Abstract：
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.
摘要：
复制缺陷型干扰RNA是麻疹病毒和其他负链RNA病毒的病毒原液制剂的主要污染物。它们是正义反基因组和负义基因组的杂合分子。它们具有完全互补的末端,允许形成极其稳定的双链RNA泛柄结构。3'-末端启动子的存在允许这些分子通过病毒聚合酶复制。因此,它们负面地干扰标准基因组的复制。此外,双链RNA茎结构具有高度免疫刺激性,可激活抗病毒细胞固有的先天免疫应答.因此，复制缺陷的干扰RNA严重影响麻疹病毒种群的毒力和发病机理。我们描述了两种生化方法来分析病毒感染样品中的复制回缺陷干扰RNA，或纯化的病毒RNA。首先,我们提出了我们的Northern印迹方案,该方案可以准确确定有缺陷的干扰RNA分子的大小,并估计病毒制剂的相对污染水平.第二,我们描述了一种PCR方法来扩增有缺陷的干扰RNA，这允许详细的序列分析。
