Division of Veterinary Medicine, Paul-Ehrlich-Institute, Langen, Germany.
Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA.
Methods Mol Biol. 2024;2808:71-88. doi: 10.1007/978-1-0716-3870-5_6.
Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.
回文缺陷干扰 RNA 是副黏病毒和其他负链 RNA 病毒病毒株制备物的主要污染物。它们是正链抗原基因组和负链基因组的杂交分子。它们具有完全互补的末端，允许形成极其稳定的双链 RNA 柄状结构。3'-末端启动子的存在允许病毒聚合酶复制这些分子。因此，它们会对标准基因组的复制产生负干扰。此外，双链 RNA 茎结构具有高度的免疫刺激性，并激活抗病毒细胞固有先天免疫反应。因此，回文缺陷干扰 RNA 严重影响副黏病毒株的毒力和发病机制。我们描述了两种生化方法来分析感染病毒的样本或纯化的病毒 RNA 中的回文缺陷干扰 RNA。首先，我们介绍了 Northern 印迹技术，该技术可准确确定缺陷干扰 RNA 分子的大小，并估计病毒制剂的相对污染水平。其次，我们描述了一种专门扩增缺陷干扰 RNA 的 PCR 方法，该方法允许进行详细的序列分析。
