PCV2复制起始区DNA互作蛋白的筛选及PARP1蛋白对病毒复制的调控

doi:10.11843/j.issn.0366-6964.2022.10.023

摘要/Abstract

摘要： 旨在筛选PK-15细胞中与猪圆环病毒2型（porcine circovirus type 2，PCV2）复制起始区（origin of replication，Ori）互作的细胞蛋白谱，并初步探究PARP1蛋白对PCV2复制的调控。利用DNA-protein pull down结合LC-MS/MS方法筛选PCV2复制起始区DNA互作蛋白；对互作蛋白进行GO分析以及KEGG通路富集分析，并绘制蛋白互作网络图。在对互作蛋白进行分析的基础上，选择聚（ADP-核糖）聚合酶1（PARP1）蛋白，开展了深入研究。构建了PARP1蛋白以及PCV2复制蛋白Rep真核表达质粒，并利用DNA-protein pull down及DNA IP试验验证PARP1蛋白与PCV2 Ori的互作；通过Co-IP试验，研究了PARP1蛋白与PCV2 Rep蛋白的互作；在细胞中过表达或沉默PARP1蛋白，通过qPCR方法检测对PCV2复制的影响。本研究共鉴定到130种可能与Ori互作的宿主蛋白，其中，与DNA功能相关的蛋白共有45种，主要涉及宿主DNA损伤修复及DNA复制功能；验证了PARP1蛋白与PCV2基因组Ori以及Rep蛋白互作。用siRNA沉默PARP1蛋白表达后，PCV2基因组复制效率显著下降，瞬时过表达PARP1蛋白，PCV2基因组复制效率显著提高。综上所述，本研究筛选到130种可能与PCV2 Ori互作的细胞蛋白，发现PARP1蛋白可结合PCV2基因组Ori及Rep蛋白并促进PCV2的复制，为PCV2药物靶点选择及PCV2复制起始过程研究奠定了基础。

关键词: PCV2, 复制起始区, 细胞互作蛋白, PARP1, DNA损伤修复

Abstract: The purpose of this study was to gain a cell protein profile that interact with the porcine circovirus type 2 (PCV2) replication initiation region (Ori) in PK-15 cells and the regulation of PARP1 protein on PCV2 replication was preliminarily investigated. To gain a protein profile that interact with the PCV2 Ori in PK-15 cells, we performed a biotinylated DNA-protein pull down assay in conjunction with LC-MS/MS analysis; GO analysis and KEGG pathway enrichment analysis were performed and the protein interaction network was mapped; subsequently, we constructed eukaryotic plasmids of PARP1 and viral protein Rep, and the interaction between PARP1 protein and Ori region was verified by DNA pull down and IP experiments; Co-IP assay was used to detect the binding of PARP1 protein to Rep proteins; finally, the effect of over-expression or silencing of PARP1 protein on virus replication was detected. A total of 130 host proteins were identified, in which 45 were related to DNA function, mainly involved in host DNA damage repair and replication function; the interaction between PARP1 protein and PCV2 genome Ori or Rep protein was verified; after siRNA silenced the expression of PARP1 protein, the copy number of PCV2 genome was significantly decreased, and PARP1 protein was overexpressed instantly, and the copy number of PCV2 genome was significantly increased. In conclusion, the replication initiation of PCV2 in host cells involves the repair of host DNA damage repair and replication function. PARP1 protein could bind to Ori and Rep proteins of PCV2 and promote the replication of PCV2, which lays a foundation for the study of drug targets of PCV2 and the replication initiation of PCV2.

Key words: porcine circovirus type 2, replication initiation region, host interaction proteins, poly (ADP-ribose) polymerase 1, DNA damage repair

中图分类号:

S852.659.2

贾含笑, 袁红根, 李振, 关帅印, 张金花, 宋云峰. PCV2复制起始区DNA互作蛋白的筛选及PARP1蛋白对病毒复制的调控[J]. 畜牧兽医学报, 2022, 53(10): 3511-3521.

JIA Hanxiao, YUAN Honggen, LI Zhen, GUAN Shuaiyin, ZHANG Jinhua, SONG Yunfeng. Screening of Proteins Interacting with PCV2 Replication Initiation Region and Study on the Regulation of Virus Replication by PARP1 Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3511-3521.

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