stabilizes the Venus gene. Here, we explored the mechanisms by which Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Deep sequencing analysis revealed that PB2-E712D does not cause an appreciable change in the mutation rate, suggesting that the stability of the Venus gene is not affected by polymerase fidelity. We found by using quantitative real-time PCR that WT-Venus-PR8 induces high-level interferon beta (IFN-β) expression. The induction of IFN-β expression seemed to result from the reduced transcription/replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription/replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. Loss of the Venus gene in WT-Venus-PR8 appeared to be caused by internal deletions in the NS segment. Moreover, to further our understanding of the Venus stabilization mechanisms, we identified additional amino acid mutations in the virus polymerase complex that stabilize the Venus gene. We found that some of these amino acids are located near the template exit or the product exit of the viral polymerase, suggesting that these amino acids contribute to the stability of the Venus gene by affecting the binding affinity between the polymerase complex and the RNA template and product. IMPORTANCE The reverse genetics method of influenza virus generation has enabled us to generate recombinant viruses bearing modified viral proteins. Recombinant influenza viruses expressing foreign genes have become useful tools in basic research, and such viruses can be utilized as efficient virus vectors or multivalent vaccines. However, the insertion of a foreign gene into the influenza virus genome often impairs virus replication, and the inserted genes are unstable. Elucidation of the mechanisms of foreign gene stabilization will help us to establish useful recombinant influenza viruses.Yuri FurusawaShinya YamadaTiago Jose da Silva LopesJayeeta DuttaZenab KhanDivya KritiHarm van BakelYoshihiro KawaokaAmerican Society for Microbiologyarticleforeign gene insertiongenetic stabilityinfluenzarecombinant virusvirus polymeraseMicrobiologyQR1-502ENmBio, Vol 10, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	foreign gene insertion genetic stability influenza recombinant virus virus polymerase Microbiology QR1-502
spellingShingle	foreign gene insertion genetic stability influenza recombinant virus virus polymerase Microbiology QR1-502 Yuri Furusawa Shinya Yamada Tiago Jose da Silva Lopes Jayeeta Dutta Zenab Khan Divya Kriti Harm van Bakel Yoshihiro Kawaoka Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
description	ABSTRACT We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venus-PR8, we discovered that the PB2-E712D mutation stabilizes the Venus gene. Here, we explored the mechanisms by which Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Deep sequencing analysis revealed that PB2-E712D does not cause an appreciable change in the mutation rate, suggesting that the stability of the Venus gene is not affected by polymerase fidelity. We found by using quantitative real-time PCR that WT-Venus-PR8 induces high-level interferon beta (IFN-β) expression. The induction of IFN-β expression seemed to result from the reduced transcription/replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription/replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. Loss of the Venus gene in WT-Venus-PR8 appeared to be caused by internal deletions in the NS segment. Moreover, to further our understanding of the Venus stabilization mechanisms, we identified additional amino acid mutations in the virus polymerase complex that stabilize the Venus gene. We found that some of these amino acids are located near the template exit or the product exit of the viral polymerase, suggesting that these amino acids contribute to the stability of the Venus gene by affecting the binding affinity between the polymerase complex and the RNA template and product. IMPORTANCE The reverse genetics method of influenza virus generation has enabled us to generate recombinant viruses bearing modified viral proteins. Recombinant influenza viruses expressing foreign genes have become useful tools in basic research, and such viruses can be utilized as efficient virus vectors or multivalent vaccines. However, the insertion of a foreign gene into the influenza virus genome often impairs virus replication, and the inserted genes are unstable. Elucidation of the mechanisms of foreign gene stabilization will help us to establish useful recombinant influenza viruses.
format	article
author	Yuri Furusawa Shinya Yamada Tiago Jose da Silva Lopes Jayeeta Dutta Zenab Khan Divya Kriti Harm van Bakel Yoshihiro Kawaoka
author_facet	Yuri Furusawa Shinya Yamada Tiago Jose da Silva Lopes Jayeeta Dutta Zenab Khan Divya Kriti Harm van Bakel Yoshihiro Kawaoka
author_sort	Yuri Furusawa
title	Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
title_short	Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
title_full	Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
title_fullStr	Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
title_full_unstemmed	Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment
title_sort	influenza virus polymerase mutation stabilizes a foreign gene inserted into the virus genome by enhancing the transcription/replication efficiency of the modified segment
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/89b8272724554fa485028d1ea5512c61
work_keys_str_mv	AT yurifurusawa influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT shinyayamada influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT tiagojosedasilvalopes influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT jayeetadutta influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT zenabkhan influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT divyakriti influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT harmvanbakel influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment AT yoshihirokawaoka influenzaviruspolymerasemutationstabilizesaforeigngeneinsertedintothevirusgenomebyenhancingthetranscriptionreplicationefficiencyofthemodifiedsegment
_version_	1718427039049973760

Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment

Ejemplares similares