Phelan A, Dunlop J, Patel A H, Stow N D, Clements J B
Institute of Virology, University of Glasgow, Scotland.
J Virol. 1997 Feb;71(2):1124-32. doi: 10.1128/JVI.71.2.1124-1132.1997.
We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.
我们通过直接标记方法，观察了单纯疱疹病毒1型（HSV-1）在感染的HeLa细胞内复制和转录位点的细胞内定位。病毒转录灶的数量以有限的方式增加；然而，在整个感染过程中，复制位点的数量以近乎指数的方式增加，并且复制和转录位点都位于细胞核内部深处。病毒转录灶和复制灶的同时观察表明，这两个过程在早期共定位，但在感染后期，有额外的位点专门用于复制。这与腺病毒感染细胞的情况形成对比，在腺病毒感染细胞中，在整个复制过程中，转录位点与病毒DNA复制位点相邻但不共定位。HSV转录位点增加的数据表明，输入基因组的复制存在一个初始阶段，然后进行转录。HSV复制位点与病毒DNA复制和包装蛋白共定位，但与小核核糖核蛋白颗粒的点状分布有很大不同。非常高的感染复数显示每个细胞核约有18个病毒转录灶的上限，这表明细胞对转录位点形成有限制。使用病毒复制突变体证实，标记的灶是病毒RNA和DNA合成的位点；在没有病毒DNA复制功能的情况下，没有复制灶，只有有限数量的转录灶。缺乏包装功能对转录或复制位点的形成没有明显影响，这说明DNA包装不是正在进行的DNA合成的先决条件。此外，重要的HSV蛋白IE63在感染后期高效复制位点形成中是必需的，但转录灶形成不需要它。
