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. A deno-associated viruses (AAV) are helper-dependent members of the parvovirus group that require coinfection with an unrelated helper virus, particularly adenovirus (Ad) for productive replication (1). Despite of several identified AAV serotypes most research has been done with prototype AAV type 2. The AAV2 genome consists of a linear, single-stranded DNA of 4.7 kb. Both ends carry identical inverted terminal repeats (ITR) of 145 bp (2), which flank the two major open reading frames (ORF), called rep and cap. The rep ORF codes for four nonstructural proteins, Rep78 and a C-terminally spliced variant, Rep68. In addition, N-terminally truncated versions thereof are expressed, called Rep52 and Rep40, respectively. The Rep proteins are required as regulators for various steps of the AAV life cycle. AAV cap harbors the ORF for the capsid proteins (VP1 to -3) and a separate ORF for the assembly-activating protein (AAP) (3).The current knowledge of AAV transcription dates back to early research in the 1980s when the three AAV2 promoters, p5, p19, or p40 and major transcripts derived thereof were characterized (4, 5). All AAV transcripts identified since then map to only one DNA plus strand (6), which led to the assumption that the complementary AAV minus strand was not transcribed. Furthermore, spliced AAV transcripts were identified displaying a single, shared intron located in the center of the AAV2 genome (4, 7). Initially, a single splice donor site at AAV nucleotide 1906 and a single splice acceptor site located at nucleotide (nt) 2228 were described. Later, an alternative splice acceptor site at nt 2201 was detected (8), allowing the expression of VP1 protein ...

Mapping and Engineering Functional Domains of the Assembly-Activating Protein of Adeno-associated Viruses

“…Adeno-associated virus (AAV) encodes different proteins within its 4.7-kb genome using alternative splicing, alternative start codons, and overlapping reading frames (23). AAP is expressed from an alternative reading frame overlapping the VP2/3 sequences (5).…”

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys

“…All parvoviral genomes comprise two major genes encoding a non-structural replication protein NS1 (gene rep) and a capsid protein VP (gene cap). Alternative splicing or alternative translation initiation sites can allow the production of truncated forms of VP; all sharing the same C-terminal region [1,2]. Open reading frames (ORFs) usually overlapping the NS1 or VP reading frames encode smaller genus-specific accessory proteins.…”

Subgenomic satellite particle generation in recombinant AAV vectors results from DNA lesion/breakage and non-homologous end joining

“…As suggested in Figure 7, subgenome particles with a promoter can produce dsRNA which will be detrimental to long term gene expression. Although dsRNA was investigated in AAV vectors (10,11), here we identified SBG as the true source for such dsRNA formation. In addition, the SBG containing only the promoter can potentially cause tumorigenesis events in the host cells or in human patients (12).…”