Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy
Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy
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DOI：
10.1016/j.bbagen.2019.02.015
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发表时间：
2020-02-01
影响因子：
3
通讯作者：
Wong, Richard W.
中科院分区：
文献类型：
--
作者：
Lim, Kee Siang;Mohamed, Mahmoud Shaaban;Wong, Richard W.
背景：甲型流感病毒血凝素（HA）是介导病毒组分在宿主细胞中释放的关键毒力因子之一。HA最初合成为三聚体前体（HA 0），然后被蛋白酶切割成为功能性HA。低pH诱导HA 0和HA两者的不可逆构象变化，但仅HA是融合相容的。在这里，我们使用高速原子力显微镜（HS-AFM）记录HA 0三聚体（H5 N1）从中性到酸性条件下的构象变化在毫秒scale.Methods：纯化HA 0蛋白稀释中性Tris-HCl（pH 7.4）或乙酸滴定的Tris-HCl（pH 5.0），然后加载到裸云母。中性或酸性Tris-HCl用作扫描缓冲液。结果：HS-AFM显示的HA 0（中性）的构象与PDB数据和AFM模拟器中描述的HA三聚体结构相当。在低pH条件下，HA 0的构象发生了迅速的变化。HA 0（酸性）的环度和面积显著高于HA 0（中性）。相比之下，HA 0（酸）的高度显着低于HA 0（中性）。结论：我们已经捕获实时图像的本地HA 0三聚体结构在生理条件下使用HS-AFM。通过分析图像，我们确认HA 0三聚体是敏感的酸性conditions.General意义：HA结构的动态性质，特别是在宿主内体，是H5 N1感染性所必需的。了解这种酸性行为对于设计针对H5 N1的治疗策略至关重要。本文报道了一个复杂的新工具，用于研究时空动态的HA前体蛋白。
Background: 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.Methods: 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.Results: The conformation of HA0(neutral) 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(acid) were significantly higher than in HA0(neutral). In contrast, the height of HA0(acid) was significantly lower than in HA0(neutral).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.General significance: 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.