Chaperone-mediated in vitro assembly of Polyomavirus capsids
Chaperone-mediated in vitro assembly of Polyomavirus capsids
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DOI：
10.1073/pnas.1832245100
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发表时间：
2003-09-02
影响因子：
11.1
通讯作者：
Garcea, RL
中科院分区：
文献类型：
--
作者：
Chromy, LR;Pipas, JM;Garcea, RL
多瘤病毒外壳蛋白病毒蛋白 1 (VP1) 具有在添加钙后在体外自组装成多态性衣壳样结构的内在能力。相比之下，多瘤病毒在体内的组装受到严格控制，因此大小均匀的病毒体仅在细胞核中形成。在病毒感染期间，72 kDa 细胞伴侣热休克同源蛋白 (hsc70) 与 VP1 翻译后结合，并与 VP1 共定位于细胞核，从而表明大约 70 kDa 热休克蛋白 (hsp70) 家族伴侣在调节衣壳组装的质量和位置中发挥作用。我们发现，在大肠杆菌中表达重组 VP1 后，原核 hsp70 分子伴侣 DnaK 与连接组装衣壳中五聚体的 VP1 C 端结构域共纯化。当与 VP1 稳定结合时，DnaK 抑制钙诱导的体外组装。然而，在 ATP 存在的情况下，由 DnaK、DnaJ 和 GrpE 组成的 hsp70 伴侣系统将 VP1 组装成均匀的衣壳，而不需要钙。伴侣介导的组装类似地由真核 hsc70 蛋白与猿猴病毒 40 大 T 抗原蛋白的 J 结构域功能结合催化。因此，可以使用原核或真核 hsp70 伴侣系统在体外高保真地重现多瘤病毒衣壳组装，从而支持细胞伴侣在病毒粒子组装的体内调节中的作用。
The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium. In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus. During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximate to70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly. We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid. When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium. However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium. Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly.