| Interferon(IFN) regulatory factors(IRFs) are an important family of transcription mediators that have been identified to play diverse roles in initiating antiviral responses, regulating inflammatory cytokine expression, controlling the cell transformation and apoptosis. IRF3 and IRF7 have been belived to be the pivotal IRFs that induce the expression of IFNs and IFN-stimulated genes(ISGs), all of which play central roles in initiating host innate immune responses to virus infection. The latest research indicates that IRF1 is also invovled in antiviral responses, IRF1 may mediate a new pathway of regulating the expression of IFNs and ISGs, which is IRF3- and IRF7-independent. The antiviral pathway mediated by IRF1 will be especially important for resisting the viruses who have evolved some antagonistic strategies to depress the effect of IRF3 or IRF7. At present, the function of porcine IRF1 is still unknown, in order to discovery the role of porcine IRF1 in antiviral response, several experiments were carried out as follows.To determine the tissue distribution of porcine IRF1, total RNA was extracted from 8 tissues from healthy swine, including heart, liver, spleen, lung, kidney, brain, lymph gland and bladder, respectively. The expression levels of porcine IRF1 in different tissues were determined by real-time quantitative RT-PCR. The results showed that porcine IRF1 m RNA was detected in all tissues examined, and it was expressed strongly in spleen, lymph gland and liver, while the IRF1 m RNA level was low in the heart, brean and bladder. The high expression level of porcine IRF1 in the immune related organizations indicated that porcine IRF1 may be involved in host defence.To investigate the transcript pattern of porcine IRF1 regulated by different porcine viruses, q RT-PCR was performed to examine the expression profiles of porcine IRF1 m RNA in two DNA-containing virus(PRV and PPV) and two RNA-containing virus(TGEV and VSV) infected PK-15 cells. The results showed that porcine IRF1 transcript was increased significantly during all infection, except the infection of the virus strain PRV-QXX, compared with mock-infected cells. These data suggested that porcine IRF1 might be involved in antiviral response.To determine the effect of porcine IRF1 on the expression of porcine IFN-β, porcine IRF1 gene was amplificated by RT-PCR from the total RNA of peripheral blood lymphocytes. An eukaryotic expression plasmid PCAGGS-HA-p IRF1 was constructed and transfected into PK-15 cells. Under the condition of no stimulation or treatment with poly(I:C), the promoter activity of IFN-β was tested by dual-luciferase reporter system and the transcriptional level of IFN-β was dectected by q RT-PCR. The results revealed that overexpression of porcine IRF1 promoted the promoter activity of IFN-β and up-regulated the transcription of IFN-β in the absence of stimulation, and the promotion was more remarkable in the treatment of poly(I:C), even stronger than porcine IRF3 and IRF7. When IRF1 gene of PK-15 cells was knocked down by si RNA which targeted the DNA banding domain, the promoter activity of IFN-β unchanged in the treatment of poly(I:C). These data suggested that porcine IRF1 could regulae the expression of IFN-β, but it is not the dominating regulator.To elucidate the antiviral roles of porcine IRF1 in vitro, the plasmid PCAGGS-HA-p IRF1 was transfected into PK-15 cells and the virus production of PRV and TGEV were detected in porcine IRF1 overexpressing PK-15 cells. The results revealed that overexpression of porcine IRF1 could suppress the replication of PRV and TGEV, respectively. In detail, the viral titer of PRV reduced by ten times, and that of TGEV reduced by twenty times. Thesse date indicated that porcine IRF1 were able to inhibite virus replication and facilitate the host antiviral response.To establish a PK-15 cell line stably expressing porcine IRF1, porcine IRF1 gene was transfected into PK-15 cells using Piggy Bac transposon system. The positive cells were screened by puromycin selection and fluorescence detection. A single clone of transfected PK-15 cells expressing porcine IRF1 was generated by several rounds of cloning and sub-cloning and identified by q RT-PCR assay and fluorescence detection. The PK-15 cell line stably expressing porcine IRF1 provides a useful tool for the study on the other functions else of porcine IRF1 as well as on its mechanism. |
