ChlR1蛋白直接与HPV16 E2相互作用并增强E2依赖性DNA复制

ChlR1 Protein Directly Interacts with HPV16 E2 and Enhances E2-dependent DNA Replication

为探讨HPV16 E2和ChlR1的相互作用,阐明HPV基因组在复制过程中两个蛋白因子间的调控机制,本研究通过先构建HPV16 E2蛋白和带有血凝素(HA)标签的ChlR1蛋白的C33a细胞表达系统,利用免疫共沉淀分析蛋白间相互作用;通过双胸腺嘧啶阻断使细胞同步化,借助免疫荧光和流式细胞术分析ChlR1和E2的亚细胞定位,与细胞周期和DNA复制起点Ori间的相关性,以及Mre11和Nbs1与E2间的共定位;通过qPCR定量不同浓度梯度HA-ChlR1与HPV16 E2共转染条件下p160OriM的复制情况。免疫共沉淀结果显示,ChlR1是E2蛋白的直接靶向效应因子,并能形成复合物;E2表达使ChlR1从细胞核周围向核内积累。流式细胞术检测结果表明,E2和ChlR1在整个病毒感染周期内共定位,并在S期中期达到峰值;同时,HPV16 E2表达质粒转染的C33a细胞中,观察到明显的E2-NBS1和E2-MRE11共定位。而当Ori存在时,内源性ChlR1与E1/E2共定位,表明ChlR1可能被募集参与病毒DNA复制。此外,qPCR定量分析结果显示,ChlR1过表达明显增强了HPV E2依赖性DNA复制(p<0.001)。HPV E2通过靶向ChlR1蛋白,介导Mre11/Nbs1 DNA损伤应答复合物募集到HPV复制起点,以促进病毒基因组复制。本试验结果为研究HPV新型抑制剂提供思路。

To characterize the interact ion between HPV16 E2 and ChlR1,and to elucidate the regulation mechanism between the two protein factors in the process of HPV genome replication.This study firstly constructed the expression system of HPV16 E2 protein and the C33 a cell of ChlR1 protein labeled with hemagglutinin(HA),and analyzed the protein interaction by immunoprecipitation.The cells were synchronized by blocking double thymidine,and the subcellular localization of ChlR1 and E2 was analyzed by immunofluorescence and flow cytometry,which correlation of ChlR1 and E2 was analyzed by immunofluorescence and flow cytometry,the correlation between the cell cycle and Ori of DNA replication origin,and the co-localization between Mre11 and Nbs1 and E2,The replication of p160 OriM plasmid under different co-transfection conditions of HA-ChlR1 and HPV16 E2 was quantified by qPCR.The results of co-immunoprecipitation showed that ChlR1 was a direct targeting effector of E2 protein and can form complexes.E2 expression causes ChlR1 to accumulate from around the nucleus to the nucleus.Flow cytometry results showed that E2 and ChlR1 co-localized throughout the viral life cycle and peaked in the mid-S phase.At the same time,significant E2-NBS1 and E2-MRE11 colocalization were observed in C33 a cells transfected with HPV16 E2 expression plasmid.When Ori is present,endogenous ChlR1 colocalizes with E1/E2,suggesting that ChlR1 may be recruited to participate in viral DNA replication.In addition,qPCR quantitative analysis showed that ChlR1 overexpression significantly enhanced HPV E2-dependent DNA replication(p<0.001).HPV E2 promotes viral genome replication by targeting the ChrR1 protein,which mediates the Mre11/Nbs1 DNA damage response complex to the HPV origin of replication.The results of this experiment provide ideas for studying new inhibitors of HPV.