Abstract：
Rift Valley fever virus (RVFV; genus Phlebovirus, family Phenuiviridae, order Bunyavirales) is a mosquito-borne zoonotic pathogen endemic in Africa. Its negative-stranded genomic RNA (vRNA) is divided into three segments termed L, M, and S. Both vRNAs and antigenomic cRNAs are encapsidated by viral nucleoprotein (N) to form nucleocapsids, which constitute the template for genome transcription and replication. Based on a number of electron microscopy and structural studies, the viral RNAs of negative-strand RNA viruses, including phleboviruses, are commonly considered to be entirely and uniformly covered by N protein. However, high resolution data supporting this notion was missing to date.Here, we describe a method how to globally map all N-RNA interactions of RVFV by using iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation). The protocol is based on covalent cross-linking of direct protein-RNA interactions by UV irradiation. Following sample lysis, a selective isolation of N in complex with its RNA targets is achieved by immunoprecipitation. Then, N-RNA complexes are separated by SDS-PAGE, and after membrane transfer, RNA is isolated and subjected to library preparation and high-throughput sequencing. We explain how the standard iCLIP protocol can be adapted to RVFV N-RNA interaction studies. The protocol describes mapping of all N interactions with the vRNAs and cRNAs derived either from RVFV particles or from infected cells.
摘要：
裂谷热病毒（RVFV；Phlebovirus属，Phenuiviridae科，目Bunyavirales）是非洲特有的蚊子传播的人畜共患病原体。它的负链基因组RNA（vRNA）分为三个片段，称为L，M,和S。vRNA和反基因组cRNA都被病毒核蛋白（N）包裹，形成核衣壳，构成基因组转录和复制的模板。基于许多电子显微镜和结构研究，负链RNA病毒的病毒RNA，包括静脉病毒,通常认为被N蛋白完全和均匀地覆盖。然而,迄今为止，支持这一概念的高分辨率数据仍然缺失。这里,我们描述了如何通过使用iCLIP（单个核苷酸分辨率UV交联和免疫沉淀）全局映射RVFV的所有N-RNA相互作用的方法。该方案基于通过UV照射的直接蛋白质-RNA相互作用的共价交联。样品裂解后，通过免疫沉淀实现了与RNA靶标复合的N的选择性分离。然后，通过SDS-PAGE分离N-RNA复合物,膜转移后，分离RNA并进行文库制备和高通量测序。我们解释了标准iCLIP协议如何适应RVFVN-RNA相互作用研究。该方案描述了所有N相互作用与源自RVFV颗粒或感染细胞的vRNA和cRNA的映射。
