Abstract

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.

摘要

人类呼吸道合胞病毒 (hRSV) 是全球儿童急性呼吸道疾病（如细支气管炎和肺炎）的最常见原因之一。在病毒蛋白中，核蛋白 (N) 因形成核衣壳 (NC) 而脱颖而出，该核衣壳作为病毒聚合酶复合物复制和转录的模板。NC/聚合酶识别由磷蛋白 (P) 介导，磷蛋白 (P) 建立其 C 末端残基与 N (N-NTD) 的 N 末端结构域中的疏水袋的相互作用。本研究包括 N-NTD 的生物物理表征和使用实验和计算方法研究与该域结合的黄酮类化合物。饱和转移差 (STD)-NMR 测量表明，在研究的类黄酮中，只有橙皮素 (Hst) 与 N-NTD 结合。Hst的结合表位作图表明其稠合的芳环埋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 N-NTD 中的 hRSV 核蛋白/磷蛋白 (N/P) 相互作用位点，表明 Hst 与该结构域中的疏水袋结合。分子对接和动力学的计算模拟证实了实验结果，表明 Hst 与 N/P 结合位点建立了稳定的相互作用。本文提出的结果揭示了描述 Hst 对 hRSV 的显着抗复制活性的文献报道，揭示了可以提供对抗这种病毒的新策略的分子细节。