INTERMONOMER DISULFIDE BONDS IMPAIR THE FUSION ACTIVITY OF INFLUENZA-VIRUS HEMAGGLUTININ
INTERMONOMER DISULFIDE BONDS IMPAIR THE FUSION ACTIVITY OF INFLUENZA-VIRUS HEMAGGLUTININ
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DOI：
10.1128/jvi.66.8.4940-4950.1992
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发表时间：
1992-08-01
影响因子：
5.4
通讯作者：
WHITE, JM
中科院分区：
文献类型：
--
作者：
KEMBLE, GW;BODIAN, DL;WHITE, JM
在低pH下，流感病毒血凝素（HA）经历促进膜融合的构象变化。虽然融合肽从三聚体界面释放的关键作用已经在之前被证明，但球状头部解离在整体融合机制中的作用仍然不清楚。为了研究这个问题，我们详细分析了突变体Cys-HA的融合活性和低pH诱导的构象变化，其中球状头部结构域通过改造的单体间二硫键锁定在一起（L. Godley，J. Pfeifer，D.施泰因豪尔湾伊利，G.肖河Kaufmann，E.苏查内克角Pabo，J. J. Skehel，D. C. Wiley和S. Wharton，Cell 68：635 - 645，1992）。在本文中，我们表明，Cys-HA细胞表面上表达的主要是一个二硫键结合的三聚体。细胞表面Cys-HA的膜融合活性受损，如内容物混合和脂质混合融合测定所示。它在低pH下改变构象的能力也受损，如通过蛋白酶K敏感性所评估的。然而，在单体间二硫键还原后，细胞表面Cys-HA的融合活性和低pH诱导的构象变化恢复到接近野生型水平。通过使用一组构象特异性单克隆抗体和抗肽抗体，我们发现纯化的Cys-HA三聚体在球状头部结构域界面发生的变化中受损。此外，在离工程化单体间二硫键很远的地方发生的变化，特别是融合肽的释放，也受到损害。我们的研究结果进行了讨论，相对于目前的看法的HA三聚体的融合活性构象。
At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.