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Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein
The fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase pr...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8475503/ https://www.ncbi.nlm.nih.gov/pubmed/34379500 http://dx.doi.org/10.1128/JVI.01205-21 |
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author | Tang, Wei Li, Yueyue Song, Qiaoyun Wang, Ziqin Li, Manmei Zhang, Qiwei Wang, Ying Ye, Wencai Li, Yaolan |
author_facet | Tang, Wei Li, Yueyue Song, Qiaoyun Wang, Ziqin Li, Manmei Zhang, Qiwei Wang, Ying Ye, Wencai Li, Yaolan |
author_sort | Tang, Wei |
collection | PubMed |
description | The fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase protocol for RSV fusion inhibitor discovery. A small-molecule inhibitor, salvianolic acid R (LF-6), was identified to inhibit virus-cell and cell-cell fusion mediated by the RSV F protein. Sequence analysis of the resultant resistant viruses identified a K394R mutation in the viral F protein. The K394R mutant virus also conferred cross-resistance to multiple RSV fusion inhibitors, including several inhibitors undergoing clinical trials. Our study further showed that K394R mutation not only increased the triggering rate of F protein in prefusion conformation but also enhanced the fusion activity of F protein, both of which were positively correlated with resistance to fusion inhibitors. Moreover, the K394R mutation also showed cooperative effects with other escape mutations to increase the fusion activity of F protein. By substitution of K394 into different amino acids, we found that K394R or K394H substitution resulted in hyperfusiogenic F proteins, whereas F variants with other substitutions exhibited less fusion activity. Both K394R and K394H in F protein exhibited cross-resistance to RSV fusion inhibitors. Collectively, these findings reveal a positive correlation between the membrane fusion activity of F protein and the resistance of corresponding inhibitors. All of the results demonstrate that K394R in F protein confers cross-resistance to fusion inhibitors through destabilizing F protein and increasing its membrane fusion activity. IMPORTANCE Respiratory syncytial virus (RSV) causes serious respiratory tract disease in children and the elderly. Therapeutics against RSV infection are urgently needed. This study reports the discovery of a small-molecule inhibitor of RSV fusion glycoprotein by using a dual-luciferase protocol. The escape mutation (K394R) of this compound also confers cross-resistance to multiple RSV fusion inhibitors that have been reported previously, including two candidates currently in clinical development. The combination of K394R with other escape mutations can increase the resistance of F protein to these inhibitors through destabilizing F protein and enhancing the membrane fusion activity of F protein. By amino acid deletion or substitution, we found that a positively charged residue at the 394th site is crucial for the fusion ability of F protein, as well as for the cross-resistance against RSV fusion inhibitors. These results reveal the mechanism of cross-resistance conferred by the K394R mutation and the possible cross-resistance risk of RSV fusion inhibitors. |
format | Online Article Text |
id | pubmed-8475503 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-84755032021-10-01 Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein Tang, Wei Li, Yueyue Song, Qiaoyun Wang, Ziqin Li, Manmei Zhang, Qiwei Wang, Ying Ye, Wencai Li, Yaolan J Virol Virus-Cell Interactions The fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase protocol for RSV fusion inhibitor discovery. A small-molecule inhibitor, salvianolic acid R (LF-6), was identified to inhibit virus-cell and cell-cell fusion mediated by the RSV F protein. Sequence analysis of the resultant resistant viruses identified a K394R mutation in the viral F protein. The K394R mutant virus also conferred cross-resistance to multiple RSV fusion inhibitors, including several inhibitors undergoing clinical trials. Our study further showed that K394R mutation not only increased the triggering rate of F protein in prefusion conformation but also enhanced the fusion activity of F protein, both of which were positively correlated with resistance to fusion inhibitors. Moreover, the K394R mutation also showed cooperative effects with other escape mutations to increase the fusion activity of F protein. By substitution of K394 into different amino acids, we found that K394R or K394H substitution resulted in hyperfusiogenic F proteins, whereas F variants with other substitutions exhibited less fusion activity. Both K394R and K394H in F protein exhibited cross-resistance to RSV fusion inhibitors. Collectively, these findings reveal a positive correlation between the membrane fusion activity of F protein and the resistance of corresponding inhibitors. All of the results demonstrate that K394R in F protein confers cross-resistance to fusion inhibitors through destabilizing F protein and increasing its membrane fusion activity. IMPORTANCE Respiratory syncytial virus (RSV) causes serious respiratory tract disease in children and the elderly. Therapeutics against RSV infection are urgently needed. This study reports the discovery of a small-molecule inhibitor of RSV fusion glycoprotein by using a dual-luciferase protocol. The escape mutation (K394R) of this compound also confers cross-resistance to multiple RSV fusion inhibitors that have been reported previously, including two candidates currently in clinical development. The combination of K394R with other escape mutations can increase the resistance of F protein to these inhibitors through destabilizing F protein and enhancing the membrane fusion activity of F protein. By amino acid deletion or substitution, we found that a positively charged residue at the 394th site is crucial for the fusion ability of F protein, as well as for the cross-resistance against RSV fusion inhibitors. These results reveal the mechanism of cross-resistance conferred by the K394R mutation and the possible cross-resistance risk of RSV fusion inhibitors. American Society for Microbiology 2021-09-27 /pmc/articles/PMC8475503/ /pubmed/34379500 http://dx.doi.org/10.1128/JVI.01205-21 Text en Copyright © 2021 Tang et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Virus-Cell Interactions Tang, Wei Li, Yueyue Song, Qiaoyun Wang, Ziqin Li, Manmei Zhang, Qiwei Wang, Ying Ye, Wencai Li, Yaolan Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title | Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title_full | Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title_fullStr | Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title_full_unstemmed | Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title_short | Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein |
title_sort | mechanism of cross-resistance to fusion inhibitors conferred by the k394r mutation in respiratory syncytial virus fusion protein |
topic | Virus-Cell Interactions |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8475503/ https://www.ncbi.nlm.nih.gov/pubmed/34379500 http://dx.doi.org/10.1128/JVI.01205-21 |
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