Zhong L, Hayward G S
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Virol. 1997 Apr;71(4):3146-60. doi: 10.1128/JVI.71.4.3146-3160.1997.
Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.
在单纯疱疹病毒（HSV）裂解周期早期或存在DNA合成抑制剂的情况下，核心病毒复制机制蛋白会在核内斑点状的前复制焦点中积累，其中一些与许多被称为复制体的宿主细胞DNA合成起始位点共定位。在后期，在没有抑制剂的情况下，会形成几个球状或大的不规则形状的复制区室；这些区室还包含子代病毒DNA，并将IE175（ICP4）转录因子与几种参与DNA复制和修复的细胞蛋白结合在一起。在本研究中，我们证明，在接受编码所有七种必需HSV复制叉蛋白的基因以及oriS靶质粒DNA的DNA转染细胞的瞬时试验中，可以成功组装几种形式的前复制焦点和活跃的病毒复制区室，其外观与HSV感染细胞中的区室相似。此外，在这些复制区室中，溴脱氧尿苷（BrdU）脉冲标记的DNA合成起始位点与HSV单链DNA结合蛋白（SSB）共定位，这意味着可能正在进行活跃的病毒DNA复制。用膦甲酸（PAA）处理以及缺失七种病毒复制蛋白（UL5、UL8、UL9、UL42、UL52、SSB和Pol）中的任何一种（这些蛋白对病毒DNA复制至关重要），都会消除完整HSV复制区室的组装以及BrdU的掺入。与HSV IE175和IE63（ICP27）都定位于HSV感染细胞的复制区室内这一事实一致，组装好的HSV复制区室也能够募集这两种必需的调节蛋白。用PAA阻断病毒DNA合成，但不缺失oriS，可阻止IE175与前复制结构的结合。组装好的HSV复制区室还将共转染的细胞p53重新分布到病毒复制区室中。然而，另外两种HSV立即早期核蛋白IE110（ICP0）和IE68（ICP22）在感染或转染的细胞中都不会进入复制区室。
