Full-Length Isoform Sequencing Reveals Novel Transcripts and Substantial Transcriptional Overlaps in a Herpesvirus

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

全转录组研究（whole transcriptome studies）已成为解析遗传调控复杂性的核心手段。然而，常规应用的短读长测序平台（short-read sequencing platforms）无法可靠区分多数转录本异构体（transcript isoforms）。太平洋生物科学（Pacific Biosciences, PacBio）RS II测序平台可在单次测序反应（sequencing run）中读取长核酸序列片段（long nucleic acid stretches）。伪狂犬病病毒（pseudorabies virus, PRV）是研究疱疹病毒基因表达及转录单元间潜在互作的优良模型系统。本研究采用无扩增及扩增型转录本测序方案（non-amplified and amplified isoform sequencing protocols），对PRV裂解期转录组（lytic transcriptome）的多聚腺苷酸化（poly(A+)）组分进行表征，旨在完成该病毒基因的完整转录注释（transcriptional annotation）。分析结果揭示了此前未被认知的PRV转录组复杂性，包括新型编码蛋白基因与非编码基因的发现、新型单顺反子及多顺反子转录单元（mono- and polycistronic transcription units）的鉴定，以及相邻乃至远端基因间广泛存在的转录重叠现象（transcriptional overlaps）。本研究鉴定出覆盖PRV全部三个复制起点（replication origins）的非编码转录本，这类转录本可能在DNA合成调控中发挥作用。此外，我们还确定了基因产物的相对表达水平（relative expression levels）。研究结果显示，PRV全基因组均存在转录活性，涵盖所有编码区及基因间区的两条DNA链。全基因组范围内的转录重叠现象表明，基因间可通过由互作转录机器（transcriptional machineries）构成的网络实现串扰（crosstalk），该网络可能在基因表达调控中具备潜在功能。