In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli
In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli
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DOI：
10.1128/jvi.73.7.5309-5319.1999
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发表时间：
1999-07-01
影响因子：
5.4
通讯作者：
Kuhn, RJ
中科院分区：
文献类型：
--
作者：
Tellinghuisen, TL;Hamburger, AE;Kuhn, RJ
甲病毒病毒粒子的产生是一个多步骤事件，需要在细胞质中组装核衣壳核心以及在内质网和高尔基体中成熟糖蛋白。这些组分在出芽过程中结合以产生成熟的病毒体。辛德毕斯病毒和罗斯河病毒的核衣壳蛋白已经在基于T7的大肠杆菌表达系统中产生并纯化。在存在单链而非双链核酸的情况下，蛋白质在体外寡聚成类似于天然病毒核衣壳核心的核心样颗粒。尽管辛德毕斯病毒和罗斯河病毒的衣壳蛋白有相似之处，但它们并不形成混合的核样颗粒。辛德毕斯衣壳蛋白的截短形式用于建立组装的氨基酸需求。从残基19开始的衣壳蛋白[CP（19-264）]完全胜任体外组装，而具有进一步N-末端截短的蛋白不能支持组装。然而，在残基32或81处开始的衣壳蛋白能够在CP（19-264）存在下掺入颗粒中，或者如果其相对于CB（19-264）的摩尔比大于1：1，则可以抑制组装。该系统为甲病毒核心装配的分子解剖提供了基础。
The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CB(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.