Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.
本研究旨在探究猪圆环病毒2 型 (Porcine circovirus type 2, PCV2) 能否借助rep 基因启动子区的转录因子结合位点调控自身基因转录，并寻找参与这一调控过程的宿主因子。凝胶迁移实验证实rep 基因启动子具有核蛋白结合活性。DNA-pull down 联合液相-串联质谱分析鉴定到了可与rep 基因启动子结合的猪源转录因子AP-2δ(Porcine transcription factor AP-2δ, poTFAP2δ)。双荧光素酶报告基因试验、实时荧光定量PCR、免疫印迹及间接免疫荧光等试验证明poTFAP2δ 不仅可以特异性地增强rep 基因启动子活性，而且可以在PCV2 感染过程中促进rep 和cap 基因的转录、翻译及PCV2 病毒滴度。文中揭示了PCV2 利用宿主蛋白促进自身增殖的分子机制，为进一步从病毒与宿主互作角度阐明PCV2 的致病机制提供新的研究思路，亦为PCV2 高效疫苗的研制提供理论基础。.
Keywords: porcine circovirus type 2; porcine transcription factor AP-2δ; rep gene promoter; transcription factor binding sites.