Department of Medical Biology, Albert Szent-Györgyi Medical School, University of Szeged, Somogyi u. 4, 6720 Szeged, Hungary.
Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungária krt. 21, 1143 Budapest, Hungary.
Viruses. 2022 Jun 13;14(6):1289. doi: 10.3390/v14061289.
In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the gene is expressed with immediate-early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene () for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.
在这项工作中，我们使用基于牛津纳米孔技术 MinION 平台的长读测序（LRS）技术，在 12 小时的感染周期内定量分析和动力学表征牛α疱疹病毒 1（BoHV-1）裂解转录组的多聚（A）片段。基于扩增的 LRS 技术经常产生人为转录读段，并且偏向于产生较短的扩增子。为了避免这些不良影响，我们应用了无扩增的直接 cDNA 测序技术。在这里，我们展示了单个启动子可以产生多个转录起始位点，其分布模式在不同的病毒基因之间存在差异，但在同一基因的不同时间点相似。我们的研究表明， 基因通过利用另一个立即早期（IE）基因（）的启动子进行转录控制，以 IE 动力学方式表达。此外，我们检测到 DNA 复制的起始与 基因转录之间存在重叠，这表明这两种分子机制之间存在相互作用。这项研究开发了一种普遍适用的基于 LRS 的方法，用于对任何生物体的转录组进行时间过程特征分析。
