Penfold M E, Mocarski E S
Department of Microbiology and Immunology, Stanford University, California 94305-5124, USA.
Virology. 1997 Dec 8;239(1):46-61. doi: 10.1006/viro.1997.8848.
To characterize the formation of replication compartments in human cytomegalovirus-infected cells, and to determine the fate of newly synthesized DNA, we localized viral replication proteins and DNA synthesis at early and late times during infection. As expected, ppUL57 (single-stranded DNA binding protein) and ppUL44 (DNA polymerase processivity factor) both localized to replication compartments beginning at 48 hpi. BrdU was incorporated into viral DNA in these compartments that was found to mature into progeny virus based on our ability to chase the label into the cytoplasm and out of the cell over the ensuing 72-h period. Although the pattern of BrdU incorporation at early times (20 or 24 hpi) was punctate, and distinct from the replication compartment that formed later during infection, viral DNA synthesized at this time also matured into progeny virus during a chase. Interestingly, sites of ppUL57 localization did not overlap completely with sites of BrdU incorporation at early times. Products from the UL112-113 gene localized to subnuclear regions by 6 hpi, earlier than ppUL57. Between 12 and 24 hpi, both ppUL57 and ppUL44 joined UL112-113 gene products at sites that subsequently developed into replication compartments. When infection was carried out in the presence of phosphonoformate or ganciclovir, replication compartment formation was blocked. A viral mutant deficient in uracil DNA glycosidase, previously shown to exhibit a delay in the initial phase of DNA replication, also exhibited delayed formation of replication compartments. These results raise the possibility that subnuclear sites defined by UL112-113 localization orchestrate the assembly of the CMV replication compartment and implicate punctate sites of BrdU incorporation as sites of early viral DNA replication that precedes the formation of the replication compartment.
为了表征人巨细胞病毒感染细胞中复制区室的形成，并确定新合成DNA的去向，我们在感染的早期和晚期对病毒复制蛋白和DNA合成进行了定位。正如预期的那样，ppUL57（单链DNA结合蛋白）和ppUL44（DNA聚合酶持续合成因子）在感染后48小时开始均定位于复制区室。基于我们在随后72小时内将标记物追踪到细胞质并排出细胞的能力，BrdU被掺入这些区室中的病毒DNA中，这些DNA后来成熟为子代病毒。尽管早期（感染后20或24小时）BrdU掺入的模式是点状的，且与感染后期形成的复制区室不同，但此时合成的病毒DNA在追踪过程中也成熟为子代病毒。有趣的是，早期ppUL57的定位位点与BrdU掺入位点并不完全重叠。UL112 - 113基因的产物在感染后6小时定位于亚核区域，比ppUL57更早。在感染后12至24小时之间，ppUL57和ppUL44都在随后发展为复制区室的位点与UL112 - 113基因产物会合。当在膦甲酸或更昔洛韦存在的情况下进行感染时，复制区室的形成被阻断。先前已证明缺乏尿嘧啶DNA糖基化酶的病毒突变体在DNA复制的初始阶段表现出延迟，它也表现出复制区室形成的延迟。这些结果增加了一种可能性，即由UL112 - 113定位定义的亚核位点协调了CMV复制区室的组装，并暗示BrdU掺入的点状位点是在复制区室形成之前的早期病毒DNA复制位点。
