Role of spike protein endodomains in regulating coronavirus entry.
Role of spike protein endodomains in regulating coronavirus entry.
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DOI：
10.1074/jbc.m109.043547
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发表时间：
2009-11-20
期刊：
影响因子：
--
通讯作者：
Gallagher T
中科院分区：
文献类型：
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作者：
Shulla A;Gallagher T
有包膜病毒通过病毒糖蛋白介导的与宿主细胞的结合以及随后病毒与宿主细胞膜的融合而进入细胞。对于冠状病毒，病毒刺突 (S) 蛋白执行这些细胞进入功能。 S 蛋白与其他病毒和细胞膜融合蛋白的区别在于其广泛的棕榈酰化膜相关尾部。棕榈酸酯加合物通常是蛋白质介导的融合所必需的，但它们在该过程中的确切作用尚不清楚。为了获得对 S 介导的膜融合过程的更多了解，我们重点关注这些酰化的羧基末端病毒颗粒内尾部。用丙氨酸取代进行棕榈酰化的半胱氨酸对 S 掺入病毒粒子和 S 介导的膜融合都有影响。在专门剖析内域突变对融合过程的影响时，我们使用了仅与 S 介导的融合过程中的折叠中间体结合的抗病毒七肽重复肽，发现缺乏三个棕榈酰化半胱氨酸的突变体保持过渡折叠状态的时间比天然 S 蛋白长近 10 倍。这种较慢的重折叠也反映在突变体 S 蛋白中融合后六螺旋束构型的缺乏。棕榈酰化S蛋白半胱氨酸较少的病毒进入细胞缓慢，并且特异性感染性降低。这些发现表明，将 S 蛋白锚定到病毒膜上的脂质加合物对于快速、高效的 S 蛋白重折叠事件（最终导致膜融合）是必需的。这些研究揭示了病毒蛋白内域上共价连接的脂质引发膜融合反应的先前未被认识到的作用。
Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions.