Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication

Ariffianto, Adi and Deng, Lin and Abe, Takayuki and Matsui, Chieko and Ito, Masahiko and Ryo, Akihide and Aly, Hussein Hassan and Watashi, Koichi and Suzuki, Tetsuro and Mizokami, Masashi and Matsuura, Yoshiharu and Shoji, Ikuo (2023) Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication. Journal of Virology, 97 (10). ISSN 0022538X

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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. Copyright © 2023 American Society for Microbiology. All Rights Reserved.

Item Type: Article
Additional Information: Cited by: 4; All Open Access, Green Open Access
Uncontrolled Keywords: Antioxidant Response Elements; Hepatitis B; Hepatitis B virus; Humans; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Oxidative Stress; Signal Transduction; Virus Replication; hepatitis B virus X protein; kelch like ECH associated protein 1; reactive oxygen metabolite; transcription factor Nrf2; ubiquitin; kelch like ECH associated protein 1; transcription factor Nrf2; antioxidant activity; antioxidant responsive element; Article; chromatin immunoprecipitation; coimmunoprecipitation; enzyme activity; enzyme linked immunosorbent assay; flow cytometry; gene expression; gene knockdown; gene overexpression; genetic transfection; Hepatitis B virus; homeostasis; immunoblotting; immunofluorescence; liver disease; luciferase assay; nonhuman; Nrf2 signaling; oxidative stress; protein expression; protein phosphorylation; reverse transcription polymerase chain reaction; RNA extraction; signal transduction; ubiquitination; upregulation; virus replication; Western blotting; antioxidant responsive element; genetics; hepatitis B; human; metabolism; oxidative stress; virus replication
Subjects: R Medicine > RB Pathology
Divisions: Faculty of Medicine, Public Health and Nursing > Public Health and Nutrition
Depositing User: Sri JUNANDI
Date Deposited: 06 Nov 2024 07:24
Last Modified: 06 Nov 2024 07:24
URI: https://ir.lib.ugm.ac.id/id/eprint/10791

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