Proinflammatory cytokine TNF-α increases the stability of hepatitis B virus X protein through NF-κB signaling

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV-induced hepatocellular carcinoma (HCC). HBx interacts with several cell signaling molecules, leading to activation of various transcription factors including nuclear factor-kappaB (NF-kappa B). Activated NF-kappa B signaling is implicated in many human cancers including HCC. Here, we present evidence that the NF-kappa B signaling activator, tumor necrosis factor (TNF)-alpha, induces the accumulation of HBx in cells by increasing protein stability due to reduced proteasomal degradation. The effects of TNF-alpha on HBx protein stability are mediated via activated NF-kappa B effector kinases IKK alpha and IKK beta and p65. The non-IKK-phosphorylable p65-S534A mutant did not induce HBx protein stability; hence, phosphorylation of p65 by IKK is a key step in TNF-alpha-induced stabilization of HBx. Phospho-p65 showed higher affinity to HBx compared with the non-phosphorylable p65 mutant, suggesting that the interaction of phospho-p65 with HBx might be important for HBx stabilization. We also show that the increased level of HBx in cells cooperates with TNF-alpha toward activation of NF-kappa B and expression of NF-kappa B-regulated genes, indicating a positive feedback loop between HBx and NF-kappa B signaling. Overall, our study provides evidence for interplay between HBx and NF-kappa B signaling, which may account for HBV-mediated liver carcinogenesis.