Newcomb W W, Trus B L, Cheng N, Steven A C, Sheaffer A K, Tenney D J, Weller S K, Brown J C
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
J Virol. 2000 Feb;74(4):1663-73. doi: 10.1128/jvi.74.4.1663-1673.2000.
Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.
1型单纯疱疹病毒（HSV-1）的衣壳蛋白在体外组装成球形原衣壳，其结构和稳定性与成熟的多面体衣壳有显著差异，但可转化为成熟形式。间接证据表明，体内组装遵循类似的原衣壳组装和成熟途径，这一途径类似于双链DNA噬菌体的途径。我们通过从HSV-1感染的细胞中分离原衣壳并对其形态、热敏感性和蛋白质组成进行表征，证实了上述途径。实验使用了一种HSV-1突变体（m100），该突变体缺乏成熟蛋白酶，预计原衣壳（通常是寿命短暂的中间体）会在感染细胞中积累。从m100感染的细胞中分离出的颗粒被发现具有体外组装的原衣壳的特征性质。例如，通过电子显微镜观察，发现它们呈球形而非多面体形状，并且在0摄氏度时会解体，这与成熟衣壳不同，成熟衣壳在该温度下是稳定的。从冷冻电子显微照片以18埃分辨率计算的三维重建显示，m100原衣壳在结构上与体外组装的原衣壳无法区分。在这两种情况下，它们的主要成分都是四种必需的衣壳蛋白：主要衣壳蛋白（VP5）、支架蛋白（前VP22a）和三聚体蛋白（VP19C和VP23）。VP26是一种小的、丰富但非必需的衣壳蛋白，未发现与m100原衣壳相关，这表明它仅在衣壳成熟为多面体形式后才与衣壳结合。原衣壳也从在非允许温度下用HSV-1突变体tsProt.A（一种蛋白酶具有热可逆损伤的突变体）感染的细胞中分离出来，其作为原衣壳的身份通过冷冻电子显微镜得到证实。该分析揭示了原衣壳支架核心内表面的密度，这可能与成熟蛋白酶的位置相对应。在允许温度下孵育时，tsProt.A原衣壳转化为多面体的成熟衣壳，进一步证实了它们作为前体的状态。
