Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan.
Doctoral Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung, 40227, Taiwan.
Virol J. 2023 Oct 12;20(1):232. doi: 10.1186/s12985-023-02201-0.
In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo.
To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains.
In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions.
The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.
除了众所周知的冠状病毒基因组和亚基因组 mRNAs 外，还提出了其他冠状病毒 RNA 物种的存在，这些 RNA 统称为非规范转录物；然而，它们的生物学特性尚未在体外和体内得到实验验证。
为了全面确定牛冠状病毒在 HRT-18 细胞和小鼠肝炎病毒 A59（一种小鼠冠状病毒）中的非规范转录物的数量、种类和结构，采用纳米孔直接 RNA 测序。为了实验验证常规感染下非规范转录物的合成，进行了 Northern 印迹。Northern 印迹和纳米孔直接 RNA 测序也用于检验非规范转录物的重现性。此外，Northern 印迹还用于确定不同感染条件下非规范转录物的调控特征，包括不同的细胞、感染复数（MOI）和冠状病毒株。
在本研究中，我们（i）实验确定了冠状病毒非规范转录物大量合成，（ii）根据结构和潜在的合成机制将非规范转录物分为七类，（iii）表明非规范转录物在常规感染过程中的种类和数量具有重现性，但在改变的感染环境中受到调节，（iv）揭示了冠状病毒可能采用多种机制来合成非规范转录物，（v）发现了非规范冠状病毒转录物在体外和体内条件下的生物学特性相似。
首次对非规范冠状病毒转录物的生物学特性进行了实验验证。在丰度、重现性和多样性方面，非规范转录物的鉴定特征扩展了冠状病毒基因表达的当前模型。冠状病毒调节非规范转录物种类和数量的能力可能有助于病毒在感染过程中的发病机制，对疾病控制构成潜在挑战。因此，这里体外和体内的非规范转录物生物学可以为生物研究提供数据库，有助于抗病毒策略的发展。
