In Vitro Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain
In Vitro Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain
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DOI：
10.1128/jvi.00942-10
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发表时间：
2010-09-15
影响因子：
5.4
通讯作者：
Boulanger, Pascale
中科院分区：
文献类型：
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作者：
Huet, Alexis;Conway, James F.;Boulanger, Pascale
Siphoviridae大肠杆菌噬菌体T5与该家族的其他成员的不同之处在于其基因组的大小（121 kbp）和其大的二十面体衣壳（90 nm），其以T=13几何结构组织。T5不编码单独的支架蛋白，但其头部蛋白pb8含有159个残基的氨基末端支架结构域（Delta结构域），其为成熟衣壳。我们已经从纯化的pb8及其Delta结构域（pb8p）开始破译了T5壳组装的早期事件。pb8p的自组装受盐条件的调节，并导致具有不同形态的结构。在NaCl存在下形成膨胀管，而Ca（2+）促进pb8p结合到收缩管和前衣壳中。原衣壳显示角组织和20 nm长的内部放射状结构确定为三角洲域。T5头部成熟蛋白酶pb11特异性切割收缩和扩张管的Delta结构域。Ca（2+）不是蛋白水解活性所必需的，而是Delta结构域的组织所必需的。综上所述，这些数据表明，pb8p携带其一级序列中的所有信息，在体外组装，而不需要门户和辅助蛋白。此外，Ca（2+）在引入允许形成稳定的原衣壳的构象多样性中起关键作用。噬菌体T5是由准等价的六聚体和五聚体组成的病毒衣壳的第一个例子，其组装可以在体外进行，从主要头部蛋白质及其支架结构域开始，并且其终点是二十面体T=13颗粒。
The Siphoviridae coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Delta domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Delta domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca(2+) promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Delta domain. The T5 head maturation protease pb11 specifically cleaves the Delta domain of contracted and expanded tubes. Ca(2+) is not required for proteolytic activity but for the organization of the Delta domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble in vitro without the requirement of the portal and accessory proteins. Furthermore, Ca(2+) plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out in vitro, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.