Adeno-associated virus type 2 transcriptome analysis by Illumina-based RNA sequencing (RNA-Seq). adeno-associated virus 2

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.

腺相关病毒（Adeno-associated virus, AAV）以其双相生命周期著称：其增殖性复制依赖于腺病毒（adenovirus, Ad）的共感染，而在无辅助病毒存在时可建立潜伏感染。从潜伏状态转向腺病毒依赖型增殖复制的过程，主要在转录层面受到调控。现有AAV转录图谱所展示的，均为与腺病毒共感染时所检测到的高丰度转录本。截至目前，AAV转录本仅在其单链DNA基因组的正链上得到了表征，而负链被认为不具备转录活性。本研究采用基于Illumina平台的RNA测序（RNA-Seq）技术，对有无腺病毒共感染条件下的完整AAV2转录组进行系统表征。我们不仅鉴定出了已知的AAV转录本，还发现了全新的转录本类型，包括额外的可变剪接变体；其中丰度最高的可变剪接变体可编码一种全新的18kDa Rep/VP融合蛋白。此外，我们首次在AAV负链上检测到转录现象：其测序读段在p5启动子上游成簇分布，该结果通过5'端cDNA末端快速扩增技术（5'-RACE）与RNase保护实验得到了验证。p5启动子在双向均展现出显著的转录活性，这一发现表明该启动子存在双向转录现象。仅感染AAV时，可检测到AAV两条链的低水平转录；当与腺病毒共感染时，该转录水平会得到显著上调。