JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

Abstract

Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.