The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface–bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

中文翻译：

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膜融合 pH 下流感血凝素前体的可逆结构变化

流感血凝素 (HA) 三聚体的亚基被合成为单链前体 (HA0s)，它们被蛋白水解切割成二硫键连接的多肽 HA1 和 HA2。在低 pH 条件下激活膜融合需要切割，这发生在细胞表面结合病毒转移到核内体后感染开始时。激活导致裂解 HA 的构象发生广泛变化。为了确定裂解对 HA 介导的膜融合机制的总体贡献，我们使用低温电子显微镜 (cryo-EM) 在中性和低 pH 值下直接成像 HA0。我们发现了广泛的 pH 诱导的结构变化，其中一些与低 pH 下裂解 HA 重折叠中的中间体描述的相似。它们涉及通过熔化先前存在的二级结构形成的长中央盘绕线圈的部分延伸，将其穿入膜远端区域之间，然后重新折叠为延伸的螺旋。融合肽在其 N 末端共价连接，在其部分长度上采用两亲性螺旋构象，并重新定位并堆积在保守残基的互补表面凹槽上。此外，与裂解的 HA 相比，低 pH 下 HA0 结构的变化在中性 pH 下再孵育时是可逆的。我们讨论了共价限制性 HA0 重折叠对介导膜融合的裂解 HA 构象变化的影响，以及对抗病毒候选药物和可阻断流感传染性的交叉反应性抗 HA 抗体的作用的影响。