Contribution in an anthology

Authors list: Shalamova, Lyudmila; Lorenzo, Gema; Brun, Alejandro; Rossbach, Oliver; Weber, Friedemann

Appeared in: Rift Valley Fever Virus

Editor list: Lozach, Pierre-Yves

Publication year: 2024

Pages: 281-318

ISBN: 978-1-0716-3925-2

eISBN: 978-1-0716-3926-9

DOI Link: https://doi.org/10.1007/978-1-0716-3926-9_18

Title of series: Methods in Molecular Biology

Number in series: 2824

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.

Harvard Citation style: Shalamova, L., Lorenzo, G., Brun, A., Rossbach, O. and Weber, F. (2024) Nucleotide Resolution Mapping of Rift Valley Fever Virus Nucleoprotein-Genome RNA Interactions, in Lozach, P. (ed.) Rift Valley Fever Virus. New York, NY: Humana , pp. 281-318. https://doi.org/10.1007/978-1-0716-3926-9_18

APA Citation style: Shalamova, L., Lorenzo, G., Brun, A., Rossbach, O., & Weber, F. (2024). Nucleotide Resolution Mapping of Rift Valley Fever Virus Nucleoprotein-Genome RNA Interactions. In Lozach, P. (Ed.), Rift Valley Fever Virus (pp. 281-318). Humana . https://doi.org/10.1007/978-1-0716-3926-9_18