Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication
Authors: Ariffianto Adi , Deng Lin , Abe Takayuki , Matsui Chieko , Ito Masahiko , Ryo Akihide , Aly Hussein Hassan , Watashi Koichi , Suzuki Tetsuro , Mizokami Masashi , Matsuura Yoshiharu , Shoji Ikuo 
Published: October 2023

ABSTRACT
Hepatitis B virus (HBV) infection promotes reactive oxygen species production while paradoxically inducing the expression of antioxidant enzymes. HBV-induced disorders of redox homeostasis are closely associated with the development of hepatic diseases. However, the molecular mechanisms underlying the HBV-induced antioxidant response are poorly understood. The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an intrinsic defense mechanism against oxidative stress. We here aim to elucidate the role of the Nrf2/ARE signaling pathway in the HBV life cycle. ARE-driven reporter assays revealed that expression of HBV X protein (HBx), but not HBV core, large HBV surface, or HBV polymerase, strongly enhanced ARE-luciferase activity, suggesting that HBx plays an important role in the HBV-induced antioxidant response. Knockdown of Nrf2 resulted in a marked decrease in HBx-induced ARE-luciferase activity. Immunoblot analysis and immunofluorescence staining suggested that HBx activates Nrf2 by increasing Nrf2 protein levels and enhancing Nrf2 nuclear localization. The oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) is required for the ubiquitin-dependent degradation of Nrf2. Coimmunoprecipitation analysis revealed that HBx interacted with Keap1, suggesting that HBx competes with Nrf2 for interaction with Keap1. A cell-based ubiquitylation assay showed that HBx promoted polyubiquitylation of Nrf2 via K6-linked polyubiquitin chains, and that this action may be associated with Nrf2 stabilization. A chromatin immunoprecipitation assay suggested that Nrf2 interacts with the HBV core promoter. Overexpression of Nrf2 strongly suppressed HBV core promoter activity, resulting in a marked reduction in viral replication. Based on the above, we propose that Keap1 recognizes HBx to activate the Nrf2/ARE signaling pathway upon HBV infection, thereby inhibiting HBV replication.

IMPORTANCE
The Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.