In vitro disassembly of a parvovirus capsid and effect on capsid stability of heterologous peptide insertions in surface loops
In vitro disassembly of a parvovirus capsid and effect on capsid stability of heterologous peptide insertions in surface loops
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DOI：
10.1074/jbc.m307662200
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发表时间：
2004-02-20
影响因子：
4.8
通讯作者：
Mateu, MG
中科院分区：
文献类型：
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作者：
Carreira, A;Menéndez, M;Mateu, MG
我们分析了小鼠微小病毒衣壳的体外解体，以及携带异源表位插入的衣壳嵌合体的稳定性。在生理缓冲液中加热后，由60个拷贝的蛋白质VP 2形成的空衣壳首先经历可逆的构象变化，其中通过荧光检测到小的焓变化。这种变化与但不限于VP 2 N末端的外部化有关。在更高的温度下，通过荧光、血凝活性和电泳迁移率的变化检测到不可逆的衣壳解离。在相同条件下的差示扫描量热法表明，解离/变性转变涉及高焓变化，并通过一个或多个中间体进行。与此相反，在1.5 m氯化胍的存在下，热诱导拆卸安装一个两态不可逆过程。热和化学诱导的解离/变性都产生了失去部分三级结构但仍保留天然二级结构的形式。来自化学解离的数据表明这种形式可能对应于衣壳蛋白的熔融球状单体状态。尝试在暴露的环中插入所有五种抗原肽，尽管可能是破坏性最小的，但会导致衣壳折叠/组装缺陷，并且在大多数情况下，会降低衣壳对热解离的稳定性。最简单的病毒衣壳之一的结果揭示了一个复杂的解体途径，以及在插入肽后衣壳组装和稳定性的降低，即使在最暴露的衣壳环内。
We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 m guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.