Pombo A, Ferreira J, Bridge E, Carmo-Fonseca M
Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, Portugal.
EMBO J. 1994 Nov 1;13(21):5075-85. doi: 10.1002/j.1460-2075.1994.tb06837.x.
We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse-chase experiments indicate that newly synthesized viral double-stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single-stranded DNA replication intermediates accumulate in adjacent sites. Double-labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single-stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single-stranded DNA accumulation. These data indicate that newly synthesized double-stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co-localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co-transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.
我们已经观察到感染的HeLa细胞中腺病毒复制和转录的核内拓扑结构。结果表明，病毒DNA复制发生在核质中高度组织化的多个病灶中。脉冲追踪实验表明，新合成的病毒双链DNA分子从复制病灶中移出并扩散到整个核质中，而单链DNA复制中间体则积聚在相邻位点。双标记实验和共聚焦显微镜显示，复制发生在单链DNA积累位点周边的病灶中。病毒复制和转录的同时观察表明，转录位点主要与复制位点分开。在复制病灶附近检测到转录，并围绕单链DNA积累位点延伸。这些数据表明，新合成的双链DNA分子从复制病灶中移出并在周围的核质中扩散，在那里它们被用作转录模板。剪接snRNP显示与转录位点共定位，并被排除在复制位点之外。这提供了证据，表明病毒RNA的剪接在转录过程中发生，并且病毒DNA复制位点在空间上与RNA转录和加工位点不同。
