Bonner P H
J Virol. 1974 Nov;14(5):1152-68. doi: 10.1128/JVI.14.5.1152-1168.1974.
The in vivo process of virion assembly was studied in rifampin-treated, MS2-infected Escherichia coli during late times of infection-after 18 min postinfection. Differential sucrose gradient sedimentation of infected-cell lysates taken at various times after radioactive labeling indicated a definite temporal order of appearance of phage-specific protein in assembly-related structures. Labeled MS2 protein appears first as a low-molecular-weight peak at the tops of gradients, then as a peak at 40S and as a large number of almost unseparable structures between 40 and 80S, and finally as 80S mature phage particles. During the chase of a short labeling period, radioactive phage protein was found to disappear from gradients in the same temporal order as it appeared; the soluble peak disappears first, followed by the 40 to 70S region. The chased label appears quantitatively in the 80S phage peak. Labeled phage RNA was found to appear first in the 40S peak, then in the structures between 40 and 70S, and finally in 80S phage particles. The order of disappearance of labeled phage RNA during a chase is the same as its appearance. Resedimentation of the 40 to 70S region indicated the presence of distinct structures at 60 and 70S and many indistinct ones between 40 and 60S. The smaller intermediates exhibit separable maturation protein-rich and coat protein-rich segments, indicating nonrandom binding of the two proteins during the initial steps of assembly. Larger, discrete intermediates appear at 60 and 70S. Treatment of the various structures with pancreatic RNase results in destruction of those from 40 through 60S; treatment of the 70S structure results in the conversion of some of it to a 45S peak, presumably the complete capsid. A small fraction of the 80S phage peak is also sensitive to RNase, resulting in a similar 45S peak. Pulse-chase experiments indicate that structures from 40 through 60S as well as the RNase-sensitive 70S structure are assembly intermediates, but that the RNase-insensitive 70S and the RNase-sensitive 80S structures are not.
在感染后期（感染后18分钟），对利福平处理的、被MS2感染的大肠杆菌中病毒粒子组装的体内过程进行了研究。对放射性标记后不同时间采集的感染细胞裂解物进行蔗糖密度梯度离心分析，结果表明噬菌体特异性蛋白在组装相关结构中出现具有明确的时间顺序。标记的MS2蛋白首先以低分子量峰的形式出现在梯度的顶部，然后出现在40S处的峰以及40至80S之间大量几乎无法分离的结构中，最后以80S成熟噬菌体颗粒的形式出现。在短时间标记后的追踪过程中，发现放射性噬菌体蛋白从梯度中消失的时间顺序与其出现顺序相同；可溶性峰首先消失，随后是40至70S区域。追踪标记定量地出现在80S噬菌体峰中。发现标记的噬菌体RNA首先出现在40S峰中，然后出现在40至70S之间的结构中，最后出现在80S噬菌体颗粒中。在追踪过程中标记噬菌体RNA消失的顺序与其出现顺序相同。对40至70S区域进行再离心，结果表明在60和70S处存在明显的结构，在40至60S之间存在许多不明显的结构。较小的中间体表现出可分离的富含成熟蛋白和富含衣壳蛋白的片段，表明在组装的初始步骤中这两种蛋白的结合是非随机的。较大的离散中间体出现在60和70S处。用胰核糖核酸酶处理各种结构会导致40至60S的结构被破坏；处理70S结构会使其部分转化为45S峰，推测为完整的衣壳。80S噬菌体峰的一小部分对核糖核酸酶也敏感，导致出现类似的45S峰。脉冲追踪实验表明，40至60S的结构以及对核糖核酸酶敏感的70S结构是组装中间体，但对核糖核酸酶不敏感的70S结构和对核糖核酸酶敏感的80S结构不是。
