Pye Sarah E, Achouri Emna, Yang Yanling, Musa Abdulafiz, López Carolina B
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri, USA.
J Virol. 2025 Jul 31:e0089425. doi: 10.1128/jvi.00894-25.
Copyback viral genomes (cbVGs) are truncated viral genomes with complementary ends produced when the negative-sense RNA virus polymerase detaches from the replication template and resumes elongation from the nascent strand. Despite advances in methods to identify cbVGs based on the site of polymerase break and rejoin, PCR-based tools cannot provide full-length sequences of most cbVGs and/or can introduce errors and artifacts during cbVG amplification. These limitations have painted an incomplete picture of the diverse population of cbVGs generated during infection. To improve our ability to obtain native full-length sequences of cbVGs, we optimized direct RNA sequencing (DRS) as a fast and simple tool to sequence full-length cbVGs and harnessed a BLAST-based analysis approach to identify cbVGs from long-read sequencing data. We analyzed the DRS outputs of multiple Sendai virus (SeV) stocks to highlight both the utility and limitations of this tool. We found that to capture the dominant 546 nt cbVG produced by SeV strain Cantell, the length of complementarity between the virus trailer and the DRS oligonucleotide should optimally be increased to up to 32 nt. We also demonstrate comparable quality of cbVG sequences by DRS from as little RNA as 17.6 ng from the media fraction or 50 ng from the cellular fraction of cells infected with SeV, in contrast to the recommended 1,000 ng. Importantly, we validated different cbVG species from two recombinant SeV stocks, including cbVGs whose break positions occurred at or near position one in the reference genome.IMPORTANCEMost viruses of the order Mononegavirales have been demonstrated to naturally generate copyback viral genomes. These genomes are critical determinants of infection outcomes; they interfere with standard virus replication by competing for viral resources, activate antiviral responses, and inhibit protein translation. Despite their critical roles in infection, current tools to study copyback viral genomes rely either on preexisting knowledge of the sequence of a target RNA or require reverse transcription and amplification of the target RNA, biasing toward short copyback genomes and introducing relatively high rates of errors. Here, we detail the optimization of direct RNA sequencing to validate native full-length copyback viral genomes, including species that have not been validated previously.
回环病毒基因组（cbVGs）是负链RNA病毒聚合酶从复制模板上脱离并从新生链重新开始延伸时产生的具有互补末端的截短病毒基因组。尽管基于聚合酶断裂和重新连接位点识别cbVGs的方法取得了进展，但基于PCR的工具无法提供大多数cbVGs的全长序列，并且/或者在cbVG扩增过程中会引入错误和假象。这些局限性使得我们对感染过程中产生的多样化cbVGs群体的了解并不完整。为了提高我们获取cbVGs天然全长序列的能力，我们优化了直接RNA测序（DRS），将其作为一种快速简便的工具来对全长cbVGs进行测序，并采用基于BLAST的分析方法从长读长测序数据中识别cbVGs。我们分析了多种仙台病毒（SeV）毒株的DRS输出结果，以突出该工具的实用性和局限性。我们发现，为了捕获由SeV Cantell毒株产生的占主导地位的546 nt cbVG，病毒拖尾序列与DRS寡核苷酸之间的互补长度应最佳地增加至32 nt。我们还证明，与推荐的1000 ng相比，从感染SeV的细胞的培养基组分中仅17.6 ng或细胞组分中50 ng的RNA通过DRS获得的cbVG序列质量相当。重要的是，我们验证了来自两种重组SeV毒株的不同cbVG种类，包括那些断裂位置在参考基因组中第1位或其附近的cbVGs。
重要性
大多数单股负链RNA病毒目病毒已被证明会自然产生回环病毒基因组。这些基因组是感染结果的关键决定因素；它们通过竞争病毒资源干扰标准病毒复制，激活抗病毒反应，并抑制蛋白质翻译。尽管它们在感染中起关键作用，但目前研究回环病毒基因组的工具要么依赖于对目标RNA序列的现有了解，要么需要对目标RNA进行逆转录和扩增，这偏向于短回环基因组并引入相对较高的错误率。在这里，我们详细介绍了直接RNA测序的优化，以验证天然全长回环病毒基因组，包括以前未经验证的种类。
