WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kakuma-machi, Kanazawa, Japan.
Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan.
Biochim Biophys Acta Gen Subj. 2020 Feb;1864(2):129313. doi: 10.1016/j.bbagen.2019.02.015. Epub 2019 Feb 27.
Hemagglutinin (HA) of influenza A is one of the key virulence factors that mediates the release of viral components in host cells. HA is initially synthesized as a trimeric precursor (HA0) and then it is cleaved by proteases to become a functional HA. Low pH induces irreversible conformational changes in both HA0 and HA but only HA is fusion compatible. Here, we used high-speed atomic force microscopy (HS-AFM) to record conformational changes in HA0 trimers (H5N1) from neutral to acidic conditions at a millisecond scale.
Purified HA0 protein was diluted with either neutral Tris-HCl (pH 7.4) or acetic acid-titrated Tris-HCl (pH 5.0) and then loaded onto bare mica. Neutral or acidic Tris-HCl was used as the scanning buffer. HS-AFM movies were recorded and processed using Image J software.
The conformation of HA0 visualized using HS-AFM was comparable to the HA trimer structures depicted in the PDB data and the AFM simulator. HA0 underwent rapid conformational changes under low pH condition. The circularity and area of HA0 were significantly higher than in HA0. In contrast, the height of HA0 was significantly lower than in HA0 CONCLUSIONS: We have captured real-time images of the native HA0 trimer structure under physiological conditions using HS-AFM. By analyzing the images, we confirm that HA0 trimer is sensitive to acidic conditions.
The dynamic nature of the HA structure, particularly in the host endosome, is essential for H5N1 infectivity. Understanding this acidic behavior is imperative for designing therapeutic strategies against H5N1. This article reports a sophisticated new tool for studying the spatiotemporal dynamics of the HA precursor protein.
甲型流感的血凝素（HA）是介导病毒成分在宿主细胞中释放的关键毒力因子之一。HA 最初作为三聚体前体（HA0）合成，然后被蛋白酶切割成为有功能的 HA。低 pH 值诱导 HA0 和 HA 发生不可逆的构象变化，但只有 HA 具有融合兼容性。在这里，我们使用高速原子力显微镜（HS-AFM）在毫秒尺度上记录 H5N1 的 HA0 三聚体从中性到酸性条件的构象变化。
纯化的 HA0 蛋白用中性 Tris-HCl（pH 7.4）或乙酸滴定的 Tris-HCl（pH 5.0）稀释，然后加载到裸云母上。中性或酸性 Tris-HCl 用作扫描缓冲液。使用 Image J 软件记录和处理 HS-AFM 电影。
使用 HS-AFM 可视化的 HA0 构象与 PDB 数据和 AFM 模拟器中描绘的 HA 三聚体结构相当。HA0 在低 pH 条件下迅速发生构象变化。HA0 的圆形度和面积明显高于 HA0。相比之下，HA0 的高度明显低于 HA0。
我们使用 HS-AFM 在生理条件下捕获了天然 HA0 三聚体结构的实时图像。通过分析图像，我们确认 HA0 三聚体对酸性条件敏感。
HA 结构的动态特性，特别是在宿主内体中，对于 H5N1 的感染力至关重要。了解这种酸性行为对于设计针对 H5N1 的治疗策略至关重要。本文报道了一种研究 HA 前体蛋白时空动力学的复杂新工具。
