Sarisky R T, Hayward G S
Molecular Virology Laboratories, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
J Virol. 1996 Nov;70(11):7398-413. doi: 10.1128/JVI.70.11.7398-7413.1996.
The protein products of 11 viral genomic loci cooperate in a transient cotransfection assay to mediate lytic-phase DNA replication of oriLyt, the human cytomegalovirus (HCMV) origin of replication. Six of these genes have homology with the well-characterized herpes simplex virus replication genes and encode core replication machinery proteins that are typically essential for DNA synthesis. The remaining five HCMV gene loci, initially referred to as auxiliary components, include several known immediate-early (IE) transcriptional regulatory proteins as well as genes encoding functionally uncharacterized polypeptides. Some or all of the auxiliary components may be necessary in trans to replicate the HCMV oriLyt only because they are required for efficient expression or transactivation of the native early promoters and 3' processing elements included in the genomic clones. Therefore, we reassessed the requirements for the auxiliary components by adding constitutive heterologous promoters and control signals to the coding regions and carrying out transient DpnI replication assays in cotransfected Vero cells. The results revealed that in the presence of the UL69 posttranscriptional activator and the remaining auxiliary polypeptides, UL84 was the only auxiliary component that could not be omitted to obtain oriLyt-dependent DNA replication. Nevertheless, in human diploid fibroblasts, some additional auxiliary loci as well as UL84 were critical. There was also an obligatory requirement for UL84, in cooperation with two other auxiliary factors, UL112-113 and IE2, and the core machinery, to constitute the minimal HCMV proteins necessary to direct oriLyt-dependent DNA amplification. However, the Epstein-Barr virus core replication genes could substitute for the HCMV core genes, and in these circumstances, UL84 alone directed amplification of HCMV oriLyt. Moreover, there was also an absolute requirement for UL84 along with the core and other auxiliary factors for the formation of intranuclear replication compartments as assayed by immunofluorescence in transient DNA cotransfection assays. These compartments were typical of those associated with active viral DNA replication in HCMV-infected cells, they incorporated pulse-labeled bromodeoxyuridine, and their formation was both phosphonoacetic acid sensitive and oriLyt dependent. These results demonstrate that UL84 is obligatory for both intranuclear replication compartment formation and origin-dependent DNA amplification and suggest that it is a key viral component in promoting the initiation of HCMV oriLyt-directed DNA replication.
在瞬时共转染实验中，11个病毒基因组位点的蛋白质产物协同作用，介导人巨细胞病毒（HCMV）复制起点oriLyt的裂解期DNA复制。其中6个基因与特征明确的单纯疱疹病毒复制基因具有同源性，编码核心复制机制蛋白，这些蛋白通常对DNA合成至关重要。其余5个HCMV基因位点最初被称为辅助成分，包括几个已知的立即早期（IE）转录调节蛋白以及编码功能未明多肽的基因。部分或所有辅助成分可能仅在反式作用中对复制HCMV oriLyt是必需的，因为它们是基因组克隆中天然早期启动子和3'加工元件有效表达或反式激活所必需的。因此，我们通过在编码区添加组成型异源启动子和控制信号，并在共转染的Vero细胞中进行瞬时DpnI复制实验，重新评估了对辅助成分的需求。结果显示，在存在UL69转录后激活剂和其余辅助多肽的情况下，UL84是唯一不能省略以获得依赖oriLyt的DNA复制的辅助成分。然而，在人二倍体成纤维细胞中，一些额外的辅助位点以及UL84也很关键。与另外两个辅助因子UL112 - 113和IE2以及核心机制协同作用时，UL84也是构成指导依赖oriLyt的DNA扩增所需的最小HCMV蛋白的必需成分。然而，爱泼斯坦 - 巴尔病毒核心复制基因可以替代HCMV核心基因，在这些情况下，单独的UL84就能指导HCMV oriLyt的扩增。此外，在瞬时DNA共转染实验中通过免疫荧光检测，对于形成核内复制区室，UL84与核心及其他辅助因子同样是绝对必需的。这些区室是HCMV感染细胞中与活跃病毒DNA复制相关的典型区室，它们掺入脉冲标记的溴脱氧尿苷，其形成对膦甲酸敏感且依赖oriLyt。这些结果表明，UL84对于核内复制区室形成和依赖起点的DNA扩增都是必需的，并提示它是促进HCMV oriLyt指导的DNA复制起始的关键病毒成分。
