Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

腺病毒（Adenovirus）是一类常见的人类病原体，其转录过程、病毒RNA加工及蛋白质合成都依赖宿主细胞的生命活动机制。尽管此前已有研究通过低通量生化技术或基于短读长cDNA的测序技术，对腺病毒启动子、剪接连接位点以及切割与多聚腺苷酸化位点完成了表征，但这些技术无法完全揭示腺病毒转录组的复杂性。本研究结合Illumina短读长测序与纳米孔（Nanopore）长读长直接RNA测序技术，在全腺病毒基因组范围内绘制了转录起始位点与切割及多聚腺苷酸化位点的图谱。在确认已知经典病毒早期与晚期RNA转录单元的基础上，本研究通过对长读段RNA中的剪接连接位点进行分析，额外发现了35种新型病毒转录本。这些RNA包含14个可介导经典开放阅读框（open reading frame, ORF）表达的新型剪接连接位点、6种携带新型ORF的转录本，以及15种转录本，其编码的mRNA可通过截短或融合经典ORF的方式潜在改变蛋白质功能。此外，本研究还检测到一类可绕过经典切割位点的RNA，它们通过连接独立的基因转录单元，编码潜在的嵌合蛋白质。其中，研究人员检测到一种进化保守的蛋白质，其包含E4orf6的N端与下游DBP/E2A ORF融合的序列。这种新型蛋白质E4orf6/DBP的缺失，会导致病毒复制中心形态异常以及病毒扩散能力受损。本研究凸显了长读长测序技术在揭示病毒转录组更深层次复杂性方面的重要价值。