Influenza Virus Hemagglutinin: Acid-Induced Changes in T

血凝素（HA）是流感病毒与靶细胞中含唾液酸的受体结合的主要表面膜糖蛋白。病毒和内体膜的融合是由酸诱导的HA构象变化触发的，这发生在低pH值的内体中。结果，病毒转录酶复合物被转移到细胞中，并启动病毒复制。HA是由相同亚基的胞外域组成的三聚体整合膜糖蛋白（MW 220，000），每个亚基含有通过二硫键连接的两个多肽HA 1和HA 2。虽然HA 1是受体结合亚基，但HA 2通过低pH依赖性构象变化介导融合，该构象变化导致高度疏水的N-末端（融合肽）暴露。由于病毒融合是由酸化引发的，因此使用差示扫描量热法（DSC）、分析超离心、色氨酸荧光和圆二色性（CD）研究了流感病毒（X31株）血凝素（HA）的构象稳定性作为pH的函数。HA在7.4至5.4的pH范围内作为由5-6个三聚体（31-35 S）组成的球形复合物（玫瑰花结）沉淀。在pH 7.4下，在不存在（和存在1%辛基-β-葡糖苷）的情况下，HA的单个DSC吸热显示三个区域，其转变温度为66.0 +/-0.2（65.0）C，总焓变为800 +/-80 kcal/（mol三聚体），即使HA还原物在洗涤剂中解离成三聚体（10.3 S）。因此，在pH 7.4的罗丹明钠中，三聚体之间的分子间相互作用最多贡献约1 C的稳定性。当pH从pH7.4降低到5.4时，转变温度从66 ℃降低到45 ℃，解折叠温度从880 kcal/（mol三聚体）降低到260 kcal/（mol三聚体），在每个pH下仅观察到一个吸热。量热与vant霍夫热的相应比率从3.0（在pH7.4下）降低到约1.3（在pH5.4下）。在整个pH范围内保持了总体二级和三级结构（如远紫外和近紫外CD光谱所示），并且充分记录了酸化后HA结构的变化。破坏HA 1/HA 1接触的三聚体帐户观察到的质子诱导的表面疏水性增加和降低的内在色氨酸荧光低于pH 6.0，以及热展开的转变温度的大幅下降。在热展开的质子化，解离的HA 1区域，与其他部分展开的HA 1结构域内的三聚体的相互作用明显降低协同比（量热货车？t霍夫表位）。病毒融合的最佳温度（例如，37 ℃，pH5.4）低于HA在不同酸性pH值下的转变温度，这表明解离的HA 1远端结构域必须折叠以进行有效的病毒融合。

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.