Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein.
Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein.
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DOI：
10.1128/jvi.01205-21
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发表时间：
2021-09-27
影响因子：
5.4
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Tang W;Li Y;Song Q;Wang Z;Li M;Zhang Q;Wang Y;Ye W;Li Y
融合糖蛋白 (F) 对于呼吸道合胞病毒 (RSV) 的进入至关重要，并已成为抗 RSV 药物开发的一个有吸引力的靶标。尽管RSV F抑制剂前景广阔，但耐药性问题仍然具有挑战性。在本研究中，我们建立了用于 RSV 融合抑制剂发现的双荧光素酶方案。一种小分子抑制剂丹酚酸 R (LF-6) 被鉴定可抑制 RSV F 蛋白介导的病毒-细胞和细胞-细胞融合。对产生的抗性病毒进行序列分析，发现病毒 F 蛋白中存在 K394R 突变。 K394R突变病毒还对多种RSV融合抑制剂产生交叉耐药性，其中包括几种正在进行临床试验的抑制剂。我们的研究进一步表明，K394R突变不仅增加了F蛋白融合前构象的触发率，而且增强了F蛋白的融合活性，两者均与融合抑制剂的耐药性呈正相关。此外，K394R突变还表现出与其他逃逸突变的协同作用，以增加F蛋白的融合活性。通过将 K394 替换为不同的氨基酸，我们发现 K394R 或 K394H 替换导致了高度融合的 F 蛋白，而具有其他替换的 F 变体表现出较低的融合活性。 F蛋白中的K394R和K394H均表现出对RSV融合抑制剂的交叉耐药性。总的来说，这些发现揭示了 F 蛋白的膜融合活性与相应抑制剂的耐药性之间呈正相关。所有结果都表明F蛋白中的K394R通过破坏F蛋白的稳定性并增加其膜融合活性而赋予融合抑制剂交叉抗性。重要性 呼吸道合胞病毒 (RSV) 会导致儿童和老年人出现严重的呼吸道疾病。迫切需要针对 RSV 感染的治疗方法。本研究报告通过使用双荧光素酶方案发现了 RSV 融合糖蛋白的小分子抑制剂。该化合物的逃逸突变 (K394R) 还对先前报道的多种 RSV 融合抑制剂产生交叉耐药性，其中包括目前正在临床开发的两种候选药物。 K394R与其他逃逸突变的组合可以通过使F蛋白不稳定并增强F蛋白的膜融合活性来增加F蛋白对这些抑制剂的抵抗力。通过氨基酸缺失或取代，我们发现第394位点的带正电残基对于F蛋白的融合能力以及对RSV融合抑制剂的交叉耐药性至关重要。这些结果揭示了K394R突变带来的交叉耐药机制以及RSV融合抑制剂可能存在的交叉耐药风险。
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.