The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N-RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

中文翻译：

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呼吸道合胞病毒的RNA特异性核衣壳的体外可追踪组装。

呼吸道合胞病毒（RSV）聚合酶转录和复制的模板是螺旋核衣壳（NCs），由核蛋白（N）衣壳化的病毒RNA形成。正确的NC组装对于RSV聚合酶与RNA模板结合以进行RNA合成至关重要。先前对来自RSV和其他非分段负义RNA病毒的NCs或核衣壳样颗粒（NCLP）的研究为整体NC体系结构提供了见识。但是，在这些研究中，RNA是随机细胞RNA或平均病毒基因组RNA。由于缺乏可追踪NC或NCLP装配的体外检测方法，因此无法对NC进行深入的机械理解。在这里，我们建立了一个获取不含RNA的N蛋白（N0）的协议，并成功展示了一种新方法的实用性，该新方法可用于跟踪N与RNA寡核苷酸组装成NCLP的过程。我们发现，NCLP（N-RNA）组装的效率取决于掺入NCLPs中的RNA的长度和序列。这项工作提供了一个生成纯化的N0的框架，并以可控制的方式将其与RNA结合到NCLP中。我们预计，我们的体外可追踪RSV特异性核衣壳装配的检测方法可能会对此过程进行深入的机械分析。这项工作提供了一个生成纯化的N0的框架，并以可控制的方式将其与RNA结合到NCLP中。我们预计，我们的体外可追踪RSV特异性核衣壳装配的检测方法可能会对此过程进行深入的机械分析。这项工作提供了一个生成纯化的N0的框架，并以可控制的方式将其与RNA结合到NCLP中。我们预计，我们的体外可追踪RSV特异性核衣壳装配的检测方法可能会对此过程进行深入的机械分析。