P53 And Interferon-α In Host Cells Regulated By VIR Protein From Orf Virus

Orf virus(ORFV), the protype species of parapoxvirus genus of poxviridae family, causes a contagious zoonotic infectious disease called Contagious Ecthyma or orf. ORFV primarily infects sheep and goats, and the main clinical characters are proliferative lesions of the lips and muzzle, around the nostrils, and in theoral mucosa. Generally the disease has a high morbidity and a low mortality, however, the mortality could increase when there is other pathogens co-infection or secondary infection. The disease has a worldwide distribution, and has both brought badly damage to the development of the goat industry and threatens the health of people.Because of immunosuppressive function of various proteins encoded by its genome, ORFV infects the same host repeatedly although the host responds to the infection with a vigorous immune and inflammatory reaction. VIR is one of the immune regulatory proteins encoded by ORFV, and has the activity of inhibiting interferon(IFN). However, the mechanism VIR functions by is not clear yet. p53 is a multi-functional protein, and play an important role in antiviral innate immunity. There have been several studies showed that p53 inhibits replication of many viruses, and has important contact with type I IFN pathway. Therefore, this study mainly explored the effects of VIR encoded by ORFV on p53 and IFN-α in host cells. The main researches were as follows:1. The regulatory effect of VIR on the production of IFN-αTo investigate the influence of VIR on IFN-α expression in host cells, goat skin fibroblast(GSF) cells and quantitative ELISA were used to detect the concentration of IFN-α in cell-free supernatant of GSF cells after the stimulation of SeV or SeV and VIR together. Results showed that VIR significantly reduced the expression of IFN-α in host cells.2. The regulatory role of VIR in p53 expressionFirst, the mRNA level of p53 was detected after ORFV infection. Results showed that the expression of p53 was inhibited during the early phase of ORFV infection. Then a eukaryotic expression vector of VIR was transfected to BHK-21 cells to study the effect on expression of p53. The results showed that the expression of p53 both at transcriptional and translational level significantly decreased. Results indicated that the inhibitory effect of ORFV on p53 expression related to VIR.3. The impact of p53 on downstream genes of type I interferon during ORFV infectionA method to detected copy-numbers of ORFV focus on VIR gene was developed to detect levels of ORFV replication after p53 was inhibited. ORFV growth curves of p53-down-regulated group and the control group were portrayed. Results showed that replication levels of ORFV in host cells were increase after p53 was inhibited. Our study also confirmed that after p53 was inhibited the mRNA levels of ISGs including IRF7, IRF9, ISG15, ISG20, GBP1, OAS1 induced by ORFV infection or IFN-α were significantly reduced. Then a eukaryotic expression vector of p53 was constructed and transfected to BHK-21 cells. And the mRNA levels of IRF7, IRF9, ISG15, ISG20, GBP1, OAS1 gene were quantified by real-time PCR. Results showed that the mRNA levels of these genes were up-regulated. Eukaryotic expression vectors of p53 and VIR were co-transfected to BHK-21 cells. The the mRNA levels of IRF7, IRF9, ISG15, ISG20, GBP1, OAS1 gene were significantly lower than cells only p53 were transfected. This suggested that VIR inhibited the ability of p53 to induce transcript of ISGs.The full study showed that the antagonistic activity toward to the interferon of VIR protein encoded by ORFV functions through inhibiting p53 gene at transcription and proteion level, the expression of IFN-α and the transcription of IRF7, IRF9, ISG15, ISG20, GBP1, OAS1 genes.