一种新的干扰素调节因子拼接异构体IRF-3b的结构及功能

Structure and function of a new spliced isoform of interferon regulatory factor IRF-3b

目的 干扰素调节因子 (interferonregulatoryfactors ,IRF) 3是病毒感染后调节干扰素基因转录重要的转录因子 ,寻找IRF 3新的基因拼接异构体 ,研究其结构及功能。方法 抽提人类细胞RNA ,用IRF 3已发表序列设计引物 ,作cDNA末端快速放大 (RACE)及RT PCR ,生物信息学方法比较新序列 ,亚克隆IRF 3b至 pcDNA3.1 flag ,转染人胚胎肾上皮 2 93细胞 ,用抗flag抗体作Westernblot分析 ,用荧光素酶功能分析的方法观测IRF 3b对病毒诱导的干扰素 β启动子荧光素酶活性的影响。结果 发现了一种新的IRF 3拼接异构体IRF 3b ,IRF 3b用了紧接第七外显子前的 1 6个第六内含子的碱基 ,导致阅读框架移位 ,相应蛋白质的C端第 32 8～ 4 52位为不同于IRF 3的新C末端。Westernblot出现预期的相对分子质量 (Mr)为 57.75× 1 0 3的IRF 3b蛋白强阳性条带。新的外显子序列可在小鼠的表达序列标签 (EST)表达库中找到同源序列 ,提示这种新的异构体在生物演进中有其保守功能 ,IRF 3b荧光素酶功能分析显示 ,该同分异构体能抑制病毒诱导的干扰素 β启动子活性至对照组的 4 0 %～ 50 %。结论 新的异构体的发现为IRF 3这一重要分子的功能调节提供了新的线索 ,它可能是病毒感染通路中干扰素的显性负性抑制剂 ,提示其功能为干扰素产生的?

Objective Interferon regulatory factors 3 (IRF 3) is a key transcription factor to regulate gene expression of interferon after virus infection. This study aims to look for new spliced isoforms of IRF 3 and to investigate their structures and functions. Methods RNA extracts from human embryonic kidney 293 cells were amplified by RACE and RT PCR. New sequences were compared with the published sequences of IRF 3 and murine EST database using bioinformatics method. A new sequence, IRF 3b, was subcloned into pcDNA3.1 flag. The IRF 3b/pcDNA3.1 flag plasmid was transfected in HEK 293 cells. Whole cell extract was analyzed by Western blot and then probed with monoclonal Flag antibody. Luciferase assay was carried out by co transfection with reporter plasmid, pGL2B with interferon β promoter, and IRF 3b cDNA expression plasmid. At 16th hour of transfection, cells were infected with Sendai virus for 8 h. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF 3, named IRF 3b was discovered. The new isoform is almost the same as IRF 3, except for the utilization of the 16 bp bases in intron 6 adjacent to exon 7. This additional 16 bp sequence lead to reading frame shift, which may produce a protein with a different C terminal in amino acids 328 452. Western blot analysis confirmed an expected 57.75 kD strong band. The new inserted bases and new C terminal amino acids was found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF 3b inhibited the IFN β promoter activity to around 40% 50% as that of control after Sendai virus infection. Conclusion The discovery of a new isoform of IRF 3 provides a new insight into the functional regulation of IRF 3 family. It is a dominant negative inhibitor for interferon β promoter activity in the virus infection pathway, demonstrates a mechanism for the fine tuning of the virus induced activation of the interferon response, and prevents interferon β from its overexpression and its toxic effects. It is worthwhile to explore the role of IRF 3b in the pathogenesis of human diseases.