| Avian leukosis virus subgroup J(ALV-J) can cause growth retardation, immunosuppression and neoplasia in chickens, causing significant economic losses in the poultry industry. At present, there is no effective vaccine and drug for control the ALV-J infection, therefore, the infection pathogenic mechanisms need to further in-depth study. Chicken sodium hydrogen exchanger isoform 1(ch NHE1) is a transmembrane protein located on the plasma membrane of eukaryotic cells, catalyzing the exchange of the intracellular H+ for the extracellular Na+, regulating intracellular p H, involving in physiological and pathological processes. ch NHE1 is the receptor of ALV-J, however, its role in ALV-J infection is still unknown.It is well known that the combination of virus and host cells receptor is the first step of infection. Identifying the functional changes of ch NHE1 in ALV-J infection, as well as the effect of this change on intracellular signaling pathways and cellular biological functions, is important to clear the pathogenic mechanism and develop new drugs for control the ALV-J infection. Therefore, we design experiments to study if ALV-J can regulate ch NHE1.We used 103 TCID50 ALV-J strains to infect DF-1 cells and collected cells at different time points after infection. Then, DF-1 cells were infected with 101, 103 and 104 TCID50 ALV-J strains, and the cells were collected at 72 h after infection. The expression of ch NHE1 and ALV-J in m RNA level were detected by RT-PCR and real-time PCR. The results showed that the expression of ch NHE1 was up-regulated by ALV-J in time-dependent manner, with wave-like curve. However, the expression of ch NHE1 was not dependent on viral titer, because the expression of ch NHE1 in 104 TCID50 group was lower than 101 and 103 TCID50 group. Next, DF-1 cells were infected by ALV-J at TCID50 of 103. The intracellular p H was assessed by spectrofluorometer at different times post infection. The results showed that ALV-J infection led to rapid increase of p Hi up to 7.7 at 24 h and slightly decreased at 120 h, indicating that ALV-J up-regulated the expression of ch NHE1 and then affected p Hi. Finally, we validate the regulation of ALV-J on ch NHE1 by in vivo experiments. After establishment of ALV-J infection model, the kidney tissue of chickens was selected to extract RNA, and then the expression of ch NHE1 and ALV-J at m RNA level were detected by RT-PCR and real-time PCR. The results showed that the expression of ch NHE1 in infection group was significantly higher than the control group. We further detected the expression and distribution of ch NHE1 in different tissues of ALV-J infected chickens by immunohistochemistry analysis. The results showed that the staining intensities of ch NHE1 in ALV-J infection group were higher than the control group. The increased ch NHE1 protein was present in the kidney, liver, bone marrow and thymus tissue, not in the spleen and bursa. In conclusion, the study demonstrated that ALV-J infection could up-regulate the expression and function of ch NHE1. The results indicated that the up-regulation of ch NHE1 played a pivotal role in tumorigenesis caused by ALV-J infection.Accumulated documents demonstrated that cellular signal pathways involved in viral infection process including viral invasion, transcription, translation and release of the virus. Previous studies have shown that Wnt signaling pathway was involved in a variety of viral infections, however, the role of Wnt signaling involved in ALV-J infection is unknown. In order to prove if ALV-J could regulate the Wnt pathway, we designed the following experiment.CEF cells were infected by ALV-J at 103 TCID50, and the expression of β-catenin was detected by western blot at different time points after infection. The results showed that ALV-J infection led to the decrease of protein expression level of β-catenin. At the same time, the β-catenin expression of nucleus was also verified by western blot analysis at 120 h post infection. The results showed that the β-catenin expression of nucleus in ALV-J infection group was also significantly lower than the control group. Then, CEF cells were infected by ALV-J at 103 TCID50 and the relative expression levels of Tcf1, Tcf4 and Lef1 were detected by real-time PCR at different time points post infection. Tcf4 and Lef1 showed a significant down-regulation in ALV-J infection compared to the control, whereas no significant difference was found for Tcf1. Next we examined the downstream target genes expression of Wnt pathway. CEF cells were infected by ALV-J at 103 TCID50 and the relative expression levels of Axin2, Cyclin D1, c-myc and MMP-7 were detected by real-time PCR at different time points post infection. The results showed that the expression of Axin2 and Cyclin D1 showed a significant down-regulation, however, expression of c-myc and MMP-7 showed a significant up-regulation. We further detected the cell cycle and cell proliferation after ALV-J infection. CEF cells were infected by ALV-J at 103 TCID50, and the cell cycle was detected by flow cytometry at 48 h after infection. The results showed that the proportion of G2/M phase cells increased significantly, indicating that the cell cycle was blocked by ALV-J infection. CEF cells were infected by ALV-J at 103 TCID50. Cell proliferation was tested by CCK-8 cell counting kit at different time points post infection. The results showed ALV-J infection significantly inhibited the cell proliferation compared to the control, especially at 5d and 7d. Finally, the expression and distribution of β-catenin in muscle, bursa and spleen were detected by immunohistochemistry analysis. The results showed that the expression of β-catenin in muscle was significantly lower in ALV-J infection. For bursa tissue, the increased β-catenin protein was present in the reticular cells between the cortex and medulla in the control group, while the expression of β-catenin was significantly decreased after ALV-J infection. For spleen tissue, the increased β-catenin protein was present in vascular endothelial cells, sinusoidal endothelial cells and red marrow in the control group, while the expression of β-catenin was significantly decreased after ALV-J infection. These results demonstrated that ALV-J infection could down-regulate the Wnt signaling pathway, and the down-regulated Wnt pathway might be one of the mechanisms resulting in growth retardation and immunosuppression in chickens.In order to study how ch NHE1 down-regulate Wnt signaling pathway in ALV-J infection, we used 103 TCID50 ALV-J strains to infect CEF cells and detected the expression of GSK3β, p-GSK3β-ser9 and p-GSK3β-tyr216 by western blot analysis at different time points after infection. The results showed that the protein expression level of GSK3β did not change significantly. The protein expression of p-GSK3β-ser9 also did not change significantly in the early stage of ALV-J infection, while up-regulated at 120 h. However, the protein expression of p-GSK3β-tyr216 increased significantly after ALV-J infection. In order to study the mechanism caused p-GSK3β-tyr216 up-regulation, we used 103 TCID50 ALV-J strains to infect CEF cells and detected the expression of p-PYK2 and ERK by western blot analysis at different time points after infection. The results showed that the protein expression level of p-PYK2 was decreased after ALV-J infection, while the expression of ERK did not change significantly. Next, we used 103 TCID50 ALV-J strains to infect CEF cells, and then detected the cytoplasmic Ca2+ concentration by Live cell Imaging System at 6 h post infection. The results showed that the cytoplasmic Ca2+ concentration was significantly increased by ALV-J infection. Finally, we designed the in vivo experiment. The expression and distribution of p-GSK3β-tyr216 in muscle tissue was detected by immunohistochemistry analysis. The results showed that the expression of p-GSK3β-tyr216 was significantly higher in ALV-J infection group. These results demonstrated that ALV-J infection could lead to the down-regulation of Wnt signaling pathway by up-regulating the intracellular Ca2+ concentration and p-GSK3β-tyr216 expression, and suggested that down-regulation of Wnt signaling pathway mediated by ch NHE1 might play a pivotal role in growth retardation caused by ALV-J infection. |
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