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
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 b...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-09-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/18/13862 |
| _version_ | 1797579746485207040 |
|---|---|
| author | Cédric Diot Charles-Adrien Richard Jennifer Risso-Ballester Davy Martin Jenna Fix Jean-François Eléouët Christina Sizun Marie-Anne Rameix-Welti Marie Galloux |
| author_facet | Cédric Diot Charles-Adrien Richard Jennifer Risso-Ballester Davy Martin Jenna Fix Jean-François Eléouët Christina Sizun Marie-Anne Rameix-Welti Marie Galloux |
| author_sort | Cédric Diot |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T22:40:34Z |
| format | Article |
| id | doaj.art-b770b3ae919c41c5ad614c12d9cb1e03 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T22:40:34Z |
| publishDate | 2023-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-b770b3ae919c41c5ad614c12d9cb1e032023-11-19T11:04:17ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-09-0124181386210.3390/ijms241813862Hardening of Respiratory Syncytial Virus Inclusion Bodies by Cyclopamine Proceeds through Perturbation of the Interactions of the M2-1 Protein with RNA and the P ProteinCédric Diot0Charles-Adrien Richard1Jennifer Risso-Ballester2Davy Martin3Jenna Fix4Jean-François Eléouët5Christina Sizun6Marie-Anne Rameix-Welti7Marie Galloux8Institut Pasteur, Université Paris Cité, M3P, F-75015 Paris, FranceINRAE, Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay-Versailles St. Quentin, F-78350 Jouy-en-Josas, FranceINSERM, UMR 1173 (2I), Université Paris-Saclay-Versailles St. Quentin, M3P, F-78180 Versailles, FranceINRAE, Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay-Versailles St. Quentin, F-78350 Jouy-en-Josas, FranceINRAE, Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay-Versailles St. Quentin, F-78350 Jouy-en-Josas, FranceINRAE, Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay-Versailles St. Quentin, F-78350 Jouy-en-Josas, FranceInstitut de Chimie des Substances Naturelles, CNRS, Université Paris-Saclay, F-91190 Gif-sur-Yvette, FranceINSERM, UMR 1173 (2I), Université Paris-Saclay-Versailles St. Quentin, M3P, F-78180 Versailles, FranceINRAE, Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay-Versailles St. Quentin, F-78350 Jouy-en-Josas, FranceRespiratory 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.https://www.mdpi.com/1422-0067/24/18/13862RSVcyclopamineM2-1-P interactionantiviral mechanisminclusion bodies |
| spellingShingle | Cédric Diot Charles-Adrien Richard Jennifer Risso-Ballester Davy Martin Jenna Fix Jean-François Eléouët Christina Sizun Marie-Anne Rameix-Welti Marie Galloux 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 International Journal of Molecular Sciences RSV cyclopamine M2-1-P interaction antiviral mechanism inclusion bodies |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | RSV cyclopamine M2-1-P interaction antiviral mechanism inclusion bodies |
| url | https://www.mdpi.com/1422-0067/24/18/13862 |
| work_keys_str_mv | AT cedricdiot hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT charlesadrienrichard hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT jenniferrissoballester hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT davymartin hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT jennafix hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT jeanfrancoiseleouet hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT christinasizun hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT marieannerameixwelti hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein AT mariegalloux hardeningofrespiratorysyncytialvirusinclusionbodiesbycyclopamineproceedsthroughperturbationoftheinteractionsofthem21proteinwithrnaandthepprotein |