Hardening of Respiratory Syncytial Virus Inclusion Bodies by Cyclopamine Proceeds through Perturbation of the Interactions of the M2-1 Protein with RNA and the P Protein

• Published in: International journal of molecular sciences 2023-09, Vol.24 (18), p.13862
• Main Authors: Diot, Cédric, Richard, Charles-Adrien, Risso-Ballester, Jennifer, Martin, Davy, Fix, Jenna, Eléouët, Jean-François, Sizun, Christina, Rameix-Welti, Marie-Anne, Galloux, Marie
• Format: Article
• Language: English
• Subjects: antiviral mechanism; cyclopamine; inclusion bodies; Infections; Life Sciences; M2-1-P interaction; Morphogenesis; Mutation; Protein-protein interactions; Proteins; Respiratory syncytial virus; RNA; RNA polymerase; RSV; Synthesis; Transcription factors; Viral proteins; Viruses
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms241813862

Respiratory syncytial virus (RSV) RNA synthesis takes place in cytoplasmic viral factories also called inclusion bodies (IBs), which are membrane-less organelles concentrating the viral RNA polymerase complex. The assembly of IBs is driven by liquid-liquid phase separation promoted by interactions between the viral nucleoprotein N and the phosphoprotein P. We recently demonstrated that cyclopamine (CPM) inhibits RSV multiplication by disorganizing and hardening IBs. Although a single mutation in the viral transcription factor M2-1 induced resistance to CPM, the mechanism of action of CPM still remains to be characterized. Here, using FRAP experiments on reconstituted pseudo-IBs both in cellula and in vitro, we first demonstrated that CPM activity depends on the presence of M2-1 together with N and P. We showed that CPM impairs the competition between P and RNA binding to M2-1. As mutations on both P and M2-1 induced resistance against CPM activity, we suggest that CPM may affect the dynamics of the M2-1-P interaction, thereby affecting the relative mobility of the proteins contained in RSV IBs. Overall, our results reveal that stabilizing viral protein-protein interactions is an attractive new antiviral approach. They pave the way for the rational chemical optimization of new specific anti-RSV molecules.