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Biochemical characterization of the respiratory syncytial virus N0-P complex in solution

Abstract : 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.
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https://hal.archives-ouvertes.fr/hal-02276899
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Submitted on : Tuesday, September 3, 2019 - 10:56:58 AM
Last modification on : Wednesday, March 3, 2021 - 12:20:03 PM
Long-term archiving on: : Wednesday, February 5, 2020 - 2:05:58 PM

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Camille Esneau, Bertrand Raynal, Pierre Roblin, Sébastien Brûlé, Charles-Adrien Richard, et al.. Biochemical characterization of the respiratory syncytial virus N0-P complex in solution. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2019, 294 (10), pp.3647-3660. ⟨10.1074/jbc.RA118.006453⟩. ⟨hal-02276899⟩

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