Effects Of PI3K-Akt Pathway On ORFV Replication And Apoptosis Of Host Cells

Orf virus is one of the representative members among Parapoxvirus, one of the eight genera of the subfamily Chordopoxvirinae of the Poxviridae, which is a viral zoonosis that primarily affects sheep, goats and human. It is the causative agent of Orf,which is a nonsystemic, highly contagious, ubiquitous disease of sheep, goats and other ruminants as well as human. The disease is characterized by maculopapular and proliferative lesions of affecting mouth, lips, tongue, nose, breasts and other oarl mucosa with papules, blisters, verrucous thick callus and pustules. In recent years, Orf frequently occurs in flocks around the world. The disease has also been reported in the Gansu province, Xinjiang province, Inner Mongolia, Jilin province and other provinces of China. The increase of Orf outbreaks may be associated to the characteristics of immune evasion of ORFV or the lack of effective prevention and control measures. Currently, the development of new vaccines or drugs for the control of Orf face the serious challenges due to understanding not deeply for the pathogenesis and immune characteristics of ORFV. Thus, it is necessary to enhance the basic research of ORFV(genes, proteins, pathogenesis, and so on), especially the molecular mechanism of ORFV infection.In order to clarify the pathogenic molecular mechanism of ORFV, we constructed the m RNA expression profiles of the different expressed genes of ovine fetal turbinate(OFTu) cells-infected with ORFV at 48 h and 96 h by Illumina / Solexa high through-put sequencing technology. The PI3K(phosphatidylinositol kinase3-kinase) showed significantly up-regulated expression in the OFTu cells infected with ORFV at 48 h and 96 h. PI3 K involves in cell proliferation, differentiation,apoptosis and so on. A variety of viruses could successfully infect host cells by the regulation of PI3K-Akt signaling pathway. However, it is not clear whether ORFVinfection could activate the PI3K-Akt signaling pathway. In addition, little is known for the role of PI3K-Akt signaling pathway in ORFV infection, and the effect on apoptosis of host cells infected with ORFV.In the study, the expression regulation of PI3 K in m RNA and protein levels from ORFV infected Hela cells in different time points(3 h, 6 h, 12 h, 24 h, 48 h, 72 h) was analysed by real-time PCR and western blot methods. Comparing to mock-infected Hela cells, the expression of PI3 K from ORFV infected Hela cells appeared to be obvious up-regulation in different time points, which was consistent with the result obtained from the m RNA expression profiles of the different expressed genes of OFTu cells-infected with ORFV. Next, we explored the activation of PI3K-Akt signaling pathway in Hela cells infected with ORFV by Western blotting using the anti-Akt(phospho-S473) antibody. The phosphorylated AKT levels at 3 hpi appeared significantly increased, and a high level of phosphorylated Akt still could be detected at 48 hpi. The results indicated that PI3K-Akt signaling pathway had been activated in Hela cells infected with ORFV. In order to further identify the effects of PI3 K gene on ORFV replication, we inoculated ORFV to Hela cells after treating with specific PI3 K inhibitor LY294002. The Hela cells inoculated ORFV after treating with specific PI3 K inhibitor LY294002 for 12 h, 24 h, 48 h and 72 h were respectively collected and used for real-time PCR detection and TCID50 assay. The virus titer in Hela cells treating with LY294002 was lower than in untreated Hela cells. The results indicated that PI3 K could accelerate the replication of ORFV in host cells.Considering the important role of PI3K-Akt signaling pathway in the regulation of cell survival and apoptosis, we further clarify the effect of PI3K-Akt pathway on apoptosis of host cells infected with ORFV. The obvious apoptosis phenomenon was found in Hela cells infected with ORFV after blocking the PI3K-Akt pathway using PI3 K inhibitor(LY294002) by immunofluorescence and flow cytometry. The results showed that PI3K-Akt signaling pathway could inhibit the apoptosis of Hela cells induced by ORFV. In order to further clarify the path of the apoptosis of Hela cells infected with ORFV via PI3K-Akt signaling pathway, we performed the detection forseveral key proteins(GSK-3β,MDM2 and Bad) in the downstream of PI3K-Akt pathway. Comparing with ORFV infected Hela cells, the ratio of Bad/β-actin in ORFV infected Hela cells treating with LY294002 at 6 hpi and 24 hpi showed significant change. However, the ratios of MDM2/β-actin and GSK-3β/β-actin were not obvious changes. The above results indicated that PI3K-Akt signaling pathway inhibited the apoptosis of ORFV infected Hela cells possibly by the Bad in the downstream of the pathway.The above results showed that PI3K-Akt signaling pathway could accelerate the replication of ORFV in infected Hela cells, and might be inhibit the apoptosis of Hela cells infected with ORFV in order to establish the successful infection. The study would not only be beneficial to understanding the molecular pathogenesis of ORFV infection, but also contribute to the prevention and treatment of the disease.