Structure and function of a truncated spliced isoform of interferon regulatory factor IRF-3c

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 published sequences of IRF-3 and murine EST database using bioinformatics method. A new sequence, IRF-3c, was subcloned into pcDNA3.1-flag. The IRF-3c/pcDNA3.1-flag plasmid was transfected in HEK 293 cells. Whole cell extract was analysed 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-3c cDNA expression plasmid. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF-3, named IRF-3c was discovered. The new isoform is almost the same as IRF-3, except for the utilization of the 180 bp bases in intron 6 adjacent to exon 6. The first 2,3 and 4 bases are a stop codon, which may produce a protein with a truncated C-terminal stoped at amino acids 327. Western blot analysis confirmed an expected 44 kDa strong band. The new inserted bases can be found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF-3c inhibited the IFNβ promoter activity to (around) 40%～50% as that of control after Sendai virus infection. Conclusions 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, provides 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-3c in the pathogenesis of human diseases using IRF-3c’s specific sequence.