Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis.
Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis.
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DOI：
10.1006/viro.1997.8848
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发表时间：
1997-12
期刊：
影响因子：
3.7
通讯作者：
M. Penfold;E. Mocarski
中科院分区：
文献类型：
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作者：
M. Penfold;E. Mocarski
为了表征人巨细胞病毒感染细胞中复制区室的形成，并确定新合成的DNA的命运，我们定位了感染过程中早期和晚期的病毒复制蛋白和DNA合成。正如预期的那样，ppUL 57（单链DNA结合蛋白）和ppUL 44（DNA聚合酶持续合成因子）都定位于48 hpi开始的复制区室。BrdU被掺入这些隔室中的病毒DNA中，基于我们在随后的72小时内将标记物追踪到细胞质中并从细胞中排出的能力，发现这些隔室成熟为子代病毒。虽然在早期（20或24 hpi）的BrdU掺入模式是点状的，并与后来在感染过程中形成的复制区室不同，但此时合成的病毒DNA也在追逐过程中成熟为子代病毒。有趣的是，ppUL 57定位的网站并没有完全重叠的BrdU掺入的网站在早期。来自UL 112 -113基因的产物在6 hpi定位于亚核区域，早于ppUL 57。在12和24 hpi之间，ppUL 57和ppUL 44都在随后发展成复制区室的位点处加入UL 112 -113基因产物。当在膦甲酸酯或更昔洛韦存在下进行感染时，复制室形成被阻断。尿嘧啶DNA糖苷酶缺陷的病毒突变体，先前显示出在DNA复制的初始阶段中表现出延迟，也表现出复制区室的延迟形成。这些结果提出了一种可能性，即由UL 112 -113定位定义的亚核位点协调CMV复制区室的组装，并暗示BrdU掺入的点状位点作为复制区室形成之前的早期病毒DNA复制的位点。
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.