1. Biochemical characterization of the respiratory syncytial virus N0-P complex in solution
- Author
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Jean-François Eléouët, Sébastien Brûlé, Charles-Adrien Richard, Pierre Roblin, Jenna Fix, Camille Esneau, Marie Galloux, Bertrand Raynal, Centre National de la Recherche Scientifique - CNRS (FRANCE), Institut National Polytechnique de Toulouse - Toulouse INP (FRANCE), Institut Pasteur (FRANCE), Université Toulouse III - Paul Sabatier - UT3 (FRANCE), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris], Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génie Chimique (LGC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), French Agence Nationale de la Recherche, specific program ANR Blanc ANR-13-IVS3-0007, Institut Pasteur [Paris] (IP), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Unité de recherche Virologie et Immunologie Moléculaires (VIM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Institut National Polytechnique de Toulouse - INPT (FRANCE), Eleouet, Jean Francois, and Galloux, Marie
- Subjects
0301 basic medicine ,Biochimie, Biologie Moléculaire ,Respiratory syncytial virus ,Biochemistry ,Virus ,03 medical and health sciences ,Human metapneumovirus ,N0-P complex ,Viral replication ,Génie chimique ,Nucleoprotein N ,Mononegavirales ,Génie des procédés ,Molecular Biology ,protein folding ,structural model ,small-angle X-ray scattering (saxs) ,mutagenesis ,analytical ultracentrifugation ,n0-p complex ,nucleoprotein n ,respiratory syncytial virus ,structure-function ,viral replication ,030102 biochemistry & molecular biology ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Structure-function ,Chemistry ,RNA ,Cell Biology ,biology.organism_classification ,Nucleoprotein ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,030104 developmental biology ,Phosphoprotein ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Protein folding - Abstract
International audience; As all the viruses belonging to the Mononegavirales order, the non-segmented negative strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1-30 of P specifically bind to N0. Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P-like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.
- Published
- 2019
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