title: A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins.
abstract: 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 5' 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.
  
  
  OBJECTIVE
  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 in vivo, combined in cellulae and in silico studies will help to forward the understanding of the unique, bipartite AAV life cycle.
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany.
authors.forename: Catrin
authors.initials: C
authors.lastname: Stutika
authors.affiliation: German Centre for Integrative Biodiversity Research, Halle-Jena-Leipzig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gogol-Döring
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Botschen
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Mietzsch
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Weger
authors.affiliation: Laboratory for Functional Genomics and Systems Biology, Berlin Institute for Medical Systems Biology, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Feldkamp
authors.affiliation: Laboratory for Functional Genomics and Systems Biology, Berlin Institute for Medical Systems Biology, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany regine.heilbronn@charite.de.
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
mesh_terms: D000256:Adenoviridae / Q000254:growth & development; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D020869:Gene Expression Profiling; D006801:Humans; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics*; D012326:RNA Splicing; D017423:Sequence Analysis, RNA; D014764:Viral Proteins / Q000235:genetics
chemical_list: D020033:Protein Isoforms; D014764:Viral Proteins