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A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

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Zeitschriftentitel: Journal of Virology
Personen und Körperschaften: Stutika, Catrin, Gogol-Döring, Andreas, Botschen, Laura, Mietzsch, Mario, Weger, Stefan, Feldkamp, Mirjam, Chen, Wei, Heilbronn, Regine
In: Journal of Virology, 90, 2016, 3, S. 1278-1289
Medientyp: E-Article
Sprache: Englisch
veröffentlicht:
American Society for Microbiology
Schlagwörter:
author_facet Stutika, Catrin
Gogol-Döring, Andreas
Botschen, Laura
Mietzsch, Mario
Weger, Stefan
Feldkamp, Mirjam
Chen, Wei
Heilbronn, Regine
Stutika, Catrin
Gogol-Döring, Andreas
Botschen, Laura
Mietzsch, Mario
Weger, Stefan
Feldkamp, Mirjam
Chen, Wei
Heilbronn, Regine
author Stutika, Catrin
Gogol-Döring, Andreas
Botschen, Laura
Mietzsch, Mario
Weger, Stefan
Feldkamp, Mirjam
Chen, Wei
Heilbronn, Regine
spellingShingle Stutika, Catrin
Gogol-Döring, Andreas
Botschen, Laura
Mietzsch, Mario
Weger, Stefan
Feldkamp, Mirjam
Chen, Wei
Heilbronn, Regine
Journal of Virology
A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
Virology
Insect Science
Immunology
Microbiology
author_sort stutika, catrin
spelling Stutika, Catrin Gogol-Döring, Andreas Botschen, Laura Mietzsch, Mario Weger, Stefan Feldkamp, Mirjam Chen, Wei Heilbronn, Regine 0022-538X 1098-5514 American Society for Microbiology Virology Insect Science Immunology Microbiology http://dx.doi.org/10.1128/jvi.02750-15 <jats:title>ABSTRACT</jats:title> <jats:p>Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.</jats:p> <jats:p> <jats:bold>IMPORTANCE</jats:bold> Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV <jats:italic>in vivo</jats:italic> , combined <jats:italic>in cellulae</jats:italic> and <jats:italic>in silico</jats:italic> studies will help to forward the understanding of the unique, bipartite AAV life cycle. </jats:p> A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins Journal of Virology
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title A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_unstemmed A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_full A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_fullStr A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_full_unstemmed A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_short A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_sort a comprehensive rna sequencing analysis of the adeno-associated virus (aav) type 2 transcriptome reveals novel aav transcripts, splice variants, and derived proteins
topic Virology
Insect Science
Immunology
Microbiology
url http://dx.doi.org/10.1128/jvi.02750-15
publishDate 2016
physical 1278-1289
description <jats:title>ABSTRACT</jats:title> <jats:p>Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.</jats:p> <jats:p> <jats:bold>IMPORTANCE</jats:bold> Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV <jats:italic>in vivo</jats:italic> , combined <jats:italic>in cellulae</jats:italic> and <jats:italic>in silico</jats:italic> studies will help to forward the understanding of the unique, bipartite AAV life cycle. </jats:p>
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author Stutika, Catrin, Gogol-Döring, Andreas, Botschen, Laura, Mietzsch, Mario, Weger, Stefan, Feldkamp, Mirjam, Chen, Wei, Heilbronn, Regine
author_facet Stutika, Catrin, Gogol-Döring, Andreas, Botschen, Laura, Mietzsch, Mario, Weger, Stefan, Feldkamp, Mirjam, Chen, Wei, Heilbronn, Regine, Stutika, Catrin, Gogol-Döring, Andreas, Botschen, Laura, Mietzsch, Mario, Weger, Stefan, Feldkamp, Mirjam, Chen, Wei, Heilbronn, Regine
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description <jats:title>ABSTRACT</jats:title> <jats:p>Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.</jats:p> <jats:p> <jats:bold>IMPORTANCE</jats:bold> Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV <jats:italic>in vivo</jats:italic> , combined <jats:italic>in cellulae</jats:italic> and <jats:italic>in silico</jats:italic> studies will help to forward the understanding of the unique, bipartite AAV life cycle. </jats:p>
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spelling Stutika, Catrin Gogol-Döring, Andreas Botschen, Laura Mietzsch, Mario Weger, Stefan Feldkamp, Mirjam Chen, Wei Heilbronn, Regine 0022-538X 1098-5514 American Society for Microbiology Virology Insect Science Immunology Microbiology http://dx.doi.org/10.1128/jvi.02750-15 <jats:title>ABSTRACT</jats:title> <jats:p>Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.</jats:p> <jats:p> <jats:bold>IMPORTANCE</jats:bold> Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV <jats:italic>in vivo</jats:italic> , combined <jats:italic>in cellulae</jats:italic> and <jats:italic>in silico</jats:italic> studies will help to forward the understanding of the unique, bipartite AAV life cycle. </jats:p> A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins Journal of Virology
spellingShingle Stutika, Catrin, Gogol-Döring, Andreas, Botschen, Laura, Mietzsch, Mario, Weger, Stefan, Feldkamp, Mirjam, Chen, Wei, Heilbronn, Regine, Journal of Virology, A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins, Virology, Insect Science, Immunology, Microbiology
title A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_full A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_fullStr A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_full_unstemmed A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_short A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
title_sort a comprehensive rna sequencing analysis of the adeno-associated virus (aav) type 2 transcriptome reveals novel aav transcripts, splice variants, and derived proteins
title_unstemmed A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins
topic Virology, Insect Science, Immunology, Microbiology
url http://dx.doi.org/10.1128/jvi.02750-15