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