PCV2利用宿主免疫系统促进自身增殖的分子机制

Porcine circovirus type 2 (PCV2) is associated with post-weaning muti-systemic wasting syndrome in young weaned pigs, it can damages the immune system. Immune stimulation was found to activate the replication of PCV2, this is associated with the interferon-stimulated response element(ISRE) which located in the rep gene promoter of PCV2, interferon response molecules produced by the host’s immune system can bind to ISRE and activate the rep gene transcription, improve replication of PCV2. The promoter of rep gene not only has ISRE, but also has some binding sites for immune-related transcription factors such as SP1, CREB1, GATA1 and GATA2. Are those binding sites can competitive combination with host proteins and activate the virus gene transcription, improve the virus replication and destroy the immune system? ISRE of PCV2 has been studied in our previous work, based on this, using the promoter activity assay to investigate if these binding sites can affect the promoter activity of rep gene; To determine if there is a relationship between binding sites and the virus replication efficiency, the virus titer and viral genome copies of wild-type and mutant viruses will be detected in vitro and in vivo. Then, using EMSA and ChIP to find out the host proteins which can bind to these binding sites, analyze the Molecular mechanism of PCV2 using host’s immune system to activate its replication.

猪圆环病毒2型（PCV2）是引起断奶仔猪多系统衰竭综合征等相关疾病的重要病原，能破坏宿主免疫系统。免疫刺激能促进PCV2的增殖，这种现象与PCV2 rep基因启动子区的干扰素刺激反应原件（ISRE）有关，宿主免疫应答中产生的干扰素效应分子能与ISRE结合，启动rep基因转录，促进病毒复制。PCV2 rep基因启动子区不仅有ISRE，还有其他如SP1, CREB1, GATA1, GATA2等免疫相关转录因子的结合元件存在，这些元件是否能竞争性结合宿主蛋白启动病毒基因转录，促进病毒增殖的同时破坏宿主免疫系统？本项目以申请者前期参与的对ISRE的研究为基础，通过启动子测试载体实验分析各元件对rep基因启动子活性的影响；在此基础上，通过比较各元件突变与不突变毒株在体内和体外的复制效率分析各元件对病毒复制能力的影响；最后寻找与各元件作用的宿主蛋白，分析其利用宿主免疫系统促进自身增殖的分子机制。

猪圆环病毒2型（PCV2）是猪圆环病毒相关性系统疾病的主要病原，能破坏宿主免疫系统。据报道，PCV2 rep基因启动子区的干扰素刺激反应原件（ISRE）能与干扰素效应分子结合，启动rep基因转录，促进病毒复制。经在线软件预测，我们发现PCV2 rep基因启动子区不仅存在ISRE，还有GATA，GRS，ARE，HRE等潜在转录因子结合位点，这些位点是否能竞争性结合宿主蛋白启动病毒基因转录，促进病毒增殖的同时破坏宿主免疫系统？本研究对存在于PCV2 rep基因启动子区的潜在转录因子结合位点进行了系统的生物学功能研究。通过结合位点定点突变、启动子活性检测、EMSA等实验验证了rep启动子区的ARE、GRS4及ISRE位点是影响rep启动子活性的重要功能位点，这些位点的突变会显著降低rep基因的启动子活性。DNA-pull down结合LC-MSMS实验鉴定出了70种可与rep启动子结合的宿主核蛋白，经启动子活性检测、qPCR、WB、TCID50等实验验证了宿主转录因子hnRNP A2/B1及TFAP2δ不仅能够促进rep基因启动子活性，而且能够在PCV2感染的整个周期中持续上调rep及cap基因mRNA和蛋白表达水平，说明这两个转录因子具有通过促进rep基因启动子活性增强rep、cap基因的转录及蛋白表达水平，从而促进病毒复制增殖的功能。这为我们揭示PCV2致病机制、研发高效的病毒疫苗奠定了坚实的理论基础。