Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization.
Int J Mol Sci
; 24(1)2022 Dec 29.
Article
en En
| MEDLINE
| ID: mdl-36614009
The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Virus Sincitial Respiratorio Humano
/
Nucleoproteínas
Idioma:
En
Revista:
Int J Mol Sci
Año:
2022
Tipo del documento:
Article
País de afiliación:
Francia
Pais de publicación:
Suiza