Building a virus from scratch: Assembly of an infectious virus using purified components in a rigorously defined biochemical assay system
Building a virus from scratch: Assembly of an infectious virus using purified components in a rigorously defined biochemical assay system
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DOI：
10.1016/j.jmb.2006.01.013
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发表时间：
2006-04-07
影响因子：
5.6
通讯作者：
Catalano, CE
中科院分区：
文献类型：
--
作者：
Gaussier, H;Yang, O;Catalano, CE
双链DNA（dsDNA）病毒如痘病毒、疱疹病毒和许多噬菌体的组装是一个复杂的过程，其需要病毒和宿主来源的许多蛋白质的协调活性。在这里，我们报告了使用纯化的蛋白质和市售的DNA的感染性野生型λ病毒的组装，以及在严格定义的生化系统中组装反应的优化。七种蛋白质、纯化的原衣壳和尾以及成熟的λ DNA是体外有效组装病毒所必需的。对反应的分析表明（i）在忠实地模拟大肠杆菌细胞内的细胞内环境的条件下，体外病毒组装是最佳的，（ii）成功完成病毒组装需要多联体DNA，（iii）几种蛋白质组分寡聚化伴随着它们逐步添加到新生病毒颗粒中，和（iv）尾部添加是以下速率：病毒装配的限制步骤。重要的是，组装的病毒可以进入预期的λ病毒体的发育途径（裂解或溶原性）。因此，我们首次证明，野生型，复杂的DNA病毒可以组装从纯化的成分在规定的生化条件下。该系统提供了一个强大的工具来表征，在分子水平上，所需的一步一步的过程，以组装一个感染性病毒颗粒。鉴于dsDNA噬菌体和真核dsDNA病毒之间的显着相似性，λ系统的表征在我们理解病毒发展在全球范围内具有广泛的生物学意义。(c)2006爱思唯尔有限公司保留所有权利。
The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level. (c) 2006 Elsevier Ltd. All rights reserved.