Koths K, Dressler D
Proc Natl Acad Sci U S A. 1978 Feb;75(2):605-9. doi: 10.1073/pnas.75.2.605.
We have monitored the development of intracellular phiX DNA forms during the course of a virus life cycle that duplicates as closely as possible the normal infection of individual cells by single virions. The viral DNA was isolated in a one-step purification procedure, and quantitative electron microscopy was performed on the samples, resulting in the following conclusions: (i) Early in the life cycle, when the cells accumulate duplex rings, two types of DNA replication intermediates are observed: a rolling circle with a single-stranded tail; and a novel form, a single-stranded circle that is partially duplex. Thus, duplex ring synthesis appears to occur in two asymmetric steps, with positive strand DNA first being processed from the tail of the rolling circle and circularized, before it acts as a template for negative strand synthesis. (ii) Late in the life cycle, as single-stranded circles are synthesized and virus particles are assembled, only one replicating intermediate is observed--the rolling circle with a single-stranded tail. At this stage, the number of rolling circles reaches a level of about 35 per cell. (iii) The net rate of polymerization in the rolling circle intermediates is about 200 nucleotides per sec.
我们监测了病毒生命周期内细胞内phiX DNA形式的发展情况，该生命周期尽可能紧密地模拟单个病毒粒子对单个细胞的正常感染过程。通过一步纯化程序分离病毒DNA，并对样品进行定量电子显微镜观察，得出以下结论：(i) 在生命周期早期，当细胞积累双链环时，观察到两种类型的DNA复制中间体：一种带有单链尾巴的滚环；以及一种新形式，即部分双链的单链环。因此，双链环的合成似乎以两个不对称步骤发生，正链DNA首先从滚环的尾巴加工并环化，然后才作为负链合成的模板。(ii) 在生命周期后期，随着单链环的合成和病毒颗粒的组装，只观察到一种复制中间体——带有单链尾巴的滚环。在这个阶段，每个细胞中滚环的数量达到约35个的水平。(iii) 滚环中间体中的聚合净速率约为每秒200个核苷酸。
