Multiuser Center for Biomolecular Innovation (CMIB), Instituto de Biociências, Letras e Ciências Exatas, UNESP, São José do Rio Preto, CEP, SP, Brazil.
Department of Physics, Instituto de Biociências, Letras e Ciências Exatas, UNESP, São José do Rio Preto, SP, Brazil.
J Biomol Struct Dyn. 2022 Mar;40(5):2156-2168. doi: 10.1080/07391102.2020.1835717. Epub 2020 Oct 20.
The human Respiratory Syncytial Virus (hRSV) is one of the most common causes of acute respiratory diseases such as bronchiolitis and pneumonia in children worldwide. Among the viral proteins, the nucleoprotein (N) stands out for forming the nucleocapsid (NC) that functions as a template for replication and transcription by the viral polymerase complex. The NC/polymerase recognition is mediated by the phosphoprotein (P), which establishes an interaction of its C-terminal residues with a hydrophobic pocket in the N-terminal domain of N (N-NTD). The present study consists of biophysical characterization of N-NTD and investigation of flavonoids binding to this domain using experimental and computational approaches. Saturation transfer difference (STD)-NMR measurements showed that among the investigated flavonoids, only hesperetin (Hst) bound to N-NTD. The binding epitope mapping of Hst suggested that its fused aromatic ring is buried in the protein binding site. STD-NMR and fluorescence anisotropy experiments showed that Hst competes with P protein C-terminal dipeptides for the hRSV nucleoprotein/phosphoprotein (N/P) interaction site in N-NTD, indicating that Hst binds to the hydrophobic pocket in this domain. Computational simulations of molecular docking and dynamics corroborated with experimental results, presenting that Hst established a stable interaction with the N/P binding site. The outcomes presented herein shed light on literature reports that described a significant antireplicative activity of Hst against hRSV, revealing molecular details that can provide the development of a new strategy against this virus.
人类呼吸道合胞病毒（hRSV）是全球儿童中引起细支气管炎和肺炎等急性呼吸道疾病的最常见原因之一。在病毒蛋白中，核蛋白（N）尤为突出，它形成核衣壳（NC），作为病毒聚合酶复合物复制和转录的模板。NC/聚合酶的识别是由磷蛋白（P）介导的，其 C 末端残基与 N 端结构域（N-NTD）中的疏水性口袋建立相互作用。本研究包括使用实验和计算方法对 N-NTD 进行生物物理特性分析以及研究类黄酮与该结构域的结合。饱和转移差异（STD）-NMR 测量表明，在所研究的类黄酮中，只有橙皮素（Hst）与 N-NTD 结合。Hst 的结合表位作图表明，其融合的芳环埋藏在蛋白质结合位点中。STD-NMR 和荧光各向异性实验表明，Hst 与 P 蛋白 C 末端二肽竞争 hRSV 核蛋白/磷蛋白（N/P）在 N-NTD 中的相互作用位点，表明 Hst 结合到该结构域中的疏水性口袋。分子对接和动力学的计算模拟与实验结果相符，表明 Hst 与 N/P 结合位点建立了稳定的相互作用。本研究结果阐明了文献报道中橙皮素对 hRSV 具有显著的抗病毒活性，揭示了可以为开发针对该病毒的新策略提供分子细节。
