1
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Wada M, Morita C, Ohsaki E, Ueda K. Cell-intrinsic regulation of HBV RNAs by the nonsense-mediated mRNA decay pathway controls viral replication. iScience 2025; 28:112460. [PMID: 40352722 PMCID: PMC12063116 DOI: 10.1016/j.isci.2025.112460] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 11/28/2024] [Accepted: 04/11/2025] [Indexed: 05/14/2025] Open
Abstract
Hepatitis B virus (HBV) is a causative agent for chronic liver hepatitis, which confers risk for liver cirrhosis and hepatocellular carcinoma. Among key viral transcripts, HBV pregenome RNA (pgRNA) is indispensable for viral replication, and therefore, quality control of pgRNA is critical for the HBV life cycle. Here, we revealed degradation of HBV RNAs by the nonsense-mediated mRNA decay (NMD) pathway, a host surveillance system of RNA quality. Degradation kinetics of the HBV RNAs indicated that pgRNA, 2.4 knt RNA, and 2.1 knt RNA were targets of the NMD pathway and also interacted robustly with phosphorylated UPF1 but not X RNA. Northern blotting showed that decay of the viral NMD candidates was also delayed in NMD-deficient cells. In contrast, NMD depletion promoted the formation of capsids containing genomic DNA and exhibiting antigen production. Our data strongly suggest that the NMD pathway inspects HBV transcripts to regulate HBV replication as an intrinsic antiviral defense.
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Affiliation(s)
- Masami Wada
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Chiharu Morita
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
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2
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Feng C, Shi J, Chen Y, Chen S, Cui J, Zhang J, Zheng X, Wang Y, Li F. A hepatitis B virus-free cccDNA-producing stable cell for antiviral screening. Antiviral Res 2025; 237:106143. [PMID: 40090467 DOI: 10.1016/j.antiviral.2025.106143] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Revised: 03/14/2025] [Accepted: 03/14/2025] [Indexed: 03/18/2025]
Abstract
The covalently closed circular DNA (cccDNA) of the Hepatitis B virus (HBV) serves as a template for producing progeny viruses in virally infected hepatocytes. Promising cccDNA-targeting antiviral agents remain unavailable and unpredictable in the research and development pipelines, making sterile HBV elimination challenging at the current stage. The major challenge of discriminating trace amounts of cccDNA from the abundant HBV relaxed circular DNA (rcDNA), which is nearly identical to cccDNA in sequence, substantially discourages efforts to discover and directly screen cccDNA-targeting drugs. Therefore, an easy cccDNA cell culture system is required for high-throughput drug screening. In this study, we designed an HBV cccDNA self-generating stable cell culture system using a functional complementary concept and successfully generated an HBV cccDNA Gaussia luciferase reporter cell line in HepG2 and Huh7 cells. This design ensures that the Gluc signal is exclusively expressed upon cccDNA formation, allowing for the accurate and easy measurement of cccDNA levels via luminescent signals. Using this system, in conjunction with a firefly luciferase reporter to monitor cell activity, we screened 2074 drugs in the HepG2-HBV-cccDNA/Firefly cell line. Four compounds were selected for further experimentation and their anti-HBV effects were confirmed. Thus, this virus-free hepatitis B cccDNA cell culture system provides a valuable and convenient platform for the high-throughput screening of anti-HBV drugs.
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Affiliation(s)
- Chengqian Feng
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Jingrong Shi
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Yunfu Chen
- State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co., Ltd, Dongguan 523871, China
| | - Sisi Chen
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Jianping Cui
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Jun Zhang
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Xiaowen Zheng
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Yaping Wang
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China
| | - Feng Li
- Institute of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, 510440, China; Scientific Research Center, Shanghai Public Health Clinical Center, Shanghai, 201508, China.
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3
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Watashi K, Shionoya K, Kobayashi C, Morita T. Hepatitis B and D virus entry. Nat Rev Microbiol 2025; 23:318-331. [PMID: 39572840 DOI: 10.1038/s41579-024-01121-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/17/2024] [Indexed: 04/17/2025]
Abstract
Hepatitis B virus (HBV) entry is the initial step of viral infection, leading to the formation of covalently closed circular DNA, which is a molecular reservoir of viral persistence and a key obstacle for HBV cure. The restricted entry of HBV into specific cell types determines the nature of HBV, which has a narrow host range in tissues and species. Hepatitis D virus (HDV) shares viral surface antigens with HBV and thus follows a similar entry mechanism at its early stages. In late 2012, sodium taurocholate cotransporting polypeptide was discovered as an HBV and HDV entry receptor. Since then, the mechanisms of HBV and HDV entry have been extensively analysed. These analyses have expanded our understanding of HBV and HDV host tropism and have provided new strategies for the development of antiviral agents. Notably, the structures of sodium taurocholate cotransporting polypeptide and its interaction with the 2-48 amino acid region of viral preS1 have been recently solved. These findings will stimulate further entry studies. In this Review, we summarize current understanding of HBV and HDV entry and future perspectives.
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Affiliation(s)
- Koichi Watashi
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.
- Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
| | - Kaho Shionoya
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Applied Biological Science, Tokyo University of Science, Noda, Japan
| | - Chisa Kobayashi
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Applied Biological Science, Tokyo University of Science, Noda, Japan
| | - Takeshi Morita
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
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4
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Chen F, Wettengel JM, Gegenfurtner F, Moosmüller J, Bunse T, Jeske SD, Hagen P, Ni Y, Urban S, Protzer U. Identification of NTCP animal orthologs supporting hepatitis B virus binding and infection. J Virol 2025; 99:e0183324. [PMID: 40042307 PMCID: PMC11998539 DOI: 10.1128/jvi.01833-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2024] [Accepted: 01/27/2025] [Indexed: 04/16/2025] Open
Abstract
Hepatitis B virus (HBV) infection is a significant global health threat, resulting in more than 800,000 deaths annually. Since HBV naturally infects only humans and chimpanzees, the development and evaluation of new therapies for chronic HBV infection are hindered by the lack of suitable animal models. Human sodium-taurocholate cotransporting polypeptide (NTCP) is a critical factor for HBV binding and entry, exhibiting species-specific differences in the amino acid sequences. This study investigated NTCP orthologs from various species to determine their capability to support HBV binding and infection. We demonstrate that nonhuman NTCP orthologs from woodchuck, ferret, aardvark, horse, rabbit, whale, big brown bat, cat, and rhinoceros support HBV binding and cellular entry, thereby rendering HepG2 cells susceptible to HBV infection upon expression. NTCP orthologs from hamster, goat, and cow support HBV binding but require specific amino acid exchanges to facilitate HBV infection. We show that replacement of the functional region, amino acids (aa) 84-87, in hamster NTCP with the human counterpart allows infection of HepG2 cells expressing the chimeric NTCP variant. Furthermore, we demonstrate that aa 82 in goat and cow NTCP, close to this functional region, needs to be modified to support HBV infection. This study could help identify previously unknown HBV reservoirs and may facilitate the establishment of new animal models.IMPORTANCEThe bona fide HBV entry receptor NTCP provides a natural barrier for cross-species transmission. We identified species-specific NTCP orthologues from woodchuck, ferret, aardvark, horse, rabbit, whale, big brown bat, cat, and rhinoceros that support HBV infection. This may reveal potential HBV reservoirs and facilitate the development of new HBV animal models.
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Affiliation(s)
- Fuwang Chen
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
| | - Jochen M. Wettengel
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
| | - Florian Gegenfurtner
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
| | - Judith Moosmüller
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
| | - Till Bunse
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
| | - Samuel D. Jeske
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
| | - Philipp Hagen
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
| | - Yi Ni
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany
| | - Stephan Urban
- Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany
| | - Ulrike Protzer
- Institute of Virology, School of Medicine and Health, Technical University of Munich/Helmholtz Munich, Munich, Germany
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
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5
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Taira J, Kubo T, Nagano H, Tsuda R, Ogi T, Nakashima K, Suzuki T. Effect of Nrf2 Activators in Hepatitis B Virus-Infected Cells Under Oxidative Stress. Mar Drugs 2025; 23:155. [PMID: 40278276 PMCID: PMC12028886 DOI: 10.3390/md23040155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2025] [Revised: 03/28/2025] [Accepted: 03/30/2025] [Indexed: 04/26/2025] Open
Abstract
The liver is an active metabolic site that generates high levels of reactive oxygen species (ROS). Oxidative stress has been implicated in the chronicity of hepatitis and hepatitis B virus (HBV) infection. This study aimed to determine the involvement of oxidative stress in HBV-infected cells and the efficacy of natural Nrf2 activators. The intracellular HBV pregenomic RNA copy number relative to total RNA was measured by RT-PCR, and various protein expressions associated with oxidative stress were analyzed by a Western blot analysis. The results showed that the Nrf2, HO-1, Akt, and Bcl-xL proteins were decreased by the continuous infection, indicating that HBV-positive cells were exposed to oxidative stress. The present study evaluated the anti-HBV infection effects of the Nrf2 activator fucoxanthin (Fx), a marine carotenoid from edible biological resources, including the comparative natural Nrf2 activator pteryxin (Ptx). These Nrf2 activators suppressed the HBV pregenomic RNA production in the HBV-infected cells, thus increasing the expression of the proteins of Nrf2 and HO-1. In the persistently infected cells transfected with the HBV genome, the Bcl-xL and Keap1 proteins, which contribute to suppressing the HBx protein involved in the HBV replication, were overexpressed. In particular, the activity of these protein expressions was marked at low concentrations of Fx. This suggests that natural Nrf2 activators may play a significant role in the HBV infection and could be a valuable source for further development through the functional utilization of food resources.
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Affiliation(s)
- Junsei Taira
- Department of Bioresources Technology, National Institute of Technology, Okinawa College, Okinawa 905-2192, Japan (R.T.)
| | - Takuya Kubo
- Department of Bioresources Technology, National Institute of Technology, Okinawa College, Okinawa 905-2192, Japan (R.T.)
| | - Hiroya Nagano
- Department of Bioresources Technology, National Institute of Technology, Okinawa College, Okinawa 905-2192, Japan (R.T.)
| | - Ryuji Tsuda
- Department of Bioresources Technology, National Institute of Technology, Okinawa College, Okinawa 905-2192, Japan (R.T.)
| | - Takayuki Ogi
- Department of Environment and Natural Resources, Okinawa Industrial Technology Center, Okinawa 904-2234, Japan
| | - Kenji Nakashima
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan; (K.N.); (T.S.)
| | - Tetsuro Suzuki
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan; (K.N.); (T.S.)
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6
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Abueldahab L, Suwanmanee Y, Muriungi N, Ohsaki E, Wada M, Kimura‐Ohba S, Ueda K. Analysis of the Functional Role of TIMM29 in the Hepatitis B Virus Life Cycle. Microbiol Immunol 2025; 69:229-246. [PMID: 39956808 PMCID: PMC11973851 DOI: 10.1111/1348-0421.13206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2025] [Revised: 02/05/2025] [Accepted: 02/05/2025] [Indexed: 02/18/2025]
Abstract
Hepatitis B virus (HBV) causes chronic hepatitis B, which can progress to liver cirrhosis and hepatocellular carcinoma. HBV has complex interactions with various cell organelles and proteins that ensure effective progeny virus production. We previously reported that a mitochondrial protein, TIMM29, should regulate the HBV life cycle through interactions with the HBV preS1 protein. Here, we established Halo-TIMM29wt-, Halo-TIMM29:∆99-192-, and Halo-TIMM29:92-194-expressing cells using TIMM29-knockout HB611 (TIMM29KO/HB611) cells, a stably HBV-producing cell line based on Huh6 cells. We found that HBV antigen expression and replication were downregulated in cells stably expressing full-length TIMM29, but not in those expressing TIMM29 deletion mutants. On the other hand, in the case of TIMM29-knockout C4 (TIMM29KO/C4), which is a human NTCP-expressing HepG2 cell line that is competent for HBV infection and amplification, these phenomena were not reproduced, except in full-length TIMM29 (Halo-TIMM29wt)-expressing cells. Using gene expression microarrays, we identified downregulation of ARRDC3 and BASP1 in TIMM29KO/HB611 and TIMM29KO/C4. It was suggested that TIMM29 localized at the mitochondrial inner membrane served as a signaling hub, orchestrating the activation of ARRDC3 and BASP1 expression to restrict HBV transcription. The expression of TIMM29 mutants in TIMM29KO/HB611 and TIMM29KO/C4 cells suggested that ARRDC3 was dependent on the HBV preS1-binding region of TIMM29 (amino acids 99-189). In contrast, BASP1 expression varied according to cell type, indicating additional regulatory mechanisms. Thus, this study should significantly advance our understanding of TIMM29-mediated inhibition of HBV amplification and lead to improvements in antiviral strategies and therapeutic interventions against HBV.
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Grants
- This research was supported by Grants from the Japan Agency for Medical Research and Development (AMED) (nos. 16fk0310504h0005, 17fk0310105h0001, 18fk0310105h0002, 19fk0310105h0003, 20fk0310105h0004, 21fk310105h005, 22fk0310505h0001, 23fk0310505h0002 and 24fk0310505h0003 to K.U.).
- This research was supported by Grants from the Japan Agency for Medical Research and Development (AMED) (nos. 16fk0310504h0005, 17fk0310105h0001, 18fk0310105h0002, 19fk0310105h0003, 20fk0310105h0004, 21fk310105h005, 22fk0310505h0001, 23fk0310505h0002 and 24fk0310505h0003 to K.U.).
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Affiliation(s)
- Limia Abueldahab
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Yadarat Suwanmanee
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Nelly Muriungi
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Masami Wada
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Shihoko Kimura‐Ohba
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and ImmunologyOsaka University Graduate School of MedicineSuitaJapan
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7
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Shrilall C, Arbuthnot P, Ely A. In Vitro Transcribed Artificial Primary MicroRNA for the Inhibition of Hepatitis B Virus Gene Expression in Cultured Cells. Microorganisms 2025; 13:604. [PMID: 40142497 PMCID: PMC11946339 DOI: 10.3390/microorganisms13030604] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 02/07/2025] [Accepted: 02/26/2025] [Indexed: 03/28/2025] Open
Abstract
Available interventions for the management of chronic hepatitis B (hepB) exhibit limited efficacy and barriers to vaccination against the hepatitis B virus (HBV) have hampered prophylaxis programmes. Development of potent therapeutics capable of functional cure of chronic hepB thus remains a relevant medical objective. RNA interference (RNAi) can be exploited to effect potent and specific silencing of target genes through the introduction of RNA sequences that mimic the natural activators of the pathway. To achieve a therapeutic effect, artificial primary microRNAs (pri-miRNAs) have been used extensively to target various viruses, including HBV. To date artificial pri-miRNAs have exclusively been produced from DNA expression cassettes. Although this achieves impressive silencing, eventual translation of this platform to the clinic is complicated by the requirement for viral vectors to deliver DNA. Consequently, clinical translation has been slow. Recently, the use of in vitro transcribed RNA, specifically to produce mRNA vaccines at industrial scale, has gained significant interest. We therefore sought to evaluate the feasibility of using in vitro transcribed artificial pri-miRNAs for the inhibition of HBV gene expression. Artificial HBV-targeting pri-miR-31 sequences, which are highly effective when expressed in cells from a DNA template, demonstrated modest silencing of viral replication when incorporated into mRNA that was transcribed in vitro. Off-target effects were also observed. Characterisation revealed that intracellular processing of the artificial pri-miRNAs was inefficient and non-specific effects were caused by stimulation of the interferon response. Nevertheless, optimised nuclear delivery of the artificial pri-miRNAs should improve their processing and achieve better anti-hepB efficacy.
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Affiliation(s)
| | | | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2050, South Africa; (C.S.); (P.A.)
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8
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Choi YM, Jang J, Kim DH, Kim Z, Kim E, Choe WH, Kim BJ. PreS1 deletions in genotype C HBV leads to severe hepatic inflammation and hepatocarcinogenesis via the IRE1-JNK axis. JHEP Rep 2025; 7:101274. [PMID: 39980750 PMCID: PMC11840487 DOI: 10.1016/j.jhepr.2024.101274] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 10/23/2024] [Accepted: 11/08/2024] [Indexed: 02/22/2025] Open
Abstract
Background & Aims Deletion of 15-21 nucleotides covering the preS1 start codon frequently occurs in patients with chronic HBV (CHB) with HBV genotype C and has been reported to be related to progression to hepatocellular carcinoma (HCC). However, the underlying mechanism causing the distinct phenotype of this HBV variant remains largely unknown. We investigated the mechanism by which preS1Del is related to liver disease progression and enhanced HBV replication, focusing on endoplasmic reticulum (ER) stress. Methods The effects of HBV replicative capacity, ER stress signaling, inflammation, cell death, and tumorigenesis resulting from PreS1 deletions were investigated through in vitro and in vivo experiments. Inhibitors of the IRE1-JNK pathway and IL6 blockade were used to examine HCC tumor load induced by preS1 deletions. Results The PreS1Del variant selectively activates the IRE1 pathway, mainly via enhanced colocalization between the ER and HBsAg in infected hepatocytes. This leads to enhanced HBV replication and production of tumor-promoting inflammatory cytokines and IL6 and COX2 via the IRE1-JNK signaling pathway. Furthermore, in vivo data showed that the activation of IRE1-JNK signaling consequently leads to lipid accumulation and apoptosis within 21Del-HBV-infected hepatocytes, collectively driving severe tumorigenesis in the liver. Notably, several inhibitors of the IRE1-JNK pathway dramatically inhibited HBV replication and inflammation induced by 21Del-HBV but not by the wild-type HBV in infected hepatocytes. Furthermore, IL6 blockade significantly reduced HCC tumor load induced by 21Del-HBV. Conclusions PreS1Del leads to enhanced HBV replication and HCC development through IRE1-JNK-IL6/COX2-mediated hepatocyte proliferation and liver inflammation. Inhibitors interfering with the IRE1-JNK-IL6 pathway could selectively inhibit HBV replication and inflammation in preS1Dels, suggesting their potential for the treatment of patients with CHB with preS1-deleted HBV variants. Impact and implications Deletion of 15-21 nucleotides at the preS1 start codon is common in patients with CHB with HBV genotype C and is linked to HCC progression. However, the mechanisms underlying the distinct phenotype of this variant remain largely unknown. We found that the preS1Del variant selectively activates the IRE1 pathway, primarily through enhanced IRE1-JNK-IL6 signaling. Inhibition of either the IRE1-JNK pathway or IL6 reduced HBV replication and tumor load in in vivo HCC models. This study enhances our understanding of the mechanisms of liver disease progression caused by 5' preS1Del variants and provides new insights into treatment strategies for patients with these variants. We believe our findings will resonate with a diverse audience, including patients and their physicians, the medical community, academia, the life sciences sector, and the general public.
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Affiliation(s)
- Yu-Min Choi
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
- Institute of Endemic Disease, Seoul National University Medical Research Center, Seoul, Republic of Korea
| | - Junghwa Jang
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
| | - Dong Hyun Kim
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
| | - Ziyun Kim
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
| | - Eunseo Kim
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
| | - Won Hyeok Choe
- Department of Internal Medicine, Konkuk University School of Medicine, Seoul 05030, Republic of Korea
| | - Bum-Joon Kim
- Department of Microbiology and Immunology, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea
- Institute of Endemic Disease, Seoul National University Medical Research Center, Seoul, Republic of Korea
- Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea
- Liver Research Institute, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea
- Cancer Research Institute, College of Medicine, Seoul National University, Seoul 03080, Republic of Korea
- Seoul National University Medical Research Center (SNUMRC), Seoul 03080, Republic of Korea
- BK21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul, Republic of Korea
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9
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Mnyandu NZ, Limani SW, Ely A, Wadee R, Arbuthnot P, Maepa MB. Long-term inhibition of Hepatitis B virus gene expression by a primary microrna expressing ancestral adeno-associated viral vector. Virol J 2025; 22:41. [PMID: 39962472 PMCID: PMC11834259 DOI: 10.1186/s12985-025-02662-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Accepted: 02/12/2025] [Indexed: 02/20/2025] Open
Abstract
Current treatments for chronic infection with the hepatitis B virus (HBV) rarely cure carriers from the disease. Previously reported use of serotype 8 adeno-associated viral (AAV8) vectors to deliver expression cassettes encoding anti-HBV artificial primary microRNAs (apri-miRs) has shown promise in preclinical studies. A recently designed synthetic ancestral AAV (Anc80L65) with high liver transduction efficiency is a promising new addition to the anti-HBV vector toolbox. This study engineered Anc80L65 to express HBx-targeting apri-miRs. Single dose administration of the vectors to cultured cells and HBV transgenic mice effected reductions of secreted HBV surface antigen (HBsAg). Circulating HBV particles and HBV core antigen (HBcAg) were also significantly diminished in mice receiving the anti-HBV apri-miR-expressing ancestral AAVs. Downregulation of HBV biomarkers occurred over a period of 12 months. Absence of inflammatory responses or liver toxicity indicated that the vectors had a good safety profile. These data suggest that a single dose of apri-miR-expressing Anc80L65 is safe and capable of mediating durable suppression of HBV gene expression. Targeting HBx, which is required for transcriptional activity of covalently closed circular DNA of HBV, makes this Anc80L65-derived vector a promising candidate for functional cure from chronic HBV infection.
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Affiliation(s)
- Njabulo Ziphezinhle Mnyandu
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Private Bag X3, Johannesburg, Wits 2050, South Africa
| | - Shonisani Wendy Limani
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Private Bag X3, Johannesburg, Wits 2050, South Africa
| | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Private Bag X3, Johannesburg, Wits 2050, South Africa
| | - Reubina Wadee
- Department of Anatomical Pathology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory Services, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Private Bag X3, Johannesburg, Wits 2050, South Africa
| | - Mohube Betty Maepa
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Private Bag X3, Johannesburg, Wits 2050, South Africa.
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10
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Morita C, Wada M, Ohsaki E, Kimura-Ohba S, Ueda K. Generation of Replication-Competent Hepatitis B Virus Harboring Tagged Polymerase for Visualization and Quantification of the Infection. Microbiol Immunol 2025; 69:43-58. [PMID: 39620377 DOI: 10.1111/1348-0421.13183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2024] [Revised: 11/03/2024] [Accepted: 11/04/2024] [Indexed: 01/07/2025]
Abstract
Hepatitis B virus (HBV) infection is a serious global health problem causing acute and chronic hepatitis and related diseases. Approximately, 296 million patients have been chronically infected with the virus, leading to cirrhosis and hepatocellular carcinoma. Although HBV polymerase (HBVpol, pol) plays a pivotal role in HBV replication and must be a definite therapeutic target. The problems are that the detailed functions and intracellular dynamics of HBVpol remain unclear. Here, we constructed two kinds of tagged HBVpol, PA-tagged and HiBiT-tagged pol, and the HBV-producing vectors. Each PA tag and HiBiT tag were inserted into N-terminus of spacer region on HBVpol open reading frame. Transfection of the plasmids into HepG2 cells led to production of HBV. These tagged HBVpol were detectable in HBV replicating cells and pol-HiBiT enabled quantitative analysis. Furthermore, these recombinant HBV were infectious to primary human hepatocytes. Thus, we successfully designed infectious and replication-competent recombinant HBV harboring detectable tagged HBVpol. Such infectious recombinant HBV will provide a novel tool to study HBVpol dynamics and develop new therapeutics against HBV.
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Grants
- This research was supported by Grants from the Japan Agency for Medical Research and Development (AMED) Grants (16fk0310504h0005, 17fk0310105h0001, 18fk0310105h0002, 19fk0310105h0003, 20fk0310105h0004, 21fk0310105h005, 22fk0310505h0001, 23fk0310505h0002, and 24fk0310505h003) to K.U. and from JST SPRING, Grant Number JPMJSP2138, to C.M. and from the Osaka University Transdisciplinary Program for Biomedical Entrepreneurship and Innovation (WISE program) to C.M.
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Affiliation(s)
- Chiharu Morita
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Masami Wada
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
- Center for Infectious Disease Education and Research (CiDER), Osaka, Japan
| | - Shihoko Kimura-Ohba
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
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11
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Chuang YC, Ou JHJ. Hepatitis B virus entry, assembly, and egress. Microbiol Mol Biol Rev 2024; 88:e0001424. [PMID: 39440957 DOI: 10.1128/mmbr.00014-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2024] Open
Abstract
SUMMARYHepatitis B virus (HBV) is an important human pathogen that chronically infects approximately 250 million people in the world, resulting in ~1 million deaths annually. This virus is a hepatotropic virus and can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. The entry of HBV into hepatocytes is initiated by the interaction of its envelope proteins with its receptors. This is followed by the delivery of the viral nucleocapsid to the nucleus for the release of its genomic DNA and the transcription of viral RNAs. The assembly of the viral capsid particles may then take place in the nucleus or the cytoplasm and may involve cellular membranes. This is followed by the egress of the virus from infected cells. In recent years, significant research progresses had been made toward understanding the entry, the assembly, and the egress of HBV particles. In this review, we discuss the molecular pathways of these processes and compare them with those used by hepatitis delta virus and hepatitis C virus , two other hepatotropic viruses that are also enveloped. The understanding of these processes will help us to understand how HBV replicates and causes diseases, which will help to improve the treatments for HBV patients.
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Affiliation(s)
- Yu-Chen Chuang
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
| | - J-H James Ou
- Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, USA
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12
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Yu F, Zhu Y, Li S, Hao L, Li N, Ye F, Jiang Z, Hu X. Dysfunction and regulatory interplay of T and B cells in chronic hepatitis B: immunotherapy and emerging antiviral strategies. Front Cell Infect Microbiol 2024; 14:1488527. [PMID: 39717542 PMCID: PMC11663751 DOI: 10.3389/fcimb.2024.1488527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Accepted: 11/20/2024] [Indexed: 12/25/2024] Open
Abstract
In the context of chronic hepatitis B virus (HBV) infection, the continuous replication of HBV within host hepatocytes is a characteristic feature. Rather than directly causing hepatocyte destruction, this replication leads to immune dysfunction and establishes a state of T-B immune tolerance. Successful clearance of the HBV virus is dependent on the close collaboration between humoral and cellular immunity. Humoral immunity, mediated by B-cell subpopulations, and cellular immunity, dominated by T-cell subpopulations show varying degrees of dysfunction during chronic hepatitis B (CHB). Notably, not all T- and B-cells produce positive immune responses. This review examine the most recent developments in the mutual regulation of T-B cells during chronic HBV infection. Our focus is on the prevailing immunotherapeutic strategies, such as T cell engineering, HBV-related vaccines, PD-1 inhibitors, and Toll-like receptor agonists. While nucleos(t)ide analogues (NUCs) and interferons have notable limitations, including inadequate viral suppression, drug resistance, and adverse reactions, several HBV entry inhibitors have shown promising clinical efficacy. To overcome the challenges posed by NUCs or monotherapy, the combination of immunotherapy and novel antiviral agents presents a promising avenue for future CHB treatment and potential cure.
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Affiliation(s)
- Fei Yu
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Yue Zhu
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Shenghao Li
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Liyuan Hao
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Na Li
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Fanghang Ye
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Zhi Jiang
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
| | - Xiaoyu Hu
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China
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13
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Yamashita A, Kasai H, Maekawa S, Tanaka T, Akaike Y, Ryo A, Enomoto N, Moriishi K. Berberine promotes K 48-linked polyubiquitination of HNF4α, leading to the inhibition of HBV replication. Antiviral Res 2024; 232:106027. [PMID: 39489302 DOI: 10.1016/j.antiviral.2024.106027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Revised: 10/17/2024] [Accepted: 10/31/2024] [Indexed: 11/05/2024]
Abstract
The current antiviral agents for the treatment of chronic infection with hepatitis B virus (HBV) do not completely remove covalently closed circular DNA (cccDNA) and integrated viral DNA fragments from patients. Berberine is an isoquinoline alkaloid extracted from various plants and has been reported to inhibit the replication of various types of DNA. In this study, we tested the effects of berberine and its derivatives on HBV infection. Berberine inhibited viral core promoter activity at the highest level among the compounds tested and suppressed HBV production and cccDNA synthesis in primary human hepatocytes and HBV-infected HepG2-NTCP cells at an EC50 value of 3.6 μM and a CC50 value of over 240.0 μM. Compared with other viral promoter activities, berberine treatment potently downregulated core promoter activity and reduced protein levels, but not RNA levels, of hepatic nuclear factor 4α (HNF4α), which primarily enhances enhancer II/core promoter activity. Furthermore, berberine treatment enhanced K48-linked, but not K63-linked, polyubiquitination and subsequent proteasome-dependent degradation of HNF4α. These results suggest that berberine enhances the polyubiquitination- and proteasome-dependent degradation of HNF4α and then inhibits HBV replication via the suppression of core promoter activity. The development of antiviral agents based on berberine may contribute to the amelioration of HBV-related disorders, regardless of the presence of residual cccDNA or integrated viral DNA fragments.
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Affiliation(s)
- Atsuya Yamashita
- Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan
| | - Hirotake Kasai
- Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan
| | - Shinya Maekawa
- The First Department of Internal Medicine, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan
| | - Tomohisa Tanaka
- Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan; Division of Hepatitis Virology, Institute for Genetic Medicine, Hokkaido University, Hokkaido, 060-0808, Japan
| | - Yasunori Akaike
- Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan
| | - Akihide Ryo
- Department of Virology III, National Institute for Infectious Diseases, Tokyo, 208-0011, Japan
| | - Nobuyuki Enomoto
- The First Department of Internal Medicine, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan
| | - Kohji Moriishi
- Department of Microbiology, Faculty of Medicine, Graduate Faculty of Interdisciplinary Research, University of Yamanashi, Yamanashi, Japan; Division of Hepatitis Virology, Institute for Genetic Medicine, Hokkaido University, Hokkaido, 060-0808, Japan; Center for Life Science Research, University of Yamanashi, Yamanashi, 409-3898, Japan.
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14
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Lam AM, Mani N, Ardzinski A, Stever K, Cuconati A, Micolochick Steuer H, Thi EP, Graves IE, Espiritu CL, Mesaros E, Kultgen SG, Fan K, Cole AG, Harasym TO, Rijnbrand R, Brown J, Eley T, Varughese T, Gane E, Picchio G, Sims KD, Sofia MJ. Preclinical and clinical antiviral characterization of AB-836, a potent capsid assembly modulator against hepatitis B virus. Antiviral Res 2024; 231:106010. [PMID: 39326502 DOI: 10.1016/j.antiviral.2024.106010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Revised: 09/11/2024] [Accepted: 09/23/2024] [Indexed: 09/28/2024]
Abstract
HBV capsid assembly modulators (CAMs) target the core protein and inhibit pregenomic RNA encapsidation and viral replication. HBV CAMs also interfere with cccDNA formation during de novo infection, which in turn suppresses transcription and production of HBV antigens. In this report, we describe the antiviral activities of AB-836, a potent and highly selective HBV CAM. AB-836 inhibited viral replication (EC50 = 0.010 μM) in HepDE19 cells, and cccDNA formation (EC50 = 0.18 μM) and HBsAg production (EC50 = 0.20 μM) in HepG2-NTCP cells during de novo infection. AB-836 showed broad genotype coverage, remained active against variants resistant to nucleos(t)ide analogs, and demonstrated improved antiviral potency against core variants resistant to other CAMs. AB-836 also mediated potent inhibition of HBV replication in a hydrodynamic injection mouse model, reducing both serum and liver HBV DNA. In a Phase 1 clinical study, 28 days of once-daily AB-836 oral dosing at 50, 100, and 200 mg resulted in mean serum HBV DNA declines of 2.57, 3.04, and 3.55 log10 IU/mL from baseline, respectively. Neither on-treatment viral rebound nor the emergence of viral resistance was observed during the 28-day treatment period. Furthermore, HBV DNA sequence analysis of baseline samples from the Phase 1 study revealed that 51.4% of the chronic hepatitis B participants contained at least one core polymorphism within the CAM-binding pocket, suggesting that genetic variations exist at this site. While AB-836 was discontinued due to clinical safety findings, data from the preclinical and clinical studies could help inform future optimization of HBV CAMs.
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Affiliation(s)
| | | | | | - Kim Stever
- Arbutus Biopharma Inc., Warminster, PA, USA
| | | | | | | | | | | | | | | | - Kristi Fan
- Arbutus Biopharma Inc., Warminster, PA, USA
| | | | | | | | | | | | | | - Edward Gane
- Auckland Clinical Studies, Grafton, Auckland, New Zealand
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15
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Li S, Hao L, Deng J, Zhang J, Yu F, Ye F, Li N, Hu X. The Culprit Behind HBV-Infected Hepatocytes: NTCP. Drug Des Devel Ther 2024; 18:4839-4858. [PMID: 39494152 PMCID: PMC11529284 DOI: 10.2147/dddt.s480151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 10/16/2024] [Indexed: 11/05/2024] Open
Abstract
Hepatitis B virus (HBV) is a globally prevalent human DNA virus responsible for over 250 million cases of chronic liver infections, leading to conditions such as liver inflammation, cirrhosis and hepatocellular carcinoma (HCC). Sodium taurocholate co-transporting polypeptide (NTCP) is a transmembrane protein highly expressed in human hepatocytes and functions as a bile acid (BA) transporter. NTCP has been identified as the receptor that HBV and its satellite virus, hepatitis delta virus (HDV), use to enter hepatocytes. HBV entry into hepatocytes is tightly regulated by various signaling pathways, and NTCP plays an important role as the initial stage of HBV infection. NTCP acts as an initiation signal, causing metabolic changes in hepatocytes and facilitating the entry of HBV into hepatocytes. Thus, a comprehensive understanding of NTCP's role is crucial. In this review, we will examine the regulatory mechanisms governing HBV pre-S1 binding to liver membrane NTCP, the role of NTCP in HBV internalization, and the transcriptional and translational regulation of NTCP expression. Additionally, we will discuss clinical drugs targeting NTCP, including combination therapies involving NTCP inhibitors, and consider the safety of NTCP as a therapeutic target.
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Affiliation(s)
- Shenghao Li
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Liyuan Hao
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Jiali Deng
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Junli Zhang
- Department of Infectious Diseases, Jiangsu Province Hospital of Chinese Medicine, Nanjing, Jiangsu Province, People’s Republic of China
| | - Fei Yu
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Fanghang Ye
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Na Li
- School of Clinical Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
| | - Xiaoyu Hu
- Department of Infectious Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, People’s Republic of China
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16
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Shionoya K, Park JH, Ekimoto T, Takeuchi JS, Mifune J, Morita T, Ishimoto N, Umezawa H, Yamamoto K, Kobayashi C, Kusunoki A, Nomura N, Iwata S, Muramatsu M, Tame JRH, Ikeguchi M, Park SY, Watashi K. Structural basis for hepatitis B virus restriction by a viral receptor homologue. Nat Commun 2024; 15:9241. [PMID: 39455604 PMCID: PMC11511851 DOI: 10.1038/s41467-024-53533-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Accepted: 10/15/2024] [Indexed: 10/28/2024] Open
Abstract
Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV.
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Affiliation(s)
- Kaho Shionoya
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Graduate School of Science and Technology, Tokyo University of Science, Chiba, Japan
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Jae-Hyun Park
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
- Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | - Toru Ekimoto
- Computational Life Science Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Junko S Takeuchi
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
| | - Junki Mifune
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takeshi Morita
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Naito Ishimoto
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Haruka Umezawa
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Kenichiro Yamamoto
- Computational Life Science Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Chisa Kobayashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Graduate School of Science and Technology, Tokyo University of Science, Chiba, Japan
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Atsuto Kusunoki
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Norimichi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - So Iwata
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
- RIKEN SPring-8 Center, Hyogo, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Infectious Disease Research, Foundation for Biomedical Research and Innovation at Kobe, Hyogo, Japan
| | - Jeremy R H Tame
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Mitsunori Ikeguchi
- Computational Life Science Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
- HPC- and AI-driven Drug Development Platform Division, Center for Computational Science, RIKEN, Kanagawa, Japan
| | - Sam-Yong Park
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan.
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
- Graduate School of Science and Technology, Tokyo University of Science, Chiba, Japan.
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.
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17
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Park ES, Won J, Ahn SH, Lee AR, Lee D, Moon JY, Choi MH, Kim KH. Gender-specific alteration of steroid metabolism and its impact on viral replication in a mouse model of hepatitis B virus infection. Anim Cells Syst (Seoul) 2024; 28:466-480. [PMID: 39296537 PMCID: PMC11409417 DOI: 10.1080/19768354.2024.2403569] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Revised: 08/02/2024] [Accepted: 09/02/2024] [Indexed: 09/21/2024] Open
Abstract
Hepatitis B virus (HBV) is a sex-specific pathogen that is more severe in males than in females. Sex disparities in HBV infection have been attributed to hormonal differences between males and females. However, whether HBV infection affects the metabolic signatures of steroid hormones and how these influences viral replication remains unclear. In this study, we investigated whether HBV infection alters steroid metabolism and its effects on HBV replication. Serum samples from male and female mice obtained after the hydrodynamic injection of replication-competent HBV plasmids were subjected to quantitative steroid profiling. Serum steroid levels in mice were analyzed using an in vitro metabolism assay with the mouse liver S9 fraction. The alteration of steroids by HBV infection was observed only in male mice, particularly with significant changes in androgens, whereas no significant hormonal changes were observed in female mice. Among the altered steroids, dehydroepiandrosterone (DHEA) levels increased the most in male mice after HBV infection. An in vitro metabolism assay revealed that androgen levels were significantly reduced in HBV-infected male mice. Furthermore, the genes involved in DHEA biosynthesis were significantly upregulated in HBV-infected male mice. Interestingly, reduced dihydrotestosterone in male mice significantly inhibits viral replication by suppressing HBV promoter activity, suggesting a viral strategy to overcome the antiviral effects of steroid hormones in males. Our data demonstrated that HBV infection can cause sex-specific changes in steroid metabolism.
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Affiliation(s)
- Eun-Sook Park
- Department of Pharmacology, Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seoul, Korea
| | - Juhee Won
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Korea
| | - Sung Hyun Ahn
- Department of Pharmacology, Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seoul, Korea
| | - Ah Ram Lee
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Korea
| | - Donghyo Lee
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Korea
| | - Ju-Yeon Moon
- Center for Advanced Biomolecular Recognition, Korea Institute of Science and Technology, Seoul, Korea
| | - Man Ho Choi
- Center for Advanced Biomolecular Recognition, Korea Institute of Science and Technology, Seoul, Korea
| | - Kyun-Hwan Kim
- Department of Precision Medicine, Sungkyunkwan University School of Medicine, Suwon, Korea
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18
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Yoshita M, Funaki M, Shimakami T, Kakuya M, Murai K, Sugimoto S, Kawase S, Matsumori K, Kawane T, Nishikawa T, Nakamura A, Suzuki R, Ishida A, Kawasaki N, Sato Y, Li YY, Sumiyadorj A, Nio K, Takatori H, Kawaguchi K, Kuroki K, Kato T, Honda M, Yamashita T. High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine. Viruses 2024; 16:1346. [PMID: 39205320 PMCID: PMC11360121 DOI: 10.3390/v16081346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2024] [Revised: 07/21/2024] [Accepted: 08/20/2024] [Indexed: 09/04/2024] Open
Abstract
We developed a novel hepatitis B virus (HBV) infection-monitoring system using a luminescent, 11-amino acid reporter (HiBiT). We performed high-throughput antiviral screening using this system to identify anti-HBV compounds. After the infection of primary human hepatocytes with the recombinant virus HiBiT-HBV, which contains HiBiT at its preS1, 1262 compounds were tested in a first screening using extracellular HiBiT activity as an indicator of viral infection. Following a second screening, we focused on the compound skimmianine, which showed a potent antiviral effect. When skimmianine was added at the same time as HiBiT-HBV infection, skimmianine inhibited HiBiT activity with EC50 of 0.36 pM, CC50 of 1.67 μM and a selectivity index (CC50:EC50 ratio) of 5,100,000. When skimmianine was added 72 h after HiBiT-HBV infection, the EC50, CC50 and selectivity index were 0.19 μM, 1.87 μM and 8.79, respectively. Time-lapse fluorescence imaging analysis using another recombinant virus, ReAsH-TC155HBV, with the insertion of tetra-cysteine within viral capsid, revealed that skimmianine inhibited the accumulation of the capsid into hepatocytes. Furthermore, skimmianine did not inhibit either attachment or internalization. These results imply that skimmianine inhibits the retrograde trafficking of the virus after internalization. This study demonstrates the usefulness of the recombinant virus, HiBiT-HBV, for high-throughput screening to identify anti-HBV compounds.
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Affiliation(s)
- Mika Yoshita
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Masaya Funaki
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Tetsuro Shimakami
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Masaki Kakuya
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Kazuhisa Murai
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Saiho Sugimoto
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Shotaro Kawase
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Koji Matsumori
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Taro Kawane
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Tomoki Nishikawa
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Asuka Nakamura
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Reo Suzuki
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Atsuya Ishida
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Narumi Kawasaki
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Yuga Sato
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Ying-Yi Li
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Ariunaa Sumiyadorj
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Kouki Nio
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Hajime Takatori
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Kazunori Kawaguchi
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Kazuyuki Kuroki
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan;
| | - Masao Honda
- Department of Clinical Laboratory Medicine, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; (K.M.); (A.N.); (R.S.); (A.I.); (N.K.); (Y.S.); (M.H.)
| | - Taro Yamashita
- Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; (M.Y.); (M.F.); (M.K.); (S.S.); (S.K.); (K.M.); (T.K.); (T.N.); (Y.-Y.L.); (A.S.); (K.N.); (H.T.); (K.K.); (K.K.); (T.Y.)
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19
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Lin S, Chang P, Tsao S, Aderinwale A, Sallapalli BT, He J, Zhang Y. Oxysterol binding protein (OSBP) contributes to hepatitis E virus replication. Virol J 2024; 21:161. [PMID: 39039546 PMCID: PMC11265327 DOI: 10.1186/s12985-024-02438-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Accepted: 07/16/2024] [Indexed: 07/24/2024] Open
Abstract
Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus and causes primarily acute self-limiting infections. The ORF1 of the HEV genome encodes a polyprotein around 190 kDa, which contains several putative domains, including helicase and RNA-dependent RNA polymerase. The HEV-encoded helicase is a member of the superfamily 1 helicase family and possesses multiple enzymatic functions, such as RNA 5'-triphosphatase, RNA unwinding, and NTPase, which are thought to contribute to viral RNA synthesis. However, the helicase interaction with cellular proteins remains less known. Oxysterol binding protein (OSBP) is a lipid regulator that shuffles between the Golgi apparatus and the endoplasmic reticulum for cholesterol and phosphatidylinositol-4-phosphate exchange and controls the efflux of cholesterol from cells. In this study, the RNAi-mediated silencing of OSBP significantly reduced HEV replication. Further studies indicate that the HEV helicase interacted with OSBP, shown by co-immunoprecipitation and co-localization in co-transfected cells. The presence of helicase blocked OSBP preferential translocation to the Golgi apparatus. These results demonstrate that OSBP contributes to HEV replication and enrich our understanding of the HEV-cell interactions.
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Affiliation(s)
- Shaoli Lin
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | - Peixi Chang
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | - Shane Tsao
- National Cancer Institute, National Institute of Health, Bethesda, MD, USA
| | - Abigail Aderinwale
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | - Bhargava Teja Sallapalli
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | - Jia He
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA
| | - Yanjin Zhang
- Molecular Virology Laboratory, VA-MD College of Veterinary Medicine, University of Maryland, College Park, MD, USA.
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20
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Tu T, Wettengel J, Xia Y, Testoni B, Littlejohn M, Le Bert N, Ebert G, Verrier ER, Tavis JE, Cohen C. Major open questions in the hepatitis B and D field - Proceedings of the inaugural International emerging hepatitis B and hepatitis D researchers workshop. Virology 2024; 595:110089. [PMID: 38640789 PMCID: PMC11517827 DOI: 10.1016/j.virol.2024.110089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 04/01/2024] [Accepted: 04/12/2024] [Indexed: 04/21/2024]
Abstract
The early and mid-career researchers (EMCRs) of scientific communities represent the forefront of research and the future direction in which a field takes. The opinions of this key demographic are not commonly aggregated to audit fields and precisely demonstrate where challenges lie for the future. To address this, we initiated the inaugural International Emerging Researchers Workshop for the global Hepatitis B and Hepatitis D scientific community (75 individuals). The cohort was split into small discussion groups and the significant problems, challenges, and future directions were assessed. Here, we summarise the outcome of these discussions and outline the future directions suggested by the EMCR community. We show an effective approach to gauging and accumulating the ideas of EMCRs and provide a succinct summary of the significant gaps remaining in the Hepatitis B and Hepatitis D field.
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Affiliation(s)
- Thomas Tu
- Storr Liver Centre, The Westmead Institute for Medical Research, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia; Centre for Infectious Diseases and Microbiology, Sydney Infectious Diseases Institute, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia.
| | - Jochen Wettengel
- Division of Pathobiology and Immunology, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, 97006, USA; Institute of Virology, Technical University of Munich /Helmholtz Munich, Munich, Germany; German Center for Infection Research, Munich Partner Site, 81675, Munich, Germany
| | - Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, China; Hubei Jiangxia Laboratory, Wuhan, China; Pingyuan Laboratory, Henan, China
| | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon, Lyon, France; University of Lyon, Université Claude-Bernard, Lyon, France; Hepatology Institute of Lyon, France
| | - Margaret Littlejohn
- Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital and Department of Infectious Disease, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
| | - Nina Le Bert
- Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore
| | - Gregor Ebert
- Institute of Virology, Technical University of Munich /Helmholtz Munich, Munich, Germany
| | - Eloi R Verrier
- University of Strasbourg, Inserm, Institute for Translational Medicine and Liver Disease, UMR_S1110, Strasbourg, France
| | - John E Tavis
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine and the Saint Louis University Institute for Drug and Biotherapeutic Innovation, Saint Louis, MO, USA
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21
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Fujimuro M. The Interactions between Cells and Viruses. Int J Mol Sci 2024; 25:6886. [PMID: 38999995 PMCID: PMC11241451 DOI: 10.3390/ijms25136886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Accepted: 06/18/2024] [Indexed: 07/14/2024] Open
Abstract
Many infectious diseases are caused by life-threatening DNA and RNA viruses and have been reported worldwide, including those caused by emerging and re-emerging viruses [...].
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Affiliation(s)
- Masahiro Fujimuro
- Department of Cell Biology, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
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22
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Su PY(A, Chang CH, Yen SCB, Wu HY, Tung WJ, Hu YP, Chen YYI, Lin MH, Shih C, Chen PJ, Tsai K. Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production. Proc Natl Acad Sci U S A 2024; 121:e2400378121. [PMID: 38830096 PMCID: PMC11181118 DOI: 10.1073/pnas.2400378121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 04/20/2024] [Indexed: 06/05/2024] Open
Abstract
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
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Affiliation(s)
- Pei-Yi (Alma) Su
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Chih-Hsu Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Shin-Chwen Bruce Yen
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
- Taiwan International Graduate Program, National Yang-Ming Chiao-Tung University and Academia Sinica, Taipei115, Taiwan
| | - Hsiu-Yi Wu
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Wan-Ju Tung
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Yu-Pei Hu
- Institute of Biomedical Sciences Summer Undergraduate Internship Program, Academia Sinica, Taipei115, Taiwan
| | - Yen-Yu Ian Chen
- Institute of Biomedical Sciences Summer Undergraduate Internship Program, Academia Sinica, Taipei115, Taiwan
| | - Miao-Hsia Lin
- Department of Microbiology, National Taiwan University College of Medicine, Taipei100, Taiwan
| | - Chiaho Shih
- Graduate Institute of Cell Biology, College of Life Sciences, China Medical University, Taichung404, Taiwan
| | - Pei-Jer Chen
- National Taiwan University Center for Genomic Medicine, National Taiwan University, Taipei100, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei100, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital, Taipei100, Taiwan
| | - Kevin Tsai
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
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23
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Chen Y, Duan M, Wang X, Xu J, Tian S, Xu X, Duan A, Mahal A, Zhu Y, Zhu Q. Synthesis and evaluation of pentacyclic triterpenoids conjugates as novel HBV entry inhibitors targeting NTCP receptor. Bioorg Chem 2024; 147:107385. [PMID: 38663255 DOI: 10.1016/j.bioorg.2024.107385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Revised: 03/27/2024] [Accepted: 04/16/2024] [Indexed: 05/18/2024]
Abstract
Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157-165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.
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Affiliation(s)
- Yixin Chen
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, China; Guangdong Provincial Engineering Laboratory of Chinese Medicine Preparation Technology, Guangzhou 510515, China
| | - Meitao Duan
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Xiangwan Wang
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Jianling Xu
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Shuo Tian
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Xiaotian Xu
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Ao Duan
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
| | - Ahmed Mahal
- Department of Medical Biochemical Analysis, College of Health Technology, Cihan University-Erbil, Erbil, Kurdistan Region, Iraq
| | - Yongyan Zhu
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, China; Guangdong Provincial Engineering Laboratory of Chinese Medicine Preparation Technology, Guangzhou 510515, China.
| | - Quanhong Zhu
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, China; Guangdong Provincial Engineering Laboratory of Chinese Medicine Preparation Technology, Guangzhou 510515, China.
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24
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Liu X, Pang X, Wan Z, Zhao J, Gao Z, Deng H. Dopamine Inhibits the Expression of Hepatitis B Virus Surface and e Antigens by Activating the JAK/STAT Pathway and Upregulating Interferon-stimulated Gene 15 Expression. J Clin Transl Hepatol 2024; 12:443-456. [PMID: 38779516 PMCID: PMC11106351 DOI: 10.14218/jcth.2024.00051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Revised: 04/16/2024] [Accepted: 04/17/2024] [Indexed: 05/25/2024] Open
Abstract
Background and Aims Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and liver cancer, and its treatment continues to be difficult. We previously demonstrated that a dopamine analog inhibited the packaging of pregenomic RNA into capsids. The present study aimed to determine the effect of dopamine on the expressions of hepatitis B virus surface and e antigens (HBsAg and HBeAg, respectively) and to elucidate the underlying mechanism. Methods We used dopamine-treated HBV-infected HepG2.2.15 and NTCP-G2 cells to monitor HBsAg and HBeAg expression levels. We analyzed interferon-stimulated gene 15 (ISG15) expression in dopamine-treated cells. We knocked down ISG15 and then monitored HBsAg and HBeAg expression levels. We analyzed the expression of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway factors in dopamine-treated cells. We used dopamine hydrochloride-treated adeno-associated virus/HBV-infected mouse model to evaluate HBV DNA, HBsAg, and HBeAg expression. HBV virus was collected from HepAD38.7 cell culture medium. Results Dopamine inhibited HBsAg and HBeAg expression and upregulated ISG15 expression in HepG2.2.15 and HepG2-NTCP cell lines. ISG15 knockdown increased HBsAg and HBeAg expression in HepG2.2.15 cells. Dopamine-treated cells activated the JAK/STAT pathway, which upregulated ISG15 expression. In the adeno-associated virus-HBV murine infection model, dopamine downregulated HBsAg and HBeAg expression and activated the JAK-STAT/ISG15 axis. Conclusions Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.
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Affiliation(s)
- Xiaoquan Liu
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
| | - Xiuqing Pang
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
| | - Zhiping Wan
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
| | - Jinhua Zhao
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
| | - Zhiliang Gao
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
- Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong, China
| | - Hong Deng
- Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China
- Guangdong Key Laboratory of Liver Disease Research, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, China
- Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong, China
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25
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Willner E, Kolbe F, Momburg F, Protzer U, Dietz H. Hepatitis B Virus Neutralization with DNA Origami Nanoshells. ACS APPLIED MATERIALS & INTERFACES 2024; 16:25836-25842. [PMID: 38728653 PMCID: PMC11129107 DOI: 10.1021/acsami.4c03700] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Revised: 04/30/2024] [Accepted: 05/03/2024] [Indexed: 05/12/2024]
Abstract
We demonstrate the use of DNA origami to create virus-trapping nanoshells that efficiently neutralize hepatitis B virus (HBV) in cell culture. By modification of the shells with a synthetic monoclonal antibody that binds to the HBV envelope, the effective neutralization potency per antibody is increased by approximately 100 times compared to using free antibodies. The improvements in neutralizing the virus are attributed to two factors: first, the shells act as a physical barrier that blocks the virus from interacting with host cells; second, the multivalent binding of the antibodies inside the shells lead to stronger attachment to the trapped virus, a phenomenon known as avidity. Pre-incubation of shells with HBV and simultaneous addition of both components separately to cells lead to comparable levels of neutralization, indicating rapid trapping of the virions by the shells. Our study highlights the potential of the DNA shell system to rationally create antivirals using components that, when used individually, show little to no antiviral effectiveness.
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Affiliation(s)
- Elena
M. Willner
- Department
of Biosciences, School of Natural Sciences and Munich Institute of
Biomedical Engineering, Technical University
of Munich, Boltzmannstraße 11, 85748 Garching, Germany
| | - Fenna Kolbe
- Institute
of Virology, School of Medicine & Health, Technical University of Munich and Helmholtz Munich, Trogerstraße 30, 81675 Munich, Germany
| | - Frank Momburg
- Translational
Immunity Unit, German Cancer Research Center
(DKFZ), Im Neuenheimer Feld, 69120 Heidelberg, Germany
| | - Ulrike Protzer
- Institute
of Virology, School of Medicine & Health, Technical University of Munich and Helmholtz Munich, Trogerstraße 30, 81675 Munich, Germany
- German
Center for Infection Research (DZIF),
Munich Partner Site, 81675 Munich, Germany
| | - Hendrik Dietz
- Department
of Biosciences, School of Natural Sciences and Munich Institute of
Biomedical Engineering, Technical University
of Munich, Boltzmannstraße 11, 85748 Garching, Germany
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26
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Kato D, Choy RWY, Canales E, Dick RA, Lake AD, Shapiro ND, Chin E, Li J, Zhang JR, Wu Q, Saito RD, Metobo S, Aktoudianakis E, Schroeder SD, Yang ZY, Glatt DM, Balsitis S, Gamelin L, Yu M, Cheng G, Delaney WE, Link JO. Discovery of Hepatitis B Virus Surface Antigen Suppressor GS-8873. ACS Med Chem Lett 2024; 15:546-554. [PMID: 38628802 PMCID: PMC11017420 DOI: 10.1021/acsmedchemlett.4c00037] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Revised: 03/18/2024] [Accepted: 03/19/2024] [Indexed: 04/19/2024] Open
Abstract
Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.
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Affiliation(s)
- Darryl Kato
- Gilead
Sciences, Foster City, California 94404, United States
| | | | - Eda Canales
- Gilead
Sciences, Foster City, California 94404, United States
| | - Ryan A. Dick
- Maze
Therapeutics, South
San Francisco, California 94080, United States
| | - April D. Lake
- Gilead
Sciences, Foster City, California 94404, United States
| | | | - Elbert Chin
- Gilead
Sciences, Foster City, California 94404, United States
| | - Jiayao Li
- Gilead
Sciences, Foster City, California 94404, United States
| | | | - Qiaoyin Wu
- Gilead
Sciences, Foster City, California 94404, United States
| | - Roland D. Saito
- Gilead
Sciences, Foster City, California 94404, United States
| | - Sammy Metobo
- Circle
Pharma, South San Francisco, California 94080, United States
| | | | | | - Zheng-Yu Yang
- Gilead
Sciences, Foster City, California 94404, United States
| | - Dylan M. Glatt
- 23andMe
Therapeutics, South
San Francisco, California 94080, United States
| | - Scott Balsitis
- Gilead
Sciences, Foster City, California 94404, United States
| | - Lindsay Gamelin
- Gilead
Sciences, Foster City, California 94404, United States
| | - Mei Yu
- Gilead
Sciences, Foster City, California 94404, United States
| | - Guofeng Cheng
- AusperBio
Therapeutics Inc., San Mateo, California 94401, United States
| | | | - John O. Link
- Gilead
Sciences, Foster City, California 94404, United States
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27
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Ganbold A, Bayarsaikhan S, Chimedtseren M, Noronrenchin O, Ochirkhuree B. Hepatitis D virus reactivation in liver-transplanted patients receiving hepatitis B immunoglobulin. CLINICAL TRANSPLANTATION AND RESEARCH 2024; 38:46-51. [PMID: 38448049 PMCID: PMC11075813 DOI: 10.4285/kjt.23.0058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 01/13/2024] [Accepted: 01/31/2024] [Indexed: 03/08/2024]
Abstract
Background The utility of hepatitis B immunoglobulin (HBIg) in hepatitis D virus (HDV)-reactivation prophylaxis remains contentious. This study compared liver transplant (LT) patients based on whether they received perioperative HBIg to assess its protective effect against HDV reactivation. Methods Fifty-seven recipients with hepatitis B virus (HBV) and HBV/HDV, who were at least 1 year posttransplantation as of January 1, 2021, were enrolled in this single-center study. Tests for hepatitis B surface antigen (HBsAg), anti-HDV antibody, and quantitative reverse transcription polymerase chain reaction for HBV DNA and HDV RNA were performed. Interviews were conducted to assess compliance with the nucleos(t) ide analogue (NA) regimen and to document preoperative HBV/HDV status. Liver function tests were also carried out. The nonparametric Mann-Whitney U-test was utilized to determine statistical significance, with P<0.05 considered significant. Data analysis was conducted using GraphPad Prism software. Results The prevalence of HDV RNA, HBV DNA, HBsAg, and anti-HDV positivity in the HBIg group (n=23) was 4.3% (n=1), 17.4% (n=4), 8.7% (n=2), and 95.7% (n=22), respectively. In the non-HBIg group (n=34), these rates were 5.9% (n=2), 8.8% (n=3), 11.8% (n=4), and 97.1% (n=33), respectively. Interviews revealed that all reactivations occurred in patients who were noncompliant with their NA regimen. Eleven of the 13 patients initially reported to be monoinfected with HBV pretransplantation were anti-HDV-positive. Conclusions No HDV replication occurred in either group due to spontaneous reactivation. High-efficacy NAs appear to be effective in sustaining HDV suppression post-LT. Most recrudescent cases of chronic hepatitis D are mild and self-limiting, typically resolving after 1-2 years of replication, as evidenced by liver function tests.
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Affiliation(s)
- Anar Ganbold
- Gastroenterology Center, The First Central Hospital of Mongolia, Ulaanbaatar, Mongolia
| | - Sumiya Bayarsaikhan
- Gastroenterology Center, The First Central Hospital of Mongolia, Ulaanbaatar, Mongolia
| | | | | | - Bayarmaa Ochirkhuree
- Gastroenterology Center, The First Central Hospital of Mongolia, Ulaanbaatar, Mongolia
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28
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Asami J, Park JH, Nomura Y, Kobayashi C, Mifune J, Ishimoto N, Uemura T, Liu K, Sato Y, Zhang Z, Muramatsu M, Wakita T, Drew D, Iwata S, Shimizu T, Watashi K, Park SY, Nomura N, Ohto U. Structural basis of hepatitis B virus receptor binding. Nat Struct Mol Biol 2024; 31:447-454. [PMID: 38233573 DOI: 10.1038/s41594-023-01191-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2023] [Accepted: 11/24/2023] [Indexed: 01/19/2024]
Abstract
Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.
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Affiliation(s)
- Jinta Asami
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
| | - Jae-Hyun Park
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan
| | - Yayoi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Chisa Kobayashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Applied Biological Science, Tokyo University of Science, Noda, Japan
| | - Junki Mifune
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Naito Ishimoto
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan
| | - Tomoko Uemura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Kehong Liu
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Yumi Sato
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Zhikuan Zhang
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - David Drew
- Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
| | - So Iwata
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toshiyuki Shimizu
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
- Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.
| | - Sam-Yong Park
- Drug Design Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
| | - Norimichi Nomura
- Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
| | - Umeharu Ohto
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.
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29
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Lam AM, Dugyala RR, Sheraz M, Liu F, Thi EP, Graves IE, Cuconati A, Steuer HM, Ardzinski A, Overholt N, Mason JD, Gotchev D, Cole AG, Harasym TO, Sofia MJ. Preclinical Antiviral and Safety Profiling of the HBV RNA Destabilizer AB-161. Viruses 2024; 16:323. [PMID: 38543689 PMCID: PMC10975527 DOI: 10.3390/v16030323] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Revised: 02/13/2024] [Accepted: 02/15/2024] [Indexed: 05/23/2024] Open
Abstract
HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC90), compared to concentrations in plasma which were substantially below its EC90, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161's clinical development.
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Affiliation(s)
- Angela M. Lam
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA; (R.R.D.); (M.S.); (F.L.); (E.P.T.); (I.E.G.); (A.C.); (H.M.S.); (A.A.); (N.O.); (J.D.M.); (D.G.); (A.G.C.); (T.O.H.)
| | | | | | | | | | | | | | | | | | | | | | | | | | | | - Michael J. Sofia
- Arbutus Biopharma, Inc., 701 Veterans Circle, Warminster, PA 18974, USA; (R.R.D.); (M.S.); (F.L.); (E.P.T.); (I.E.G.); (A.C.); (H.M.S.); (A.A.); (N.O.); (J.D.M.); (D.G.); (A.G.C.); (T.O.H.)
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30
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Tian R, Yang D, Xu B, Deng R, Xue B, Wang L, Li H, Liu Q, Wang X, Tang S, Wan M, Pei H, Zhu H. Establishment of cell culture model and humanized mouse model of chronic hepatitis B virus infection. Microbiol Spectr 2024; 12:e0274523. [PMID: 38018998 PMCID: PMC10783038 DOI: 10.1128/spectrum.02745-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2023] [Accepted: 10/20/2023] [Indexed: 11/30/2023] Open
Abstract
IMPORTANCE Approximately 257 million people worldwide have been infected with hepatitis B virus (HBV), and HBV infection can cause chronic hepatitis, cirrhosis, and even liver cancer. The lack of suitable and effective infection models has greatly limited research in HBV-related fields for a long time, and it is still not possible to discover a method to completely and effectively remove the HBV genome. We have constructed a hepatocellular carcinoma cell line, HLCZ01, that can support the complete life cycle of HBV. This model can mimic the long-term stable infection of HBV in the natural state and can replace primary human hepatocytes for the development of human liver chimeric mice. This model will be a powerful tool for research in the field of HBV.
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Affiliation(s)
- Renyun Tian
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Di Yang
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Biaoming Xu
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Rilin Deng
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Binbin Xue
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Luoling Wang
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Huiyi Li
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Qian Liu
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Xiaohong Wang
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Songqing Tang
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Mengyu Wan
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
| | - Hua Pei
- Department of Pathogen Biology and Immunology, Department of Clinical Laboratory of the Second Affiliated Hospital, Key Laboratory of Tropical Translational Medicine of Ministry of Education,Institute of Pathogen Biology and Immunology,School of Basic Medicine and Life Science, The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, The Second Affiliated Hospital of Hainan Medical University, Hainan Medical University, Hainan, China
| | - Haizhen Zhu
- Institute of Pathogen Biology and Immunology, College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, China
- Department of Pathogen Biology and Immunology, Department of Clinical Laboratory of the Second Affiliated Hospital, Key Laboratory of Tropical Translational Medicine of Ministry of Education,Institute of Pathogen Biology and Immunology,School of Basic Medicine and Life Science, The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, The Second Affiliated Hospital of Hainan Medical University, Hainan Medical University, Hainan, China
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31
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Xia Y, Park SB, Liang TJ. Stem Cell-Derived Hepatocyte-Like Cells for Hepatitis B Virus Infection. Methods Mol Biol 2024; 2837:1-9. [PMID: 39044070 DOI: 10.1007/978-1-0716-4027-2_1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/25/2024]
Abstract
Hepatitis B, the leading cause of liver diseases worldwide, is a result of infection with hepatitis B virus (HBV). Due to its obligate intracellular life cycle, culture systems for efficient HBV replication are vital. Although basic and translational research on HBV has been performed for many years, conventional hepatocellular culture systems are not optimal. These studies have greatly benefited from recent improvements in cell culture models based on stem cell technology for HBV replication and infection studies. Here we describe a protocol for the differentiation of human stem cell-derived hepatocyte-like cells (HLCs) and subsequent HBV infection. HLCs are capable of expressing hepatocyte markers and host factors important for hepatic function maintenance. These cells fully support HBV infection and virus-host interactions. Stem cell-derived HLCs provide a new tool for antiviral drug screening and development.
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Affiliation(s)
| | | | - T Jake Liang
- National Institutes of Health, Bethesda, MD, USA
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32
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Chiou WC, Lyu YS, Hsia TL, Chen JC, Lin LC, Chang MF, Hsu MS, Huang C. Ergosterol peroxide blocks HDV infection as a novel entry inhibitor by targeting human NTCP receptor. Biomed Pharmacother 2024; 170:116077. [PMID: 38154274 DOI: 10.1016/j.biopha.2023.116077] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Revised: 12/08/2023] [Accepted: 12/21/2023] [Indexed: 12/30/2023] Open
Abstract
Hepatitis D virus (HDV), which co-infects or superinfects patients with hepatitis B virus, is estimated to affect 74 million people worldwide. Chronic hepatitis D is the most severe form of viral hepatitis and can result in liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Currently, there are no efficient HDV-specific drugs. Therefore, there is an urgent need for novel HDV therapies that can achieve a functional cure or even eliminate the viral infection. In the HDV life cycle, agents targeting the entry step of HDV infection preemptively reduce the intrahepatic viral RNA. Human sodium taurocholate co-transporting polypeptide (hNTCP), a transporter of bile acids on the plasma membrane of hepatocytes, is an essential entry receptor of HDV and is a promising molecular target against HDV infection. Here, we investigated the effect of ergosterol peroxide (EP) on HDV infection in vitro and in vivo. EP inhibited HDV infection of hNTCP-expressing dHuS-E/2 hepatocytes by interrupting the early fusion/endocytosis step of HDV entry. Furthermore, molecular modeling suggested that EP hinders LHBsAg binding to hNTCP by blocking access to S267 and V263. In addition, we generated hNTCP-expressing transgenic (Tg) C57BL/6 mice using the Cre/loxP system for in vivo study. EP reduced the liver HDV RNA level of HDV-challenged hNTCP-Cre Tg mice. Intriguingly, EP downregulated the mRNA level of liver IFN-γ. We demonstrate that EP is a bona fide HDV entry inhibitor that acts on hNTCP and has the potential for use in HDV therapies.
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Affiliation(s)
- Wei-Chung Chiou
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
| | - Yi-Syuan Lyu
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
| | - Tzu-Lan Hsia
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
| | - Jui-Chieh Chen
- Department of Biochemical Science and Technology, National Chiayi University, Chiayi 600355, Taiwan
| | - Lie-Chwen Lin
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112304, Taiwan
| | - Ming-Fu Chang
- Institute of Biochemistry and Molecular Biology, School of Medicine, National Taiwan University, Taipei 100233, Taiwan
| | - Meng-Shiuan Hsu
- Department of Internal Medicine, Section of Infectious Disease, Far Eastern Memorial Hospital, Taipei 220216, Taiwan.
| | - Cheng Huang
- Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan.
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33
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Zhang Z, Zhang Q, Zhang Y, Lou Y, Ge L, Zhang W, Zhang W, Song F, Huang P. Role of sodium taurocholate cotransporting polypeptide (NTCP) in HBV-induced hepatitis: Opportunities for developing novel therapeutics. Biochem Pharmacol 2024; 219:115956. [PMID: 38049009 DOI: 10.1016/j.bcp.2023.115956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 11/29/2023] [Accepted: 11/29/2023] [Indexed: 12/06/2023]
Abstract
Hepatitis B is an infectious disease caused by the HBV virus. It presents a significant challenge for treatment due to its chronic nature and the potential for developing severe complications, including hepatocirrhosis and hepatocellular carcinoma. These complications not only cause physical and psychological distress to patients but also impose substantial economic and social burdens on both individuals and society as a whole. The internalization of HBV relies on endocytosis and necessitates the involvement of various proteins, including heparin sulfate proteoglycans, epidermal growth factor receptors, and NTCP. Among these proteins, NTCP is pivotal in HBV internalization and is primarily located in the liver's basement membrane. As a transporter of bile acids, NTCP also serves as a receptor facilitating HBV entry into cells. Numerous molecules have been identified to thwart HBV infection by stifling NTCP activity, although only a handful exhibit low IC50 values. In this systematic review, our primary focus dwells on the structure and regulation of NTCP, as well as the mechanism involved in HBV internalization. We underscore recent drug breakthroughs that specifically target NTCP to combat HBV infection. By shedding light on these advances, this review contributes novel insights into developing effective anti-HBV medications.
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Affiliation(s)
- Zhentao Zhang
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Qi Zhang
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Yiwen Zhang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China; Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Hangzhou, China
| | - Yutao Lou
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Luqi Ge
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Wanli Zhang
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Wen Zhang
- Department of Pharmacology, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, China
| | - Feifeng Song
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China; Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Hangzhou, China.
| | - Ping Huang
- Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China; Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Hangzhou, China.
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34
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Fukano K, Wakae K, Nao N, Saito M, Tsubota A, Toyoshima T, Aizaki H, Iijima H, Matsudaira T, Kimura M, Watashi K, Sugiura W, Muramatsu M. A versatile method to profile hepatitis B virus DNA integration. Hepatol Commun 2023; 7:e0328. [PMID: 38051537 PMCID: PMC10697629 DOI: 10.1097/hc9.0000000000000328] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/12/2023] [Accepted: 09/26/2023] [Indexed: 12/07/2023] Open
Abstract
BACKGROUND HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome. METHODS AND RESULTS Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR. CONCLUSIONS RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.
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Affiliation(s)
- Kento Fukano
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
| | - Kousho Wakae
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Naganori Nao
- Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan
- One Health Research Center, Hokkaido University, Sapporo, Japan
- Institute for Vaccine Research and Development, HU-IVReD, Hokkaido University, Sapporo, Japan
| | - Masumichi Saito
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Center for Emergency Preparedness and Response, National Institute of Infectious Diseases, Tokyo, Japan
| | - Akihito Tsubota
- Research Center for Medical Science, The Jikei University School of Medicine, Tokyo, Japan
| | - Takae Toyoshima
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hiroko Iijima
- Department of Internal Medicine, Division of Hepatobiliary and Pancreatic Disease, Hyogo Medical University, Hyogo, Japan
| | - Takahiro Matsudaira
- Biotechnological Research Support Division, FASMAC Co., Ltd., Kanagawa, Japan
| | - Moto Kimura
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Wataru Sugiura
- Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Department of Infectious Disease Research, Foundation for Biomedical Research and Innovation at Kobe, Kobe, Japan
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35
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Li X, Nakashima K, Ito M, Matsuda M, Chida T, Sekihara K, Takahashi H, Kato T, Sawasaki T, Suzuki T. SRPKIN-1 as an inhibitor against hepatitis B virus blocking the viral particle formation and the early step of the viral infection. Antiviral Res 2023; 220:105756. [PMID: 37992764 DOI: 10.1016/j.antiviral.2023.105756] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 11/13/2023] [Accepted: 11/13/2023] [Indexed: 11/24/2023]
Abstract
New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.
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Affiliation(s)
- Xiaofang Li
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kenji Nakashima
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Masahiko Ito
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Mami Matsuda
- Department of Virology II, National Institute of Infectious Diseases, Musashi-murayama, Japan
| | - Takeshi Chida
- Department of Internal Medicine II, Hamamatsu University School of Medicine, Shizuoka, Japan; Department of Regional Medical Care Support, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Kazumasa Sekihara
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan
| | - Hirotaka Takahashi
- Division of Cell-Free Science, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tatsuya Sawasaki
- Division of Cell-Free Science, Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan
| | - Tetsuro Suzuki
- Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka, Japan.
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Ariffianto A, Deng L, Abe T, Matsui C, Ito M, Ryo A, Aly HH, Watashi K, Suzuki T, Mizokami M, Matsuura Y, Shoji I. Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication. J Virol 2023; 97:e0128723. [PMID: 37800948 PMCID: PMC10617466 DOI: 10.1128/jvi.01287-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2023] [Accepted: 08/22/2023] [Indexed: 10/07/2023] Open
Abstract
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.
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Affiliation(s)
- Adi Ariffianto
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
- Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
| | - Lin Deng
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Takayuki Abe
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Chieko Matsui
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Masahiko Ito
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Akihide Ryo
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hussein Hassan Aly
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tetsuro Suzuki
- Department of Virology and Parasitology, Hamamatsu University School of Medicine, Hamamatsu, Japan
| | - Masashi Mizokami
- Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan
| | - Yoshiharu Matsuura
- Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, Japan
- Laboratory of Virus Control, Research Institute for Microbial Diseases (RIMD), Osaka University, Osaka, Japan
| | - Ikuo Shoji
- Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan
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Michalak TI. The Initial Hepatitis B Virus-Hepatocyte Genomic Integrations and Their Role in Hepatocellular Oncogenesis. Int J Mol Sci 2023; 24:14849. [PMID: 37834296 PMCID: PMC10573506 DOI: 10.3390/ijms241914849] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 09/30/2023] [Accepted: 09/30/2023] [Indexed: 10/15/2023] Open
Abstract
Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD+, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis.
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Affiliation(s)
- Tomasz I Michalak
- Molecular Virology and Hepatology Research Group, Division of BioMedical Science, Faculty of Medicine, Health Science Center, Memorial University of Newfoundland, St. John's, NL A1B 3V6, Canada
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38
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Oshima M, Stappenbeck F, Ohashi H, Iwamoto M, Fukano K, Kusunoki A, Zheng X, Wang F, Morishita R, Aizaki H, Suzuki R, Muramatsu M, Kuramochi K, Sureau C, Parhami F, Watashi K. Selective inhibition of hepatitis B virus internalization by oxysterol derivatives. Biochem Biophys Res Commun 2023; 675:139-145. [PMID: 37473528 DOI: 10.1016/j.bbrc.2023.07.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 07/04/2023] [Accepted: 07/10/2023] [Indexed: 07/22/2023]
Abstract
Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.
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Affiliation(s)
- Mizuki Oshima
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; Department of Applied Biological Sciences, Tokyo University of Science, Noda, 278-8510, Japan
| | | | - Hirofumi Ohashi
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Masashi Iwamoto
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Kento Fukano
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo, 162-8655, Japan
| | - Atsuto Kusunoki
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Xin Zheng
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Feng Wang
- MAX BioPharma, Inc., Santa Monica, CA, 90404, USA
| | - Ryo Morishita
- CellFree Sciences. Co. Ltd., 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan
| | - Kouji Kuramochi
- Department of Applied Biological Sciences, Tokyo University of Science, Noda, 278-8510, Japan
| | - Camille Sureau
- Laboratoire de Virologie Moleculaire, Institut National de la Transfusion Sanguine, Paris, 75739, France
| | | | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; Department of Applied Biological Sciences, Tokyo University of Science, Noda, 278-8510, Japan; Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan; MIRAI, JST, Saitama, 332-0012, Japan.
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Shi Q, Zhan T, Bi X, Ye BC, Qi N. Cholesterol-autoxidation metabolites in host defense against infectious diseases. Eur J Immunol 2023; 53:e2350501. [PMID: 37369622 DOI: 10.1002/eji.202350501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 05/28/2023] [Accepted: 05/30/2023] [Indexed: 06/29/2023]
Abstract
Cholesterol plays essential roles in biological processes, including cell membrane stability and myelin formation. Cholesterol can be metabolized to oxysterols by enzymatic or nonenzymatic ways. Nonenzymatic cholesterol metabolites, also called cholesterol-autoxidation metabolites, are formed dependent on the oxidation of reactive oxygen species (ROS) such as OH• or reactive nitrogen species, such as ONOO- . Cholesterol-autoxidation metabolites are abundantly produced in diseases such as inflammatory bowel disease and atherosclerosis, which are associated with oxidative stress. Recent studies have shown that cholesterol-autoxidation metabolites can further regulate the immune system. Here, we review the literature and summarize how cholesterol-autoxidation metabolites, such as 25-hydroxycholesterol (25-OHC), 7α/β-OHC, and 7-ketocholesterol, deal with the occurrence and development of infectious diseases through pattern recognition receptors, inflammasomes, ROS production, nuclear receptors, G-protein-coupled receptor 183, and lipid availability. In addition, we include the research regarding the roles of these metabolites in COVID-19 infection and discuss our viewpoints on the future research directions.
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Affiliation(s)
- Qiwen Shi
- Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Tingzhu Zhan
- Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Xiaobao Bi
- Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Bang-Ce Ye
- Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang, China
| | - Nan Qi
- State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
- Guangzhou Laboratory, Department of Basic Research, Guangzhou International Bio-Island, Guangzhou, Guangdong, China
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40
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Duchemin NJ, Loonawat R, Yeakle K, Rosenkranz A, Bouchard MJ. Hypoxia-inducible factor affects hepatitis B virus transcripts and genome levels as well as the expression and subcellular location of the hepatitis B virus core protein. Virology 2023; 586:76-90. [PMID: 37490813 DOI: 10.1016/j.virol.2023.06.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2021] [Revised: 06/13/2023] [Accepted: 06/21/2023] [Indexed: 07/27/2023]
Abstract
Globally, a chronic-hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The transcription factor hypoxia-inducible factor 1 (HIF1) is often elevated in HCC, including HBV-associated HCC. Previous studies have suggested that the expression of the HIF1 subunit, HIF1α, is elevated in HBV-infected hepatocytes; however, whether HIF1 activity affects the HBV lifecycle has not been fully explored. We used a liver-derived cell line and ex vivo cultured primary hepatocytes as models to determine how HIF1 affects the HBV lifecycle. We observed that HIF1 elevates HBV RNA transcript levels, core protein levels, core protein localization to the cytoplasm, and HBV genome replication. Attenuating the transcription activity of HIF1 blocked HIF1-mediated effects on the HBV lifecycle. Our studies show that HIF1 regulates various stages of the HBV lifecycle in hepatocytes and could be a therapeutic target for blocking HBV replication and the development of HBV-associated diseases.
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Affiliation(s)
- Nicholas J Duchemin
- Molecular and Cellular Biology and Genetic Graduate Program, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, USA
| | - Ronak Loonawat
- Microbiology and Immunology Graduate Program, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, USA
| | - Kyle Yeakle
- Molecular and Cellular Biology and Genetic Graduate Program, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, USA
| | - Andrea Rosenkranz
- Molecular and Cellular Biology and Genetic Graduate Program, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, USA
| | - Michael J Bouchard
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19102, USA.
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41
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Shirasaki T, Murai K, Ishida A, Kuroki K, Kawaguchi K, Wang Y, Yamanaka S, Yasukawa R, Kawasaki N, Li YY, Shimakami T, Sumiyadorj A, Nio K, Sugimoto S, Orita N, Takayama H, Okada H, Thi Bich PD, Iwabuchi S, Hashimoto S, Ide M, Tabata N, Ito S, Matsushima K, Yanagawa H, Yamashita T, Kaneko S, Honda M. Functional involvement of endothelial lipase in hepatitis B virus infection. Hepatol Commun 2023; 7:e0206. [PMID: 37655967 PMCID: PMC10476801 DOI: 10.1097/hc9.0000000000000206] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2022] [Accepted: 04/05/2023] [Indexed: 09/02/2023] Open
Abstract
BACKGROUND HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. METHODS The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. RESULTS We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. CONCLUSIONS LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.
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Affiliation(s)
- Takayoshi Shirasaki
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Kazuhisa Murai
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Atsuya Ishida
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Kazuyuki Kuroki
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Kazunori Kawaguchi
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Ying Wang
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Souma Yamanaka
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Rio Yasukawa
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Narumi Kawasaki
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
| | - Ying-Yi Li
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Tetsuro Shimakami
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Ariunaa Sumiyadorj
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Kouki Nio
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Saiho Sugimoto
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Noriaki Orita
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Hideo Takayama
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Hikari Okada
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Phuong Doan Thi Bich
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Sadahiro Iwabuchi
- Department of Molecular Pathophysiology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan
| | - Shinichi Hashimoto
- Department of Molecular Pathophysiology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan
| | | | | | | | - Kouji Matsushima
- Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
| | | | - Taro Yamashita
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Shuichi Kaneko
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Masao Honda
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
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Makokha GN, Chayama K, Hayes CN, Abe-Chayama H, Abuduwaili M, Hijikata M. Deficiency of SCAP inhibits HBV pathogenesis via activation of the interferon signaling pathway. Virology 2023; 585:248-258. [PMID: 37437369 DOI: 10.1016/j.virol.2023.07.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 06/15/2023] [Accepted: 07/03/2023] [Indexed: 07/14/2023]
Abstract
Hepatitis B virus (HBV) infects the liver and is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Approaches for an effective cure are thwarted by limited knowledge of virus-host interactions. Herein, we identified SCAP as a novel host factor that regulates HBV gene expression. SCAP, sterol regulatory element-binding protein (SREBP) cleavage-activating protein, is an integral membrane protein located in the endoplasmic reticulum. The protein plays a central role in controlling lipid synthesis and uptake by cells. We found that gene silencing of SCAP significantly inhibited HBV replication; furthermore, knockdown of SREBP2 but not SREBP1, the downstream effectors of SCAP, reduced HBs antigen production from HBV infected primary hepatocytes. We also demonstrated that knockdown of SCAP resulted in activation of interferons (IFNs) and IFN stimulated genes (ISGs). Conversely, ectopic expression of SREBP2 in SCAP-deficient cells restored expression of IFNs and ISGs. Importantly, expression of SREBP2 restored HBV production in SCAP knockdown cells, suggesting that SCAP participates in HBV replication through an effect on IFN production via its downstream effector SREBP2. This observation was further confirmed by blocking IFN signaling by an anti-IFN antibody, which restored HBV infection in SCAP-deficient cells. This led to the conclusion that SCAP regulates the IFN pathway through SREBP, thereby affecting the HBV lifecycle. This is the first study to reveal the involvement of SCAP in regulation of HBV infection. These results may facilitate development of new antiviral strategies against HBV.
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Affiliation(s)
- Grace Naswa Makokha
- Laboratory of Medical Innovation, Department of Collaborative Research, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
| | - Kazuaki Chayama
- Laboratory of Medical Innovation, Department of Collaborative Research, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - C Nelson Hayes
- Department of Gastroenterology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Center for Medical Specialist Graduate Education and Research, Hiroshima University, Hiroshima, Japan
| | - Maidina Abuduwaili
- Laboratory of Medical Innovation, Department of Collaborative Research, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Makoto Hijikata
- Laboratory of Medical Innovation, Department of Collaborative Research, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
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Zhang B, Han H, Zhao X, Li AN, Wang Y, Yuan W, Yang Z, Li MD. An HBV susceptibility variant of KNG1 modulates the therapeutic effects of interferons α and λ1 in HBV infection by promoting MAVS lysosomal degradation. EBioMedicine 2023; 94:104694. [PMID: 37442062 PMCID: PMC10435766 DOI: 10.1016/j.ebiom.2023.104694] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2022] [Revised: 06/21/2023] [Accepted: 06/21/2023] [Indexed: 07/15/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) infection is one of the main causes of hepatocellular carcinoma (HCC). The relationship between HBV infection and the host genome as well as their underlying mechanisms remain largely unknown. METHODS In this study, we performed a whole-genome exon sequencing analysis of 300 sib-pairs of Chinese HBV-infected families with the goal of identifying variants and genes involved in HBV infection. A site-direct mutant plasmid was used to investigate the function of SNP rs76438938 in KNG1. The functional and mechanical studies of KNG1 were conducted with in vitro liver cell lines and a hydrodynamic injection model in vivo. The impact of KNG1 on HBV infection therapy was determined in hepatocytes treated with IFN-α/λ1. FINDINGS Our whole-exon association study of 300 families with hepatitis B infection found that SNP rs76438938 in KNG1 significantly increased the risk for HBV infection, and the rs76438938-T allele was found to promote HBV replication by increasing the stability of KNG1 mRNA. By competitively binding HSP90A with MAVS, KNG1 can inhibit the expression of types I and III IFNs by promoting MAVS lysosomal degradation. Such suppression of IFN expression and promotion of HBV replication by Kng1 were further demonstrated with an animal model in vivo. Lastly, we showed that the rs76438938-C allele can improve the therapeutic effect of IFN-α and -λ1 in HBV infection. INTERPRETATION This study identified a SNP, rs76438938, in a newly discovered host gene, KNG1, for its involvement in HBV infection and treatment effect through modulating the cellular antiviral process. FUNDING This study was supported in part by the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of the First Affiliated Hospital of Zhejiang University, the China Precision Medicine Initiative (2016YFC0906300), and the Research Center for Air Pollution and Health of Zhejiang University.
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Affiliation(s)
- Bin Zhang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Haijun Han
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Xinyi Zhao
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Andria N Li
- Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Yan Wang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Wenji Yuan
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Zhongli Yang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
| | - Ming D Li
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; Research Center for Air Pollution and Health, Zhejiang University, Hangzhou, China.
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44
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Han J, Jang KL. All- trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation. Viruses 2023; 15:1456. [PMID: 37515144 PMCID: PMC10386411 DOI: 10.3390/v15071456] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2023] [Revised: 06/22/2023] [Accepted: 06/26/2023] [Indexed: 07/30/2023] Open
Abstract
All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.
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Affiliation(s)
- Jiwoo Han
- Department of Integrated Biological Science, The Graduate School, Pusan National University, Busan 46241, Republic of Korea
| | - Kyung Lib Jang
- Department of Integrated Biological Science, The Graduate School, Pusan National University, Busan 46241, Republic of Korea
- Department of Microbiology, College of Natural Science, Pusan National University, Busan 46241, Republic of Korea
- Microbiological Resource Research Institute, Pusan National University, Busan 46241, Republic of Korea
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45
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Furuta RA, Yasui T, Minamitani T, Akiba H, Toyoda C, Tobita R, Yasui K, Aminaka R, Masaki M, Satake M. Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study. Transfusion 2023. [PMID: 37119513 DOI: 10.1111/trf.17382] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2023] [Revised: 04/09/2023] [Accepted: 04/10/2023] [Indexed: 05/01/2023]
Abstract
BACKGROUND In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.
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Affiliation(s)
- Rika A Furuta
- Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tokyo, Japan
| | - Teruhito Yasui
- Laboratory of Infectious Diseases and Immunity, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
| | - Takeharu Minamitani
- Laboratory of Infectious Diseases and Immunity, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
- Toyama Prefectural Institute for Pharmaceutical Research, Toyama, Japan
| | - Hiroki Akiba
- Laboratory of Pharmacokinetic Optimization, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan
- Laboratory of Biopharmaceutical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Chizu Toyoda
- Japanese Red Cross Kanto-Koushinetsu Block Blood Center, Tokyo, Japan
| | - Ryutaro Tobita
- Japanese Red Cross Kanto-Koushinetsu Block Blood Center, Tokyo, Japan
| | - Kazuta Yasui
- Japanese Red Cross Kinki Block Blood Center, Osaka, Japan
| | - Ryota Aminaka
- Japanese Red Cross Kinki Block Blood Center, Osaka, Japan
| | - Mikako Masaki
- Japanese Red Cross Kinki Block Blood Center, Osaka, Japan
| | - Masahiro Satake
- Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tokyo, Japan
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46
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Ouchida T, Maeda H, Akamatsu Y, Maeda M, Takamatsu S, Kondo J, Misaki R, Kamada Y, Ueda M, Ueda K, Miyoshi E. The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro. Sci Rep 2023; 13:6175. [PMID: 37061516 PMCID: PMC10105536 DOI: 10.1038/s41598-023-28572-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2022] [Accepted: 01/20/2023] [Indexed: 04/17/2023] Open
Abstract
Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.
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Affiliation(s)
- Tsunenori Ouchida
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
- Laboratory of Single Molecule Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Haruka Maeda
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yuka Akamatsu
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Megumi Maeda
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shinji Takamatsu
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
| | - Jumpei Kondo
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan
| | - Ryo Misaki
- Applied Microbiology Laboratory, International Center for Biotechnology, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Yoshihiro Kamada
- Department of Advanced Metabolic Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Masahiro Ueda
- Laboratory of Single Molecule Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Eiji Miyoshi
- Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamada-Oka, Suita, Osaka, 565-0871, Japan.
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47
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Relevance of HBx for Hepatitis B Virus-Associated Pathogenesis. Int J Mol Sci 2023; 24:ijms24054964. [PMID: 36902395 PMCID: PMC10003785 DOI: 10.3390/ijms24054964] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Revised: 02/20/2023] [Accepted: 03/02/2023] [Indexed: 03/08/2023] Open
Abstract
The hepatitis B virus (HBV) counts as a major global health problem, as it presents a significant causative factor for liver-related morbidity and mortality. The development of hepatocellular carcinomas (HCC) as a characteristic of a persistent, chronic infection could be caused, among others, by the pleiotropic function of the viral regulatory protein HBx. The latter is known to modulate an onset of cellular and viral signaling processes with emerging influence in liver pathogenesis. However, the flexible and multifunctional nature of HBx impedes the fundamental understanding of related mechanisms and the development of associated diseases, and has even led to partial controversial results in the past. Based on the cellular distribution of HBx-nuclear-, cytoplasmic- or mitochondria-associated-this review encompasses the current knowledge and previous investigations of HBx in context of cellular signaling pathways and HBV-associated pathogenesis. In addition, particular focus is set on the clinical relevance and potential novel therapeutic applications in the context of HBx.
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Suehiro Y, Tsuge M, Kurihara M, Uchida T, Fujino H, Ono A, Yamauchi M, Naswa Makokha G, Nakahara T, Murakami E, Abe-Chayama H, Kawaoka T, Miki D, Imamura M, Aikata H, Nelson Hayes C, Fujita T, Chayama K. Hepatitis B Virus (HBV) Upregulates TRAIL-R3 Expression in Hepatocytes Resulting in Escape From Both Cell Apoptosis and Suppression of HBV Replication by TRAIL. J Infect Dis 2023; 227:686-695. [PMID: 35226068 DOI: 10.1093/infdis/jiac044] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Accepted: 02/06/2022] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) evades host immunity by regulating intracellular signals. To clarify this immune tolerance mechanism, we performed gene expression analysis using HBV-infected humanized mouse livers. METHODS Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 3 (TRAIL-R3) was significantly upregulated in livers of HBV-infected human hepatocyte transplanted mice by cDNA microarray and next-generation sequencing. We analyzed the significance of TRAIL-R3 upregulation in HBV infection using human hepatocyte transplanted mice and HepG2 cell lines. RESULTS TRAIL-R3 induction by HBV infection was verified by in vitro and in vivo HBV replication models, and induction was inhibited by antiviral nucleot(s)ide analogue treatment. TRAIL-R3 transcription was regulated by the TRAIL-R3 promoter at -969 to -479 nucleotides upstream from the transcription start site, and by hepatitis B x (HBx) via activation of nuclear factor-κB (NF-κB) signal. TRAIL not only induced cell apoptosis but also inhibited HBV replication. TRAIL-R3 upregulation could inhibit both TRAIL-dependent apoptosis in HBV-infected hepatocytes and TRAIL-mediated suppression of HBV replication. CONCLUSIONS These results suggest a mechanism by which HBV persists by escaping host immunity through upregulation of TRAIL-R3. Development of novel drugs to inhibit this escape system might lead to complete HBV elimination from human hepatocytes.
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Affiliation(s)
- Yosuke Suehiro
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Masataka Tsuge
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan
| | - Mio Kurihara
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takuro Uchida
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hatsue Fujino
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Atsushi Ono
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Masami Yamauchi
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Grace Naswa Makokha
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takashi Nakahara
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Eisuke Murakami
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiromi Abe-Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Center for Medical Specialist Graduate Education and Research, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Tomokazu Kawaoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Daiki Miki
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Hiroshi Aikata
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - C Nelson Hayes
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Takashi Fujita
- Laboratory of Molecular Genetics, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Kazuaki Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.,Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.,RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
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49
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Li YY, Kuroki K, Shimakami T, Murai K, Kawaguchi K, Shirasaki T, Nio K, Sugimoto S, Nishikawa T, Okada H, Orita N, Takayama H, Wang Y, Thi Bich PD, Ishida A, Iwabuchi S, Hashimoto S, Shimaoka T, Tabata N, Watanabe-Takahashi M, Nishikawa K, Yanagawa H, Seiki M, Matsushima K, Yamashita T, Kaneko S, Honda M. Hepatitis B Virus Utilizes a Retrograde Trafficking Route via the Trans-Golgi Network to Avoid Lysosomal Degradation. Cell Mol Gastroenterol Hepatol 2023; 15:533-558. [PMID: 36270602 PMCID: PMC9868690 DOI: 10.1016/j.jcmgh.2022.10.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2021] [Revised: 10/11/2022] [Accepted: 10/12/2022] [Indexed: 02/06/2023]
Abstract
BACKGROUND & AIMS Hepatitis B virus (HBV) infection is difficult to cure owing to the persistence of covalently closed circular viral DNA (cccDNA). We performed single-cell transcriptome analysis of newly established HBV-positive and HBV-negative hepatocellular carcinoma cell lines and found that dedicator of cytokinesis 11 (DOCK11) was crucially involved in HBV persistence. However, the roles of DOCK11 in the HBV lifecycle have not been clarified. METHODS The cccDNA levels were measured by Southern blotting and real-time detection polymerase chain reaction in various hepatocytes including PXB cells by using an HBV-infected model. The retrograde trafficking route of HBV capsid was investigated by super-resolution microscopy, proximity ligation assay, and time-lapse analysis. The downstream molecules of DOCK11 and underlying mechanism were examined by liquid chromatography-tandem mass spectrometry, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS The cccDNA levels were strongly increased by DOCK11 overexpression and repressed by DOCK11 suppression. Interestingly, DOCK11 functionally associated with retrograde trafficking proteins in the trans-Golgi network (TGN), Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with HBV capsid, to open an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. Clinically, DOCK11 levels in liver biopsies from patients with chronic hepatitis B were significantly reduced by entecavir treatment, and this reduction correlated with HBV surface antigen levels. CONCLUSIONS HBV uses a retrograde trafficking route via EEs-TGN-ER for infection that is facilitated by DOCK11 and serves to maintain cccDNA. Therefore, DOCK11 is a potential therapeutic target to prevent persistent HBV infection.
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Affiliation(s)
- Ying-Yi Li
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Kazuyuki Kuroki
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Tetsuro Shimakami
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Kazuhisa Murai
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan
| | - Kazunori Kawaguchi
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Takayoshi Shirasaki
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan
| | - Kouki Nio
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Saiho Sugimoto
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Tomoki Nishikawa
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Hikari Okada
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Noriaki Orita
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Hideo Takayama
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Ying Wang
- Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan
| | - Phuong Doan Thi Bich
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Astuya Ishida
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Sadahiro Iwabuchi
- Department of Molecular Pathophysiology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan
| | - Shinichi Hashimoto
- Department of Molecular Pathophysiology, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan
| | - Takeshi Shimaoka
- Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
| | | | | | - Kiyotaka Nishikawa
- Department of Molecular Life Sciences, Doshisha University, Kyoto, Japan
| | | | - Motoharu Seiki
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Kouji Matsushima
- Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan
| | - Taro Yamashita
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Shuichi Kaneko
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
| | - Masao Honda
- Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; Department of Clinical Laboratory Medicine, Kanazawa University Graduate School of Health Medicine, Kanazawa, Japan.
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50
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Sausen DG, Shechter O, Bietsch W, Shi Z, Miller SM, Gallo ES, Dahari H, Borenstein R. Hepatitis B and Hepatitis D Viruses: A Comprehensive Update with an Immunological Focus. Int J Mol Sci 2022; 23:15973. [PMID: 36555623 PMCID: PMC9781095 DOI: 10.3390/ijms232415973] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2022] [Revised: 12/08/2022] [Accepted: 12/12/2022] [Indexed: 12/23/2022] Open
Abstract
Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are highly prevalent viruses estimated to infect approximately 300 million people and 12-72 million people worldwide, respectively. HDV requires the HBV envelope to establish a successful infection. Concurrent infection with HBV and HDV can result in more severe disease outcomes than infection with HBV alone. These viruses can cause significant hepatic disease, including cirrhosis, fulminant hepatitis, and hepatocellular carcinoma, and represent a significant cause of global mortality. Therefore, a thorough understanding of these viruses and the immune response they generate is essential to enhance disease management. This review includes an overview of the HBV and HDV viruses, including life cycle, structure, natural course of infection, and histopathology. A discussion of the interplay between HDV RNA and HBV DNA during chronic infection is also included. It then discusses characteristics of the immune response with a focus on reactions to the antigenic hepatitis B surface antigen, including small, middle, and large surface antigens. This paper also reviews characteristics of the immune response to the hepatitis D antigen (including small and large antigens), the only protein expressed by hepatitis D. Lastly, we conclude with a discussion of recent therapeutic advances pertaining to these viruses.
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Affiliation(s)
- Daniel G. Sausen
- School of Medicine, Eastern Virginia Medical School, Norfolk, VA 23507, USA
| | - Oren Shechter
- School of Medicine, Eastern Virginia Medical School, Norfolk, VA 23507, USA
| | - William Bietsch
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
| | - Zhenzhen Shi
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
| | | | - Elisa S. Gallo
- Division of Dermatology, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel
| | - Harel Dahari
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
| | - Ronen Borenstein
- The Program for Experimental and Theoretical Modeling, Division of Hepatology, Department of Medicine, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
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