ISG15 expression in response to double-stranded RNA or LPS in cultured Fetal Bovine Lung (FBL) cells

Abstract

Fetal bovine lung (FBL) cells are used in the culture of viruses which infect cattle and ISG15 plays a role in innate immunity against viral infections. However, whether the expression of ISG15 gene can be induced in FBL cells is still unknown. In this work, the expression of ISG15 in cultured FBL cells was detected after stimulated with poly I:C or LPS. Real-time PCR analyses revealed that the transcript of ISG15 can be induced by poly I:C or LPS. The increased expression of free ISG15 was confirmed via Western blotting. Furthermore, immunofluorescence assays demonstrated that IRF-3 was translocated from the cytoplasm to the nucleus in the FBL cells treated with poly I:C. Chromatin immunoprecipitation assays showed that IRF-3 can bind to the promoter of the bISG15 gene. To demonstrate IRF-3 can promote the expression of bISG15, we establish a luciferase-reporter system of bovine ISG15 gene in 293 T cells. The luciferase assay showed that the over-expression of bovine IRF-3 could activate the promoter of bISG15 gene. Taken together, these results suggest that the expression of bISG15 can be induced in FBL cells stimulated with poly I:C or LPS, and IRF-3 may play a role in inducing the expression of ISG15 in FBL cells.

Bovine Herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells

“…In our previous work it has been shown that bIRF-3 is the key factor in the induction of bISG15 in FBL cells treated with poly I:C [14] . IRF-3 commonly exists in the cytoplasm, after some stimulation by molecules such as dsRNA, then phosphorylated, dimerized and translocated to the nucleus to induce the expression of interferon stimulated genes (ISGs) [1] .…”

“…The viral protein bICP0 which encodes by the immediate early gene of BHV was the candidate protein for repressing expression. The induction of bISG15 in FBL cells treated with poly I:C is mainly stimulated by cellular transactivitor bIRF-3 [14] . 293T cells were co-transfected with a reporter plasmid pGL3-bISG15p, which has a bISG15 promoter at the upstream site of luciferase gene, bIRF-3 expressing plasmid pcDNA-bIRF-3 and bICP0 expressing plasmid pcDNA-bICP0.…”

“…There are still some questions to be answered, such as whether other BHV-1 viral patterns (Proteins and/or RNAs) take part in inhibiting expression, whether bICP0 is the key factor to repress the expression of bISG15 and what is the role of bISG15 in inhibiting BHV-1 infection. It has been reported that ISG15 could stabilize the existence of IRF-3 [17] , thus, if IRF-3 is stimulated the by the expression of ISG15 [8,14] , then ICP0 can inhibit the activation of IRF-3 [18,24] . It will be interesting to clarify the relationship among IRF-3, ISG15 and ICP0.…”

“…In our previous work, we have demonstrated that poly I:C can stimulate the expression of bISG15 robustly in FBL cells and bIRF-3 plays an important role in this process [14] . Considering the cascade relation among bISG15, the IFN pathway and BHV, in this study, we detected the transcription of bISG15 in FBL cells infected with BHV-1 and tried to find which viral gene or protein might take part in this process.…”

“…We found the expression of bISG15 in fetal bovine lung (FBL) cells could be induced after treatment with poly I : C or lipopolysaccaride (LPS) [14] . Furthermore, bISG15 expression can also be induced in bovine immunodeficiency virus (BIV) infected FBL cells, and the over-expression of bISG15 can repress the replication of BIV in FBL cells [15] .…”