GSK3介导β-catenin和N蛋白调控猪流行性腹泻病毒复制的分子机制研究

Porcine epidemic diarrhea virus is a serious threat to the healthy development of pig industry in the world. Scientific prevention and control of the virus has become an urgent problem to be solved. To analyze how PEDV uses host protein and itself encoded protein to complete its replication cycle and antagonize the molecular mechanism of natural immunity, which will provide a theoretical basis for prevention and control of the virus. The preliminary study of the applicant's laboratory showed that GSK3 mediated beta-catenin to regulate of the proliferation of the virus, and the N protein of PEDV inhibited the expression of beta-catenin. The molecular mechanism of the interaction of GSK3, N protein and beta-catenin to regulate the proliferation of PEDV is not clear. For this, this project will use Co-immunoprecipitation, mass spectrum and CRISPR-Cas9 et al, to elucidate the molecular mechanism of promoting viral proliferation by PEDV mediated PI3K/AKT/GSK3 signaling pathway regulating beta-catenin signaling pathway to inhibit cell apoptosis; to reveal the molecular mechanism of PEDV antagonizing natural immunity by N protein regulating beta-catenin signaling pathway to inhibit interferon production; to analyze molecular mechanism of GSK3 mediated phosphorylation N protein and interaction proteins regulating PEDV replication, for scientific prevention and control and treatment of PEDV to provide theoretical basis and research direction.

猪流行性腹泻病毒严重威胁世界养猪业的健康发展。科学防控该病毒成为亟待解决的科学难题。解析PEDV借用“宿主蛋白和自身编码的蛋白”完成复制周期及拮抗天然免疫分子机制，将为防控该病毒提供理论基础。申请者实验室前期研究表明：1）GSK3介导β-catenin调控病毒的增殖；2）N蛋白抑制β-catenin的表达。GSK3、N蛋白和β-catenin三者之间相互作用调控PEDV增殖的分子机制暂不清楚。为此，本项目拟通过抑制剂、免疫共沉淀、质谱和基因编辑等技术，阐明PEDV介导PI3K/AKT/GSK3信号通路调控β-catenin信号通路，抑制细胞凋亡，促进病毒增殖机理；揭示N蛋白调控β-catenin信号通路，抑制干扰素，拮抗天然免疫机制；解析GSK3介导磷酸化N蛋白及其结合转录因子调控PEDV复制的机理，为科学防控和治疗PEDV，提供理论依据和研究方向。

项目背景.猪流行性腹泻病毒（PEDV）是一种全球分布的甲型冠状病毒，可在仔猪中引起致命的水性腹泻，造成严重的经济损失。然而，目前的商业疫苗不能有效地治疗现有疾病。因此，鉴定宿主抗病毒因子和探索宿主免疫系统对PEDV感染的反应机制是非常有必要的。.主要研究内容.猪流行性腹泻病毒N蛋白是病毒复制增殖的过程中产生最多的一种RNA结合蛋白，在病毒转录、翻译和组装的过程中发挥着重要的作用。本项目以N蛋白为研究对象，通过CoIP筛选出N蛋白互作的400多个宿主蛋白，通过CoIP、激光共聚焦和GST-pull down等技术证实宿主蛋白FUBP3、TARDPB、PABPC4、IRAV、TRIM21和RALY等与N蛋白互作，阐明FUBP3、TARDPB、PABPC4、IRAV、TRIM21和RALY通过自噬途径降解N蛋白抑制PEDV增殖的分子机制，揭示转录因子TCFL5、KLF16、SP1、EGR1分别调控FUBP3、TARDPB、PABPC4、IRAV的表达机制，解析FUBP3和TARDPB调控干扰素的分子机制。.重要成果.揭示转录因子TCFL5、KLF16分别调控宿主因子FUBP3和TARDPB表达的分子机制，阐明FUBP3和TARDPB通过自噬途径降解猪流行性腹泻病毒N蛋白及上调干扰素的机制，抑制病毒增殖的机制，该成果分别发表在国际重要期刊《Journal of Virology》(2篇)；揭示转录因子EGR1调控IRAV的分子机制，阐明IRAV通过二种蛋白降解途径降解猪流行性腹泻病毒N蛋白抑制病毒增殖的分子机制，该成果发表在国际重要期刊《Journal of Virology》上；阐明RALY通过自噬途径降解猪流行性腹泻病毒N蛋白，抑制病毒增殖的机制，该成果分别发表在国际重要期刊《Journal of Biological Chemistry》上；揭示转录因子SP1调控PABPC4表达的分子机制，阐明PABPC4广谱降解冠状病毒N蛋白，抑制冠状病毒增殖的分子机制。.科学意义.解析病毒和宿主通过自噬途径相互博弈的机制，为防控病毒提供新思路和新的理论基础；揭示宿主因子FUBP3、TARDPB、IRAV、TRIM21、ATG4B和RALY抑制猪流行性腹泻病毒增殖的新机制，发现自噬激活剂在病毒感染早期可以抑制病毒增殖；发现广谱抑制冠状病毒增殖的宿主因子，解析自噬激活剂抑制冠状病毒的