ATP synthase subunit ATP5B interacts with TGEV Nsp2 and acts as a negative regulator of TGEV replication

Figure 1. Expression of TGEV Nsp2. Western blot analysis of IPEC-J2 cell lysates at different time points after overexpression of Nsp2 was performed using anti-HA antibody and anti-β-Actin antibody. Figure 2. Identification of the cellular proteins that interact with TGEV Nsp2 by immunoprecipitation (IP). (A) pCMV-HA-Nsp2 and empty vector pCMV-HA were transfected into IPEC-J2 cells. 24 h later, the cell protein samples were collected, and the immunoprecipitation (IP) test was carried out with Anti-HA Magnetic Beads. The obtained protein samples were dyed with protein glue silver. (B) Venn diagram of the identified protein candidates interacting with TGEV Nsp2 from mock infected. Figure 3. Construction and analysis of the protein-protein interactions (PPIs) network using STRING database. Figure 4. The annotation of proteins interacting with TGEV Nsp2 using Gene Ontology. Figure 5. Classification of the top 20 cellular protein-enriching KEGG pathways interacting with TGEV Nsp2. Figure 6. Confirmation of the interaction of TGEV Nsp2 with ATP5B and Citrin. (A)-(B) Plasmids expressing host cell protein were co-transfected with PCMV-HA- Nsp2 into HEK-293T cells, while plasmids co-transfected with host cell protein and pCMV-HA cells were used as negative controls. 24 h after transfection, Co-IP assay was performed using Anti-c-HA magnetic beads, followed by Western blot assay using HA and Myc monoclonal antibodies. (C)-(D) The plasmid expressing host cell protein and pCMV-HA-Nsp2 were co-transferred into IPEC-J2 cells, and the co-localization was observed by confocal laser microscopy 24 h after transfection. Figure 7. The expression of ATP5B was down-regulated after TGEV infection. The expression level of ATP5B in IPEC-J2 cells at different time points after TGEV infection was detected by QPCR (A) or Western blot (B). Figure 8. ATP5B inhibits TGEV replication. (A) Western blot and IFA (B) were used to detect ATP5B overexpression. pCMV-Myc-ATP5B and pCMV-Myc were transfected into IPEC-J2 cells for Western blot and IFA assay 24 hours later. (C) ATP5B was overexpressed in IPEC-J2 cells and TGEV was inoculated 24h later. Total cell cultures were collected at different time points (12h-72h) after infection, virus titer was detected, and a one-step growth dynamic curve was drawn. (D) Relative protein expression levels of ATP5B in cells transfected with SiRNAs against ATP5B and control SiRNA analyzed by western blot analysis with anti-ATP5B polyclonal antibodies. (E) Assay of cell viability after transfection with SiRNA. (F) SiATP5B was transfected into IPEC-J2 cells and TGEV was inoculated 24h later. Total cell cultures were collected at different time points (12h-72h) after infection, virus titer was detected, and a one-step growth dynamic curve was drawn.

图1 猪传染性胃肠炎病毒（Transmissible Gastroenteritis Virus of Swine, TGEV）非结构蛋白2（non-structural protein 2, Nsp2）的表达。采用抗HA抗体与抗β-肌动蛋白抗体，对Nsp2过表达后不同时间点的IPEC-J2细胞裂解液进行蛋白质印迹分析。
图2 采用免疫沉淀（immunoprecipitation, IP）技术鉴定与TGEV Nsp2相互作用的宿主细胞蛋白。(A) 将pCMV-HA-Nsp2与空载体pCMV-HA转染至IPEC-J2细胞中，24小时后收集细胞蛋白样品，使用抗HA磁珠进行免疫沉淀实验，所得蛋白样品采用蛋白质凝胶银染法染色。(B) 来自模拟感染组的与TGEV Nsp2相互作用的候选蛋白的韦恩图。
图3 采用STRING数据库构建并分析蛋白质相互作用（protein-protein interaction, PPI）网络。
图4 采用基因本体（Gene Ontology, GO）对与TGEV Nsp2相互作用的蛋白进行注释分析。
图5 与TGEV Nsp2相互作用的宿主细胞蛋白富集的前20条京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分类。
图6 验证TGEV Nsp2与ATP5B及Citrin的相互作用。(A)-(B) 将表达宿主细胞蛋白的质粒与pCMV-HA-Nsp2共转染至HEK-293T细胞中，以宿主细胞蛋白与pCMV-HA空载体共转染的细胞作为阴性对照。转染24小时后，采用抗HA磁珠进行免疫共沉淀（co-immunoprecipitation, Co-IP）实验，随后使用HA与Myc单克隆抗体进行蛋白质印迹检测。(C)-(D) 将表达宿主细胞蛋白的质粒与pCMV-HA-Nsp2共转染至IPEC-J2细胞中，转染24小时后采用激光共聚焦显微镜观察蛋白共定位情况。
图7 TGEV感染后ATP5B的表达下调。采用实时定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）(A) 或蛋白质印迹(B) 检测TGEV感染后不同时间点IPEC-J2细胞中ATP5B的表达水平。
图8 ATP5B抑制TGEV复制。(A) 采用蛋白质印迹与免疫荧光实验（immunofluorescence assay, IFA）(B) 检测ATP5B的过表达效果：将pCMV-Myc-ATP5B与pCMV-Myc空载体转染至IPEC-J2细胞中，24小时后分别进行蛋白质印迹与IFA检测。(C) 在IPEC-J2细胞中过表达ATP5B，24小时后接种TGEV，于感染后不同时间点（12h-72h）收集全细胞培养物，检测病毒滴度并绘制一步生长动力学曲线。(D) 采用抗ATP5B多克隆抗体进行蛋白质印迹分析，检测转染ATP5B小干扰RNA（small interfering RNA, SiRNA）及对照SiRNA的细胞中ATP5B的相对蛋白表达水平。(E) 检测转染SiRNA后的细胞活力。(F) 将SiATP5B转染至IPEC-J2细胞中，24小时后接种TGEV，于感染后不同时间点（12h-72h）收集全细胞培养物，检测病毒滴度并绘制一步生长动力学曲线。