Ning Qin, Luo Xiao-ping, Wang Zhi-mo, Han Mei-fang, Yan Wei-ming, Liu Ming-feng, Levy Gary
Division of Clinical Immunology, Tongji Hospital, Institute of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Yi Xue Za Zhi. 2003 Apr 25;83(8):678-83.
To identify the transcription factor(s) that is essential for activation of mfgl2 prothrombinase/fibroleukin gene in response to nucleocapsid protein of murine hepatitis virus type 3 (MHV-3).
Western blotting was performed to investigate whether HNF4 is expressed in macrophages of Ba1b/c mice where mfgl2 is expressed. Confocus microscope immunofluorescence was performed to show whether N protein of MHV enters into the nucleus of infected cells, which is a critical step for the N protein to facilitate its transactivation property. To facilitate the identification of three candidate factor(s) including hepatocyte nuclear factor 4 (HNF4)/liver factor A1 (LF-A1), cytomegalovirus immediate early gene 1.2 (IE1.2) regulatory element and granulocyte- macrophage colony stimulating factor (GM-CSF) in response to mfgl2 activation upon the stimulation of MHV-A59 N protein, gel mobility shift assay (GMSA), competition experiments and site directed mutagenesis were performed.
Western blotting displayed that HNF4 was constitutively expressed in macrophages and did not show significant change under the stimulation of different MHV. Confocus microscope immunofluorescence clearly showed that N protein of MHV entered into the nucleus of infected cells. GMSA and competition experiments demonstrated binding to both HNF4 and IE1.2 fragments could be competed with the cold specific oligonucleotides but not with the same amount of non-specific oligos nucleotides. A super shift band was observed when HNF4 antibody was pre-incubated with the nuclear extracts indicating the interaction between the HNF4 element and mfgl2 promoter. Site directed mutagenesis of cis-elements HNF4 (pfgl2HNF4mut) and HNF4/IE1.2 (pfgl2HNF4/IE1.2mut) mutations abolished over 75% of transcription from wild-type mfgl2 promoter. However the pfgl2IE1.2mut displayed almost wild-type promoter activity (75% approximately 80%).
The factor HNF4 binds to mfgl2 promoter and serves as an essential transcription factor for mfgl2/fibroleukin expression in response to MHV-3 N protein.
鉴定对3型鼠肝炎病毒（MHV-3）核衣壳蛋白应答时激活mfgl2凝血酶原酶/纤维白细胞介素基因至关重要的转录因子。
进行蛋白质免疫印迹法以研究HNF4是否在表达mfgl2的Balb/c小鼠巨噬细胞中表达。进行共聚焦显微镜免疫荧光法以显示MHV的N蛋白是否进入受感染细胞的细胞核，这是N蛋白发挥其反式激活特性的关键步骤。为便于鉴定在MHV-A59 N蛋白刺激下响应mfgl2激活的三种候选因子，即肝细胞核因子4（HNF4）/肝因子A1（LF-A1）、巨细胞病毒立即早期基因1.2（IE1.2）调控元件和粒细胞-巨噬细胞集落刺激因子（GM-CSF），进行了凝胶迁移率变动分析（GMSA）、竞争实验和定点诱变。
蛋白质免疫印迹显示HNF4在巨噬细胞中组成性表达，在不同MHV刺激下无显著变化。共聚焦显微镜免疫荧光清楚显示MHV的N蛋白进入受感染细胞的细胞核。GMSA和竞争实验表明，与HNF以及IE1.2片段的结合可被冷特异性寡核苷酸竞争，但不能被等量的非特异性寡核苷酸竞争。当HNF4抗体与核提取物预孵育时观察到一条超迁移带，表明HNF4元件与mfgl2启动子之间存在相互作用。对顺式元件HNF4（pfgl2HNF4mut）和HNF4/IE1.2（pfgl2HNF4/IE1.2mut）进行定点诱变消除了野生型mfgl2启动子超过75%的转录。然而，pfgl2IE1.2mut显示出几乎野生型的启动子活性（约75%至80%）。
因子HNF4与mfgl2启动子结合，是响应MHV-3 N蛋白时mfgl2/纤维白细胞介素表达的必需转录因子。
