The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor
The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor
复制标题
DOI：
10.1128/jvi.79.1.245-256.2005
复制
发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Hayward, SD
中科院分区：
文献类型：
--
作者：
Liao, GL;Huang, J;Hayward, SD
疱疹病毒编码一组核心蛋白，这些核心蛋白是其基因组裂解复制所必需的。这些蛋白质中的三种形成三重螺旋-引发酶复合物，在EB病毒（EBV）的情况下，该复合物由解旋酶BBLF 4、引发酶BSLF 1和接头蛋白BBLF 2/3组成。BBLF 2/3及其在其他疱疹病毒中的同源物的特征仍然相对较差。为了更好地理解BBLF 2/3对复制的贡献，用BBLF 2/3作为诱饵蛋白进行酵母双杂交筛选。该筛选鉴定了许多细胞复制相关蛋白质如DNA聚合酶β和DNA聚合酶δ亚基沿着EBV编码的DNA酶BGLF 5作为相互作用物。筛选还鉴定了DNA结合锌指蛋白ZBRK 1和ZBRK 1辅阻遏物KAP-1作为BBLF 2/3相互作用物。在免疫共沉淀试验中证实了BBLF 2/3与ZBRK 1和KAP-1之间的相互作用。通过电泳迁移率变动分析鉴定了EBV oriLyt增强子中ZBRK 1的结合位点。ZBRK 1、KAP-1和ZBRK 1结合蛋白BRCA 1通过间接免疫荧光显示存在于裂解诱导的D98-HR 1细胞的复制区室中，此外，染色质免疫沉淀测定确定这些蛋白与oriLyt DNA相关。在裂解诱导的D98-HR 1细胞中检查oriLyt质粒和变体oriLyt（AZBRK 1）质粒的复制。外源性ZBRK 1、KAP-1或BRCA 1增加了oriLyt复制的效率，而ZBRK 1结合位点的缺失损害了复制。这些实验将ZBRK 1鉴定为另一种细胞蛋白，其通过BBLF 2/3为EBV复制复合物提供oriLyt上的拴系点。这些数据还表明，BBLF 2/3可以作为参与EBV oriLyt复制的细胞蛋白的接触界面。
Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (AZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.