Influenza Virus Hemagglutinin: Acid-Induced Changes in T

血凝素（HA）是主要的表面膜糖蛋白，负责将流感病毒与靶细胞中含有唾液酸的受体结合。病毒和内体膜的融合是由酸诱导的HA构象变化引发的，这种变化发生在内体的低pH值中。结果，病毒转录酶复合体被转移到细胞中，病毒复制开始。HA是一种三聚体整体膜糖蛋白（分子量22万），由相同亚基的外结构域组成，每个外结构域包含两个多肽，HA1和HA2，由二硫键连接。虽然HA1是受体结合亚基，但HA2通过低ph依赖性构象变化介导融合，从而暴露高度疏水的n端，即融合肽。利用差示扫描量热法（DSC）、分析性超离心、色氨酸荧光和圆二色性（CD）研究了流感病毒（株X31）血凝素（HA）的构象稳定性，因为病毒融合是由酸化触发的。在pH 7.4 ~ 5.4范围内，HA沉积物呈5 ~ 6个三聚体（31 ~ 35s）组成的球形复合体（莲座）。当pH为7.4时，在没有（和存在1%辛基- β -葡萄糖苷）的情况下，HA的单DSC吸热显示出三个结构域，转变温度为66.0 +/- 0.2 (65.0)C，总焓变为800 +/- 80 kcal/（mol三聚体），即使HA莲座在洗洁精中解离为三聚体（10.3 S）。因此，在pH 7.4下，莲座中三聚体之间的分子间相互作用最多有助于大约1℃的稳定。当pH值从7.4降至5.4时，转变温度从66℃降至45℃，展开焓从880 kcal/（mol三聚体）降至260 kcal/（mol三聚体），每个pH值只观察到一个恒温。相应的热霍夫焓比从3.0 （pH值7.4）降至约1.3 （pH值5.4）。在整个pH范围内，二级和三级结构总体保持不变（如远紫外和近紫外CD光谱所示），并且酸化后HA结构的变化有很好的记录。三聚体中HA1/HA1接触的破坏解释了在pH值低于6.0时质子诱导的表面疏水性增加和固有色氨酸荧光的减少，以及热展开转变温度的大幅降低。在质子化解离HA1区域的热展开过程中，与三聚体中其他部分展开的HA1结构域的相互作用明显降低了协同性比(量热法与范？t霍夫焓)每三聚体表示。病毒融合的最佳温度（例如，pH值为5.4时37℃）低于HA在不同酸性pH值下的转变温度，这表明解离的HA1远端结构域必须折叠才能进行病毒融合。

Hemagglutinin (HA) is the major surface membrane glycoprotein responsible for the binding of influenza virus to sialic-acid containing receptors in target cells. Fusion of viral and endosomal membranes is triggered by an acid-induced conformational change of HA, which takes place in the low pH of the endosomes. As a result, the viral transcriptase complex is transferred to the cell, and viral replication is initiated. HA is a trimeric integral membrane glycoprotein (MW 220,000) comprised of an ectodomain of identical subunits, each of which contains two polypeptides, HA1 and HA2, linked by a disulfide bond. While HA1 is the receptor-binding subunit, HA2 mediates fusion through a low pH-dependent conformational change that leads to exposure of the highly hydrophobic N-terminus, the fusion peptide. The conformational stability of influenza virus (strain X31) hemagglutinin (HA) has been investigated using differential scanning calorimetry (DSC), analytical ultracentrifugation, Trp fluorescence, and circular dichroism (CD) as a function of pH since viral fusion is triggered by acidification. HA sediments as a spherical complex (rosette) comprised of 5-6 trimers (31-35 S) over the pH range of 7.4 to 5.4. A single DSC endotherm of HA at pH 7.4 in the absence (and presence of 1% octyl-beta-glucoside) shows three domains with a transition temperature of 66.0 +/- 0.2 (65.0) C and overall enthalpy change of 800 +/- 80 kcal/(mol trimer) even though HA rosettes are dissociated to trimers (10.3 S) in detergent. Intermolecular interactions among trimers in rosettes at pH 7.4 therefore contribute at most approximately 1 C stabilization. As the pH is decreased from pH 7.4 to 5.4, transition temperatures decrease from 66 to 45 C and unfolding enthalpies decrease from 880 to 260 kcal/(mol trimer) with only one endotherm observed at each pH. Corresponding ratios of calorimetric to vant Hoff enthalpies decrease from 3.0 (at pH 7.4) to approximately 1.3 (at pH 5.4). Overall secondary and tertiary structures are maintained throughout this pH range (as shown by far- and near-UV CD spectra), and the changes in HA structure upon acidification are well documented. Disruption of HA1/HA1 contacts in a trimer account for the observed proton-induced increase in surface hydrophobicity and reduction of intrinsic tryptophan fluorescence below pH 6.0, as well as for the large decreases in transition temperatures for thermal unfolding. During thermal unfolding of protonated, dissociated HA1 regions, interactions with other partially unfolded HA1 domains within the trimer apparently decrease cooperativity ratios (calorimetric to van?t Hoff enthalpies) expressed per trimer. Optimal temperatures for viral fusion (e.g., 37 C at pH 5.4) are lower than transition temperatures of HA at different acidic pH values, which indicates that dissociated HA1 distal domains must be folded for competent viral fusion.