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Honda T, Ishigami M, Ishizu Y, Imai N, Ito T, Yamamoto K, Yokoyama S, Muto H, Inukai Y, Kato A, Murayama A, Yoshio S, Ishikawa T, Fujishiro M, Kawashima H, Kato T. Gut microbes associated with functional cure of chronic hepatitis B. Hepatol Int 2025:10.1007/s12072-025-10776-9. [PMID: 39869245 DOI: 10.1007/s12072-025-10776-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2024] [Accepted: 01/02/2025] [Indexed: 01/28/2025]
Abstract
BACKGROUND AND AIMS Hepatitis B virus (HBV) is prevalent worldwide and is difficult to eradicate. Current treatment strategies for chronic hepatitis B ultimately seek to achieve functional cure (FC); however, the factors contributing to FC remain unclear. We aimed to investigate the gut microbiota profiles of patients with chronic hepatitis B who achieved FC. METHODS Among 105 HBeAg-negative patients with chronic hepatitis B, 70 were enrolled, after excluding patients with cirrhosis or hepatocellular carcinoma and those receiving nucleoside analogs. The gut microbiota of patients who achieved FC was assessed and compared with that of patients with high-titer of HBV DNA (HBV DNA ≥ 3.3 log IU/mL) or low-titer of HBV DNA (HBV DNA < 3.3 log IU/mL). Furthermore, we used cell culture-generated HBV (HBVcc) as a model for HBV infection to evaluate the effects of short-chain fatty acids (SCFAs) produced by the identified bacteria. RESULTS There was no difference in the alpha or beta diversity of the gut microbiota between the FC group and the other groups. However, compared with the other groups, the FC group presented a greater relative abundance of bacteria that produce SCFAs, especially butyrate. In vitro studies demonstrated that 1.0 mM butyrate reduces HBsAg production in HBVcc-infected cells. Furthermore, butyrate administration was most effective at the post-HBV infection stage. CONCLUSIONS Our findings suggest that butyrate-producing bacteria contribute to FC in HBeAg-negative patients with chronic hepatitis B through butyrate-mediated inhibition of HBV production.
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Affiliation(s)
- Takashi Honda
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan.
| | - Masatoshi Ishigami
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Yoji Ishizu
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Norihiro Imai
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Takanori Ito
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Kenta Yamamoto
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Shinya Yokoyama
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Hisanori Muto
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Yosuke Inukai
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Asuka Kato
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo, 162-8640, Japan
| | - Sachiyo Yoshio
- Department of Liver Diseases, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Kohnodai 1-7-1, Ichikawa, 272-8516, Japan
| | - Tetsuya Ishikawa
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Mitsuhiro Fujishiro
- Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hiroki Kawashima
- Department of Gastroenterology and Hepatology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo, 162-8640, Japan
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Hu K, Zai W, Xu M, Wang H, Song X, Huang C, Liu J, Chen J, Deng Q, Yuan Z, Chen J. Augmented epigenetic repression of hepatitis B virus covalently closed circular DNA by interferon-α and small-interfering RNA synergy. mBio 2024; 15:e0241524. [PMID: 39570046 PMCID: PMC11633095 DOI: 10.1128/mbio.02415-24] [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: 08/06/2024] [Accepted: 10/24/2024] [Indexed: 11/22/2024] Open
Abstract
The persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is a key obstacle for HBV cure. This study aims to comprehensively assess the effect of interferon (IFN) and small-interfering RNA (siRNA) combination on the cccDNA minichromosome. Utilizing both cell and mouse cccDNA models, we compared the inhibitory effects of IFNα, siRNA, and their combination on cccDNA activity and assessed its epigenetic state. IFNα2 treatment alone reduced HBV RNAs, HBeAg, and HBsAg levels by approximately 50%, accompanied by a low-level reconstitution of SMC5/6-a chromatin modulator that restricts cccDNA transcription. HBx-targeting siRNA (siHBx) achieved significant suppression of viral antigens and reconstitution of SMC5/6, but this effect could be reversed by the deacetylase inhibitor Belinostat. The combination of IFN with siHBx resulted in over 95% suppression of virological markers, reduction in epigenetic activation modifications (H3Ac and H4Ac) on cccDNA, and further reduced cccDNA accessibility, with the effect not reversible by Belinostat. In an extracellular humanized IFNAR C57BL/6 mouse model harboring recombinant cccDNA, the effect of combination of clinically used pegylated IFNα2 and GalNac-siHBx was further clarified, indicating a higher and more durable suppression of cccDNA activity compared to either therapy alone. In conclusion, the combination of IFNα and siRNA achieves a more potent and durable epigenetic inhibition of cccDNA activity in cell and mouse models, compared to monotherapy. These findings deepen the understanding of cccDNA modulation and strengthen the scientific basis for the potential of combination therapy. IMPORTANCE Since there are currently no approved drugs targeting and silencing covalently closed circular DNA (cccDNA), achieving a "functional cure" remains difficult. This study aims to comprehensively compare the effects of IFNα, small-interfering RNA targeting hepatitis B virus (HBV), and their combination on the activity, accessibility, and epigenetic modifications of cccDNA minichromosomes in cell models. A more durable and stable inhibition of HBV RNAs and antigens expression by IFNα and HBx-targeting siRNA (siHBx) synergy was observed, associated with augmented epigenetic repression of the cccDNA minichromosome. Besides, in an extracellular humanized IFNAR mouse model harboring recombinant cccDNA with an intact response to human IFNα, the synergistic effect of clinically used pegylated IFNα2 and in-house-developed GalNac-siHBx was further clarified.
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Affiliation(s)
- Kongying Hu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Wenjing Zai
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Mingzhu Xu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Haiyu Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai, China
| | - Xinluo Song
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Chao Huang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Jiangxia Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
| | - Juan Chen
- Key Laboratory of Molecular Biology of Infectious Diseases (MOE), Chongqing Medical University, Chongqing, China
| | - Qiang Deng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
- Shanghai Institute of Infectious Disease and Biosecurity, Shanghai, China
| | - Jieliang Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes and Infection, School of Basic Medical Sciences, Shanghai Medical College Fudan University, Shanghai, China
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Zhang Y, Cao W, Wang S, Zhang L, Li X, Zhang Z, Xie Y, Li M. Epigenetic modification of hepatitis B virus infection and related hepatocellular carcinoma. Virulence 2024; 15:2421231. [PMID: 39460469 PMCID: PMC11583590 DOI: 10.1080/21505594.2024.2421231] [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: 06/10/2024] [Revised: 09/18/2024] [Accepted: 10/21/2024] [Indexed: 10/28/2024] Open
Abstract
Hepatitis B virus (HBV) infection poses a challenge to global public health. Persistent liver infection with HBV is associated with an increased risk of developing severe liver disease. The complex interaction between the virus and the host is the reason for the persistent presence of HBV and the risk of tumor development. Chronic liver inflammation, integration of viral genome with host genome, expression of HBx protein, and viral genotype are all key participants in the pathogenesis of hepatocellular carcinoma (HCC). Epigenetic regulation in HBV-associated HCC involves complex interactions of molecular mechanisms that control gene expression and function without altering the underlying DNA sequence. These epigenetic modifications can significantly affect the onset and progression of HCC. This review summarizes recent research on the epigenetic regulation of HBV persistent infection and HBV-HCC development, including DNA methylation, histone modification, RNA modification, non-coding RNA, etc. Enhanced knowledge of these mechanisms will offer fresh perspectives and potential targets for intervention tactics in HBV-HCC.
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Affiliation(s)
- Yaqin Zhang
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Weihua Cao
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Shiyu Wang
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Lu Zhang
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Xinxin Li
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Ziyu Zhang
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Yao Xie
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
| | - Minghui Li
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
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Bächer J, Allweiss L, Dandri M. SMC5/6-Mediated Transcriptional Regulation of Hepatitis B Virus and Its Therapeutic Potential. Viruses 2024; 16:1667. [PMID: 39599784 PMCID: PMC11598903 DOI: 10.3390/v16111667] [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: 09/09/2024] [Revised: 10/16/2024] [Accepted: 10/23/2024] [Indexed: 11/29/2024] Open
Abstract
Cells have developed various mechanisms to counteract viral infections. In an evolutionary arms race, cells mobilize cellular restriction factors to fight off viruses, targeted by viral factors to facilitate their own replication. The hepatitis B virus (HBV) is a small dsDNA virus that causes acute and chronic infections of the liver. Its genome persists in the nuclei of infected hepatocytes as a covalently closed circular DNA (cccDNA) minichromosome, thus building up an episomal persistence reservoir. The chromosomal maintenance complex SMC5/6 acts as a restriction factor hindering cccDNA transcription, whereas the viral regulatory protein HBx targets SMC5/6 for proteasomal degradation, thus relieving transcriptional suppression of the HBV minichromosome. To date, no curative therapies are available for chronic HBV carriers. Knowledge of the factors regulating the cccDNA and the development of therapies involving silencing the minichromosome or specifically interfering with the HBx-SMC5/6 axis holds promise in achieving sustained viral control. Here, we summarize the current knowledge of the mechanism of SMC5/6-mediated HBV restriction. We also give an overview of SMC5/6 cellular functions and how this compares to the restriction of other DNA viruses. We further discuss the therapeutic potential of available and investigational drugs interfering with the HBx-SMC5/6 axis.
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Affiliation(s)
- Johannes Bächer
- I. Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; (J.B.); (L.A.)
| | - Lena Allweiss
- I. Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; (J.B.); (L.A.)
- German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, Germany
| | - Maura Dandri
- I. Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; (J.B.); (L.A.)
- German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, Germany
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5
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Ibrahim MK, Liu CD, Zhang L, Yu X, Kim ES, Liu Z, Jo S, Liu Y, Huang Y, Gao SJ, Guo H. The loss of hepatitis B virus receptor NTCP/SLC10A1 in human liver cancer cells is due to epigenetic silencing. J Virol 2024; 98:e0118724. [PMID: 39297647 PMCID: PMC11495020 DOI: 10.1128/jvi.01187-24] [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: 07/04/2024] [Accepted: 08/30/2024] [Indexed: 09/27/2024] Open
Abstract
Human Na+-taurocholate cotransporting polypeptide (hNTCP) is predominantly expressed in hepatocytes, maintaining bile salt homeostasis and serving as a receptor for hepatitis B virus (HBV). hNTCP expression is downregulated during hepatocellular carcinoma (HCC) development. In this study, we investigated the molecular mechanisms underlying hNTCP dysregulation using HCC tissues and cell lines, and primary human hepatocytes (PHHs). Firstly, we observed a significant reduction of hNTCP in HCC tumors compared to adjacent and normal tissues. Additionally, hNTCP mRNA levels were markedly lower in HepG2 cells compared to PHHs, which was corroborated at the protein level by immunoblotting. Sanger sequencing confirmed identical sequences for hNTCP promoter, exons, and mRNA coding sequences between PHH and HepG2 cells, indicating no mutations or splicing alterations. We then assessed the epigenetic status of hNTCP. The hNTCP promoter, with low CG content, showed no significant methylation differences between PHH and HepG2 cells. Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) revealed a loss of activating histone posttranslational modification (PTM) H3K27ac near the hNTCP transcription start site (TSS) in HepG2 cells. This loss was also confirmed in HCC tumor cells compared to adjacent and background cells. Treating HepG2 cells with histone deacetylase inhibitors enhanced H3K27ac accumulation and glucocorticoid receptor (GR) binding at the hNTCP TSS, significantly increasing hNTCP mRNA and protein levels, and rendering the cells susceptible to HBV infection. In summary, histone PTM-related epigenetic mechanisms play a critical role in hNTCP dysregulation in liver cancer cells, providing insights into hepatocarcinogenesis and its impact on chronic HBV infection. IMPORTANCE HBV is a hepatotropic virus that infects human hepatocytes expressing the viral receptor hNTCP. Without effective antiviral therapy, chronic HBV infection poses a high risk of liver cancer. However, most liver cancer cell lines, including HepG2 and Huh7, do not support HBV infection due to the absence of hNTCP expression, and the mechanism underlying this defect remains unclear. This study demonstrates a significant reduction of hNTCP in hepatocellular carcinoma samples and HepG2 cells compared to normal liver tissues and primary human hepatocytes. Despite identical hNTCP genetic sequences, epigenetic analyses revealed a loss of the activating histone modification H3K27ac near the hNTCP transcription start site in cancer cells. Treatment with histone deacetylase inhibitors restored H3K27ac levels, reactivated hNTCP expression, and rendered HepG2 cells susceptible to HBV infection. These findings highlight the role of epigenetic modulation in hNTCP dysregulation, offering insights into hepatocarcinogenesis and its implications for chronic HBV infection.
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MESH Headings
- Humans
- Organic Anion Transporters, Sodium-Dependent/metabolism
- Organic Anion Transporters, Sodium-Dependent/genetics
- Symporters/genetics
- Symporters/metabolism
- Hepatitis B virus/genetics
- Carcinoma, Hepatocellular/virology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Hep G2 Cells
- Liver Neoplasms/virology
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Epigenesis, Genetic
- Promoter Regions, Genetic
- Hepatocytes/virology
- Hepatocytes/metabolism
- DNA Methylation
- Histones/metabolism
- Gene Expression Regulation, Neoplastic
- Gene Silencing
- Receptors, Virus/metabolism
- Receptors, Virus/genetics
- Hepatitis B/virology
- Hepatitis B/genetics
- Hepatitis B/metabolism
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Affiliation(s)
- Marwa K. Ibrahim
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Cheng-Der Liu
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Liyong Zhang
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Xiaoyang Yu
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Elena S. Kim
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Zhentao Liu
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Electrical and Computer Engineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, Pennsylvania, USA
| | - Sumin Jo
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Electrical and Computer Engineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, Pennsylvania, USA
| | - Yuanjie Liu
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Yufei Huang
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Electrical and Computer Engineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, Pennsylvania, USA
- Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Shou-Jiang Gao
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Haitao Guo
- Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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Pondé RADA, Amorim GDSP. Elimination of the hepatitis B virus: A goal, a challenge. Med Res Rev 2024; 44:2015-2034. [PMID: 38528684 DOI: 10.1002/med.22030] [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: 10/06/2023] [Revised: 01/24/2024] [Accepted: 02/05/2024] [Indexed: 03/27/2024]
Abstract
The hepatitis B elimination is a goal proposed by the WHO to be achieved by 2030 through the adoption of synergistic measures for the prevention and chronic HBV infection treatment. Complete cure is characterized by the HBV elimination from the body and is the goal of the chronic hepatitis B treatment, which once achieved, will enable the hepatitis B elimination. This, today, has been a scientific challenge. The difficulty in achieving a complete cure is due to the indefinite maintenance of a covalently closed episomal circular DNA (cccDNA) reservoir and the maintenance and persistence of an insufficient and dysfunctional immune response in chronically infected patients. Among the measures adopted to eliminate hepatitis B, two have the potential to directly interfere with the virus cycle, but with limited effect on HBV control. These are conventional vaccines-blocking transmission and antiviral therapy-inhibiting replication. Vaccines, despite their effectiveness in protecting against horizontal transmission and preventing mother-to-child vertical transmission, have no effect on chronic infection or potential to eliminate the virus. Treatment with antivirals suppresses viral replication, but has no curative effect, as it has no action against cccDNA. Therapeutic vaccines comprise an additional approach in the chronic infection treatment, however, they have only a modest effect on the immune system, enhancing it temporarily. This manuscript aims to address (1) the cccDNA persistence in the hepatocyte nucleus and the immune response dysfunction in chronically infected individuals as two primary factors that have hampered the treatment and HBV elimination from the human body; (2) the limitations of antiviral therapy and therapeutic vaccines, as strategies to control hepatitis B; and (3) the possibly promising therapeutic approaches for the complete cure and elimination of hepatitis B.
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Affiliation(s)
- Robério Amorim de Almeida Pondé
- Secretaria de Estado da Saúde-SES, Superintendência de Vigilância em Saúde-SUVISA/GO, Gerência de Vigilância Epidemiológica de Doenças Transmissíveis-GVEDT/Coordenação de Análises e Pesquisas-CAP, Goiânia, Goiás, Brazil
- Department of Microbiology, Laboratory of Human Virology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Goiás, Brazil
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Geta M, Mengistu G, Yizengaw E, Manyzewal T, Hailu A, Woldeamanuel Y. Efficacy and safety of therapeutic vaccines for the treatment of chronic hepatitis B: A systematic review and meta-analysis of randomized controlled trials update. Medicine (Baltimore) 2024; 103:e39344. [PMID: 39213251 PMCID: PMC11365667 DOI: 10.1097/md.0000000000039344] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 06/08/2024] [Accepted: 07/26/2024] [Indexed: 09/04/2024] Open
Abstract
BACKGROUND Most people diagnosed with chronic hepatitis B (CHB) need treatment to help reduce the risk of liver disease and limit disease transmission. Therapeutic vaccine (TV) candidates have been under study for their clinical effects on inducing HBV-specific host immune responses. This review aimed to systematically synthesize updated evidence on the efficacy and safety of TVs in patients with CHB. METHODS This systematic review was performed by searching different databases from January to February 2021. Completed randomized controlled trials that reported TVs' efficacy and/or safety for treating CHB compared with the standard of care (SOC) or placebo were included. Efficacy and safety estimates were reported as the logarithm of the odds ratio and risk differences, respectively. I2 > 50% was considered significant heterogeneity. Significant publication bias was considered when Egger's test P value < .10. The risk of bias was assessed using the Cochrane Risk of Bias tool. The GRADE methodology was used to assess the certainty of the evidence for each outcome. RESULTS Twenty-four articles with 2889 pooled samples were included. TVs made a significant difference in hepatitis B envelope antigen (HBeAg) SC (log OR = 0.76, P = .01) and (log OR = 0.40, P = .03) compared to placebo and combination therapy, respectively. HBeAg SC was significantly affected by TVs at the end of follow up (log OR = 0.49, P = .01), with significant HBsAg mean difference (MD = -0.62, P = .00). At the end of treatment, the TVs had no significant effect on HBV DNA negativity over the SOC (log OR = 0.62, P = .09) or placebo (log OR = -0.07, P = .91). TVs do not significantly affect the risk of serious adverse events (RD 0.02, 95% CI 0.00-0.04). CONCLUSION In patients with CHB, TVs had significant effects on HBeAg SC compared to the SOC or placebo. There was no significant difference between serious adverse events. TVs are promising treatment strategy to overcome CHB.
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Affiliation(s)
- Mekuanint Geta
- Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
- Department of Medical Microbiology, School of Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia
| | - Getachew Mengistu
- Department of Medical Microbiology, School of Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia
- Department of Laboratory Science, Debre Markos University, Debre Markos, Ethiopia
| | - Endalew Yizengaw
- Department of Medical Laboratory Science, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia
- Institute of Biotechnology, Bahir Dar University, Bahir Dar, Ethiopia
| | - Tsegahun Manyzewal
- Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
| | - Asrat Hailu
- Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
| | - Yimtubeznash Woldeamanuel
- Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
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8
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Hu JL, Huang AL. Classifying hepatitis B therapies with insights from covalently closed circular DNA dynamics. Virol Sin 2024; 39:9-23. [PMID: 38110037 PMCID: PMC10877440 DOI: 10.1016/j.virs.2023.12.005] [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: 01/29/2023] [Accepted: 12/13/2023] [Indexed: 12/20/2023] Open
Abstract
The achievement of a functional cure for chronic hepatitis B (CHB) remains limited to a minority of patients treated with currently approved drugs. The primary objective in developing new anti-HBV drugs is to enhance the functional cure rates for CHB. A critical prerequisite for the functional cure of CHB is a substantial reduction, or even eradication of covalently closed circular DNA (cccDNA). Within this context, the changes in cccDNA levels during treatment become as a pivotal concern. We have previously analyzed the factors influencing cccDNA dynamics and introduced a preliminary classification of hepatitis B treatment strategies based on these dynamics. In this review, we employ a systems thinking perspective to elucidate the fundamental aspects of the HBV replication cycle and to rationalize the classification of treatment strategies according to their impact on the dynamic equilibrium of cccDNA. Building upon this foundation, we categorize current anti-HBV strategies into two distinct groups and advocate for their combined use to significantly reduce cccDNA levels within a well-defined timeframe.
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Affiliation(s)
- Jie-Li Hu
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, 400016, China.
| | - Ai-Long Huang
- Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, 400016, China.
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9
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Zhao Q, Liu H, Tang L, Wang F, Tolufashe G, Chang J, Guo JT. Mechanism of interferon alpha therapy for chronic hepatitis B and potential approaches to improve its therapeutic efficacy. Antiviral Res 2024; 221:105782. [PMID: 38110058 DOI: 10.1016/j.antiviral.2023.105782] [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: 09/16/2023] [Revised: 12/11/2023] [Accepted: 12/12/2023] [Indexed: 12/20/2023]
Abstract
Hepatitis B virus (HBV) chronically infects 296 million people worldwide and causes more than 820,000 deaths annually due to cirrhosis and hepatocellular carcinoma. Current standard-of-care medications for chronic hepatitis B (CHB) include nucleos(t)ide analogue (NA) viral DNA polymerase inhibitors and pegylated interferon alpha (PEG-IFN-α). NAs can efficiently suppress viral replication and improve liver pathology, but not eliminate or inactivate HBV covalently closed circular DNA (cccDNA). CCC DNA is the most stable HBV replication intermediate that exists as a minichromosome in the nucleus of infected hepatocyte to transcribe viral RNA and support viral protein translation and genome replication. Consequentially, a finite duration of NA therapy rarely achieves a sustained off-treatment suppression of viral replication and life-long NA treatment is most likely required. On the contrary, PEG-IFN-α has the benefit of finite treatment duration and achieves HBsAg seroclearance, the indication of durable immune control of HBV replication and functional cure of CHB, in approximately 5% of treated patients. However, the low antiviral efficacy and poor tolerability limit its use. Understanding how IFN-α suppresses HBV replication and regulates antiviral immune responses will help rational optimization of IFN therapy and development of novel immune modulators to improve the rate of functional cure. This review article highlights mechanistic insight on IFN control of HBV infection and recent progress in development of novel IFN regimens, small molecule IFN mimetics and combination therapy of PEG-IFN-α with new direct-acting antivirals and therapeutic vaccines to facilitate the functional cure of CHB.
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Affiliation(s)
- Qiong Zhao
- Baruch S. Blumberg Institute, Doylestown, PA, United States
| | - Hui Liu
- Baruch S. Blumberg Institute, Doylestown, PA, United States
| | - Liudi Tang
- Baruch S. Blumberg Institute, Doylestown, PA, United States
| | - Fuxuan Wang
- Baruch S. Blumberg Institute, Doylestown, PA, United States
| | | | - Jinhong Chang
- Baruch S. Blumberg Institute, Doylestown, PA, United States
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, PA, United States.
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10
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Olenginski LT, Attionu SK, Henninger EN, LeBlanc RM, Longhini AP, Dayie TK. Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target. Viruses 2023; 15:1913. [PMID: 37766319 PMCID: PMC10534774 DOI: 10.3390/v15091913] [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: 07/01/2023] [Revised: 09/01/2023] [Accepted: 09/05/2023] [Indexed: 09/29/2023] Open
Abstract
Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε-P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.
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Affiliation(s)
- Lukasz T. Olenginski
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Department of Biochemistry, University of Colorado, Boulder, CO 80309, USA
| | - Solomon K. Attionu
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Erica N. Henninger
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Regan M. LeBlanc
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
| | - Andrew P. Longhini
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
- Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
| | - Theodore K. Dayie
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA (R.M.L.)
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11
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Yu X, Long Q, Shen S, Liu Z, Chandran J, Zhang J, Ding H, Zhang H, Cai D, Kim ES, Huang Y, Guo H. Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription. Antiviral Res 2023; 211:105552. [PMID: 36737008 PMCID: PMC10036215 DOI: 10.1016/j.antiviral.2023.105552] [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: 12/03/2022] [Revised: 01/18/2023] [Accepted: 01/30/2023] [Indexed: 02/04/2023]
Abstract
HBV cccDNA is the persistent form of viral genome, which exists in host cell nucleus as an episomal minichromosome decorated with histone and non-histone proteins. cccDNA is the authentic viral transcription template and resistant to current antivirals. Growing evidence shows that the transcriptional activity of cccDNA minichromosome undergoes epigenetic regulations, suggesting a new perspective for anti-cccDNA drug development through targeting histone modifications. In this study, we screened an epigenetic compound library in the cccDNA reporter cell line HepBHAe82, which produces the HA-tagged HBeAg in a cccDNA-dependent manner. Among the obtained hits, a bromodomain-containing protein 4 (BRD4) inhibitor MS436 exhibited marked inhibition of cccDNA transcription in both HBV stable cell line HepAD38 and HepG2-NTCP or primary human hepatocyte infection system under noncytotoxic concentrations. Chromatin immunoprecipitation (ChIP) assay demonstrated that MS436 dramatically reduced the enrichment of H3K27ac, an activating histone modification pattern, on cccDNA minichromosome. RNAseq differential analysis showed that MS436 does not drastically change host transcriptome or induce any known anti-HBV factors/pathways, indicating a direct antiviral effect of MS436 on cccDNA minichromosome. Interestingly, the MS436-mediated inhibition of cccDNA transcription is accompanied by cccDNA destabilization in HBV infection and a recombinant cccDNA system, indicating that BRD4 activity may also play a role in cccDNA maintenance. Furthermore, depletion of BRD4 by siRNA knockdown or PROTAC degrader resulted in cccDNA inhibition in HBV-infected HepG2-NTCP cells, further validating BRD4 as an antiviral target. Taken together, our study has demonstrated the practicability of HepBHAe82-based anti-HBV drug screening system and provided a proof-of-concept for targeting HBV cccDNA with epigenetic compounds.
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Affiliation(s)
- Xiaoyang Yu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Quanxin Long
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Sheng Shen
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Zhentao Liu
- Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Electrical and Computer Engineering, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Jithin Chandran
- Department of Electrical and Computer Engineering, The University of Texas at San Antonio, San Antonio, TX, USA
| | - Junjie Zhang
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Hao Ding
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Hu Zhang
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Dawei Cai
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Elena S Kim
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Yufei Huang
- Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Electrical and Computer Engineering, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Haitao Guo
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.
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12
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A road to contemporary era of hepatitis B virus regimen replacing existing therapeutics exploiting plant secondary metabolites as emerging heroes in exploring drugs: An expedition for a functional cure. GENE REPORTS 2023. [DOI: 10.1016/j.genrep.2023.101743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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13
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Geng K, Merino LG, Wedemann L, Martens A, Sobota M, Sanchez YP, Søndergaard JN, White RJ, Kutter C. Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells. Genome Res 2022; 32:1876-1891. [PMID: 36180232 PMCID: PMC9712622 DOI: 10.1101/gr.276901.122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Accepted: 09/19/2022] [Indexed: 11/24/2022]
Abstract
The CRISPR-Cas9 system is widely used to permanently delete genomic regions via dual guide RNAs. Genomic rearrangements induced by CRISPR-Cas9 can occur, but continuous technical developments make it possible to characterize complex on-target effects. We combined an innovative droplet-based target enrichment approach with long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving the allelic co-occurrence of a genomic duplication and inversion of the target region, as well as integrations of exogenous DNA and clustered interchromosomal DNA fragment rearrangements. Furthermore, we found that these genomic alterations led to functional aberrant DNA fragments and can alter cell proliferation. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations, and introduce a data-driven workflow enabling detailed dissection of the on-target sequence content with superior resolution.
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Affiliation(s)
- Keyi Geng
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Lara G Merino
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Linda Wedemann
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Aniek Martens
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Małgorzata Sobota
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Yerma P Sanchez
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Jonas Nørskov Søndergaard
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
| | - Robert J White
- Department of Biology, University of York, York YO10 5DD, United Kingdom
| | - Claudia Kutter
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Science for Life Laboratory, 171 65, Stockholm, Sweden
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14
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Lei B, Song H, Xu F, Wei Q, Wang F, Tan G, Ma H. When does hepatitis B virus meet long-stranded noncoding RNAs? Front Microbiol 2022; 13:962186. [PMID: 36118202 PMCID: PMC9479684 DOI: 10.3389/fmicb.2022.962186] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Accepted: 07/22/2022] [Indexed: 01/16/2023] Open
Abstract
Hepatitis B virus (HBV) infection in humans and its associated diseases are long-standing problems. HBV can produce a large number of non-self-molecules during its life cycle, which acts as targets for innate immune recognition and initiation. Among these, interferon and its large number of downstream interferon-stimulated gene molecules are important early antiviral factors. However, the development of an effective antiviral immune response is not simple and depends not only on the delicate regulation of the immune response but also on the various mechanisms of virus-related immune escape and immune tolerance. Therefore, despite there being a relatively well-established consensus on the major pathways of the antiviral response and their component molecules, the complete clearance of HBV remains a challenge in both basic and clinical research. Long-noncoding RNAs (lncRNAs) are generally >200 bp in length and perform different functions in the RNA strand encoding the protein. As an important part of the IFN-inducible genes, interferon-stimulated lncRNAs are involved in the regulation of several HBV infection-related pathways. This review traces the basic elements of such pathways and characterizes the various recent targets of lncRNAs, which not only complement the regulatory mechanisms of pathways related to chronic HBV infection, fibrosis, and cancer promotion but also present with new potential therapeutic targets for controlling HBV infection and the malignant transformation of hepatocytes.
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Affiliation(s)
- Bingxin Lei
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
- Department of Anesthesiology, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Hongxiao Song
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Fengchao Xu
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Qi Wei
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
- Department of Anesthesiology, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Fei Wang
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Guangyun Tan
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Department of Immunology, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, Jilin, China
- *Correspondence: Guangyun Tan,
| | - Haichun Ma
- Department of Anesthesiology, The First Hospital of Jilin University, Changchun, Jilin, China
- Haichun Ma,
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15
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Chen D, Tan X, Chen W, Liu Y, Li C, Wu J, Zheng J, Shen HC, Zhang M, Wu W, Wang L, Xiong J, Dai J, Sun K, Zhang JD, Xiang K, Li B, Ni X, Zhu Q, Gao L, Wang L, Feng S. Discovery of Novel cccDNA Reducers toward the Cure of Hepatitis B Virus Infection. J Med Chem 2022; 65:10938-10955. [PMID: 35973101 DOI: 10.1021/acs.jmedchem.1c02215] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Chronic hepatitis B virus (HBV) infection is a worldwide disease that causes thousands of deaths per year. Currently, there is no therapeutic that can completely cure already infected HBV patients due to the inability of humans to eliminate covalently closed circular DNA (cccDNA), which serves as the template to (re)initiate an infection even after prolonged viral suppression. Through phenotypic screening, we discovered xanthone series hits as novel HBV cccDNA reducers, and subsequent structure optimization led to the identification of a lead compound with improved antiviral activity and pharmacokinetic profiles. A representative compound 59 demonstrated good potency and oral bioavailability with no cellular toxicity. In an HBVcircle mouse model, compound 59 showed excellent efficacy in significantly reducing HBV antigens, DNA, and intrahepatic cccDNA levels.
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Affiliation(s)
- Dongdong Chen
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Xuefei Tan
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Wenming Chen
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Yongfu Liu
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Chao Li
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Jun Wu
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Jiamin Zheng
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Hong C Shen
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Meifang Zhang
- Lead Discovery, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Waikwong Wu
- Lead Discovery, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Lin Wang
- pCMC, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Jing Xiong
- pCMC, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Jieyu Dai
- Pharmaceutical Sciences, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Kai Sun
- Pharmaceutical Sciences, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Jitao David Zhang
- Pharmaceutical Science, Roche Innovation Center Basel, Roche Pharma Research & Early Development, Grenzacherstrasse 124, Basel CH-4070, Switzerland
| | - Kunlun Xiang
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Baocun Li
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - XiaoJu Ni
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Qihui Zhu
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Lu Gao
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Li Wang
- Discovery Virology, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
| | - Song Feng
- Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research & Early Development, Building 5, No. 371, Lishizhen Road, Shanghai 201203, China
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16
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Bhat S, Kazim SN. HBV cccDNA-A Culprit and Stumbling Block for the Hepatitis B Virus Infection: Its Presence in Hepatocytes Perplexed the Possible Mission for a Functional Cure. ACS OMEGA 2022; 7:24066-24081. [PMID: 35874215 PMCID: PMC9301636 DOI: 10.1021/acsomega.2c02216] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/17/2023]
Abstract
Hepatitis B virus infection (HBV) is still a big health problem across the globe. It has been linked to the development of liver cirrhosis and hepatocellular carcinoma and can trigger different types of liver damage. Existing medicines are unable to disable covalently closed circular DNA (cccDNA), which may result in HBV persistence and recurrence. The current therapeutic goal is to achieve a functional cure, which means HBV-DNA no longer exists when treatment stops and the absence of HBsAg seroclearance. However, due to the presence of integrated HBV DNA and cccDNA functional treatment is now regarded to be difficult. In order to uncover pathways for potential therapeutic targets and identify medicines that could result in large rates of functional cure, a thorough understanding of the virus' biology is required. The proteins of the virus and episomal cccDNA are thought to be critical for the management and support of the HBV replication cycle as they interact directly with the host proteome to establish the best atmosphere for the virus while evading immune detection. The breakthroughs of host dependence factors, cccDNA transcription, epigenetic regulation, and immune-mediated breakdown have all produced significant progress in our understanding of cccDNA biology during the past decade. There are some strategies where cccDNA can be targeted either in a direct or indirect way and are presently at the point of discovery or preclinical or early clinical advancement. Editing of genomes, techniques targeting host dependence factors or epigenetic gene maintenance, nucleocapsid modulators, miRNA, siRNA, virion secretory inhibitors, and immune-mediated degradation are only a few examples. Though cccDNA approaches for direct targeting are still in the early stages of development, the assembly of capsid modulators and immune-reliant treatments have made it to the clinic. Clinical trials are currently being conducted to determine their efficiency and safety in patients, as well as their effect on viral cccDNA. The influence of recent breakthroughs in the development of new treatment techniques on cccDNA biology is also summarized in this review.
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Affiliation(s)
- Sajad
Ahmad Bhat
- Jamia Millia Islamia Central University, Centre for Interdisciplinary Research in Basic Sciences, New Delhi 110025, India
| | - Syed Naqui Kazim
- Jamia Millia Islamia Central University, Centre for Interdisciplinary Research in Basic Sciences, New Delhi 110025, India
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17
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IFN-α inhibits HBV transcription and replication by promoting HDAC3-mediated de-2-hydroxyisobutyrylation of histone H4K8 on HBV cccDNA minichromosome in liver. Acta Pharmacol Sin 2022; 43:1484-1494. [PMID: 34497374 PMCID: PMC9160025 DOI: 10.1038/s41401-021-00765-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2021] [Accepted: 08/12/2021] [Indexed: 02/07/2023]
Abstract
The epigenetic modification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a crucial role in cccDNA transcription and viral persistence. Interferon-α (IFN-α) is a pivotal agent against HBV cccDNA. However, the mechanism by which IFN-α modulates the epigenetic regulation of cccDNA remains poorly understood. In this study, we report that IFN-α2b enhances the histone deacetylase 3 (HDAC3)-mediated de-2-hydroxyisobutyrylation of histone H4 lysine 8 (H4K8) on HBV cccDNA minichromosome to restrict the cccDNA transcription in liver. By screening acetyltransferases and deacetylases, we identified that HDAC3 was an effective restrictor of HBV transcription and replication. Moreover, we found that HDAC3 was able to mediate the de-2-hydroxyisobutyrylation of H4K8 in HBV-expressing hepatoma cells. Then, the 2-hydroxyisobutyrylation of histone H4K8 (H4K8hib) was identified on the HBV cccDNA minichromosome, promoting the HBV transcription and replication. The H4K8hib was regulated by HDAC3 depending on its deacetylase domain in the system. The low level of HDAC3 and high level of H4K8hib were observed in the liver tissues from HBV-infected human liver-chimeric mice. The levels of H4K8hib on HBV cccDNA minichromosome were significantly elevated in the liver biopsy specimens from clinical hepatitis B patients, which was consistent with the high transcriptional activity of cccDNA. Strikingly, IFN-α2b effectively facilitated the histone H4K8 de-2-hydroxyisobutyrylation mediated by HDAC3 on the HBV cccDNA minichromosome in primary human hepatocytes and hepatoma cells, leading to the inhibition of HBV transcription and replication. Our finding provides new insights into the mechanism by which IFN-α modulates the epigenetic regulation of HBV cccDNA minichromosome.
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18
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Wang Y, Li Y, Zai W, Hu K, Zhu Y, Deng Q, Wu M, Li Y, Chen J, Yuan Z. HBV covalently closed circular DNA minichromosomes in distinct epigenetic transcriptional states differ in their vulnerability to damage. Hepatology 2022; 75:1275-1288. [PMID: 34779008 DOI: 10.1002/hep.32245] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2021] [Revised: 10/20/2021] [Accepted: 11/10/2021] [Indexed: 12/19/2022]
Abstract
BACKGROUND AND AIMS HBV covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability. APPROACHES AND RESULTS By using HBV infection cell models and in vitro and in vivo recombinant cccDNA (rcccDNA) and HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A)-mediated and CRISPR/CRISPR-associated 9 (Cas9)-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and hepatitis B x protein (HBx) deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. HBV cccDNA levels remained stable in nondividing hepatocytes; however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally active and transcriptionally repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA with low transcriptional activity exhibited an epigenetically inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A. CONCLUSIONS HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division but significantly differed in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.
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Affiliation(s)
- Yang Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Yumeng Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Wenjing Zai
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Kongying Hu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Yuanfei Zhu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Qiang Deng
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
| | - Min Wu
- Shanghai Public Health Clinical CenterFudan UniversityShanghaiChina
| | - Yaming Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
| | - Jieliang Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS)School of Basic Medical SciencesShanghai Medical CollegeFudan UniversityShanghaiChina
- Research Unit of Cure of Chronic Hepatitis B Virus InfectionChinese Academy of Medical SciencesShanghaiChina
- Shanghai Frontiers Science Center of Pathogenic Microbes and InfectionShanghaiChina
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19
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Shi Z, Zheng H, Han M, Hu J, Hu Y, Li X, Zhu W, He X, Deng H, Long Q, Huang A. Durability of Hepatitis B surface antigen seroclearance in patients experienced nucleoside analogs or interferon monotherapy: A real-world data from Electronic Health Record. Genes Dis 2022. [DOI: 10.1016/j.gendis.2022.03.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
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20
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Interaction between the Hepatitis B Virus and Cellular FLIP Variants in Viral Replication and the Innate Immune System. Viruses 2022; 14:v14020373. [PMID: 35215970 PMCID: PMC8874586 DOI: 10.3390/v14020373] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 01/28/2022] [Accepted: 02/08/2022] [Indexed: 12/10/2022] Open
Abstract
During viral evolution and adaptation, many viruses have utilized host cellular factors and machinery as their partners. HBx, as a multifunctional viral protein encoded by the hepatitis B virus (HBV), promotes HBV replication and greatly contributes to the development of HBV-associated hepatocellular carcinoma (HCC). HBx interacts with several host factors in order to regulate HBV replication and evolve carcinogenesis. The cellular FADD-like IL-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP) is a major factor that functions in a variety of cellular pathways and specifically in apoptosis. It has been shown that the interaction between HBx and c-FLIP determines HBV fate. In this review, we provide a comprehensive and detailed overview of the interplay between c-FLIP and HBV in various environmental circumstances. We describe strategies adapted by HBV to establish its chronic infection. We also summarize the conventional roles of c-FLIP and highlight the functional outcome of the interaction between c-FLIP and HBV or other viruses in viral replication and the innate immune system.
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21
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Fukutomi K, Hikita H, Murai K, Nakabori T, Shimoda A, Fukuoka M, Yamai T, Higuchi Y, Miyakawa K, Suemizu H, Ryo A, Yamada R, Kodama T, Sakamori R, Tatsumi T, Takehara T. Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration. Hepatol Commun 2022; 6:281-296. [PMID: 34558845 PMCID: PMC8793994 DOI: 10.1002/hep4.1804] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Revised: 07/12/2021] [Accepted: 07/17/2021] [Indexed: 12/18/2022] Open
Abstract
Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.
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Affiliation(s)
- Keisuke Fukutomi
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Hayato Hikita
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Kazuhiro Murai
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Tasuku Nakabori
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Akiyoshi Shimoda
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Makoto Fukuoka
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Takuo Yamai
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Yuichiro Higuchi
- Laboratory Animal Research DepartmentCentral Institute for Experimental AnimalsKawasakiJapan
| | - Kei Miyakawa
- Department of MicrobiologyYokohama City University School of MedicineYokohamaJapan
| | - Hiroshi Suemizu
- Laboratory Animal Research DepartmentCentral Institute for Experimental AnimalsKawasakiJapan
| | - Akihide Ryo
- Department of MicrobiologyYokohama City University School of MedicineYokohamaJapan
| | - Ryoko Yamada
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Takahiro Kodama
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Ryotaro Sakamori
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Tomohide Tatsumi
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
| | - Tetsuo Takehara
- Department of Gastroenterology and HepatologyOsaka University Graduate School of MedicineOsakaJapan
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22
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Zhang X, Wang Y, Yang G. Research progress in hepatitis B virus covalently closed circular DNA. Cancer Biol Med 2021; 19:j.issn.2095-3941.2021.0454. [PMID: 34931766 PMCID: PMC9088183 DOI: 10.20892/j.issn.2095-3941.2021.0454] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 11/16/2021] [Indexed: 11/28/2022] Open
Abstract
Hepatitis B virus (HBV) infections are a global public health issue. HBV covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is a key factor in the HBV replication cycle. Notably, many host factors involved in HBV cccDNA epigenetic modulation promote the development of hepatocellular carcinoma (HCC). The HBV cccDNA minichromosome is a clinical obstacle that cannot be efficiently eliminated. In this review, we provide an update on the advances in research on HBV cccDNA and further discuss factors affecting the modulation of HBV cccDNA. Hepatitis B virus X protein (HBx) contributes to HBV cccDNA transcription and the development of hepatocarcinogenesis through modulating host epigenetic regulatory factors, thus linking the cccDNA to hepatocarcinogenesis. The measurable serological biomarkers of continued transcription of cccDNA, the effects of anti-HBV drugs on cccDNA, and potential therapeutic strategies targeting cccDNA are discussed in detail. Thus, this review describes new insights into HBV cccDNA mechanisms and therapeutic strategies for cleaning cccDNA, which will benefit patients with liver diseases.
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Affiliation(s)
- Xiaodong Zhang
- Department of Gastrointestinal Cancer Biology, Liver Cancer Center, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
| | - Yufei Wang
- Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Guang Yang
- Department of Gastrointestinal Cancer Biology, Liver Cancer Center, Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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23
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Cai Q, Gan C, Tang C, Wu H, Gao J. Mechanism and Therapeutic Opportunities of Histone Modifications in Chronic Liver Disease. Front Pharmacol 2021; 12:784591. [PMID: 34887768 PMCID: PMC8650224 DOI: 10.3389/fphar.2021.784591] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Accepted: 11/08/2021] [Indexed: 02/05/2023] Open
Abstract
Chronic liver disease (CLD) represents a global health problem, accounting for the heavy burden of disability and increased health care utilization. Epigenome alterations play an important role in the occurrence and progression of CLD. Histone modifications, which include acetylation, methylation, and phosphorylation, represent an essential part of epigenetic modifications that affect the transcriptional activity of genes. Different from genetic mutations, histone modifications are plastic and reversible. They can be modulated pharmacologically without changing the DNA sequence. Thus, there might be chances to establish interventional solutions by targeting histone modifications to reverse CLD. Here we summarized the roles of histone modifications in the context of alcoholic liver disease (ALD), metabolic associated fatty liver disease (MAFLD), viral hepatitis, autoimmune liver disease, drug-induced liver injury (DILI), and liver fibrosis or cirrhosis. The potential targets of histone modifications for translation into therapeutics were also investigated. In prospect, high efficacy and low toxicity drugs that are selectively targeting histone modifications are required to completely reverse CLD and prevent the development of liver cirrhosis and malignancy.
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Affiliation(s)
- Qiuyu Cai
- Laboratory of Gastroenterology and Hepatology, West China Hospital, Sichuan University, Chengdu, China
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
| | - Can Gan
- Laboratory of Gastroenterology and Hepatology, West China Hospital, Sichuan University, Chengdu, China
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
| | - Chengwei Tang
- Laboratory of Gastroenterology and Hepatology, West China Hospital, Sichuan University, Chengdu, China
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
| | - Hao Wu
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
| | - Jinhang Gao
- Laboratory of Gastroenterology and Hepatology, West China Hospital, Sichuan University, Chengdu, China
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, China
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24
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Alvarez-Astudillo F, Garrido D, Varas-Godoy M, Gutiérrez JL, Villanueva RA, Loyola A. The histone variant H3.3 regulates the transcription of the hepatitis B virus. Ann Hepatol 2021; 21:100261. [PMID: 33007428 DOI: 10.1016/j.aohep.2020.09.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 09/17/2020] [Indexed: 02/04/2023]
Abstract
INTRODUCTION AND OBJECTIVES About 250 million people around the world are chronically infected with the hepatitis B virus (HBV). Those people are at risk of developing hepatocellular carcinoma. The HBV genome is organized as a minichromosome in the infected hepatocyte and is associated with histones and non-histone proteins. In recent years, many groups have investigated the transcriptional regulation of HBV mediated by post-translational modifications on the histones associated with the covalently closed circular DNA (cccDNA). Our aim is to investigate the role of the histone variant H3.3. MATERIALS AND METHODS An in vitro HBV replication model system based on the transfection of linear HBV genome monomeric molecules was used. We then either ectopically expressed or reduced the levels of H3.3, and of its histone chaperone HIRA. Viral intermediates were quantified and the level of H3K4me3 using Chromatin immunoprecipitation (ChIP) assay was measured. RESULTS Histone variant H3.3 ectopically expressed in cells assembles into the viral cccDNA, correlating with increasing levels of the active histone mark H3K4me3 and HBV transcription. The opposite results were found upon diminishing H3.3 levels. Furthermore, the assembly of H3.3 into the cccDNA is dependent on the histone chaperone HIRA. Diminishing HIRA levels causes a reduction in the HBV intermediates. CONCLUSIONS Histone variant H3.3 positively regulates HBV transcription. Importantly, the characterization of the viral chromatin dynamics might allow the discovery of new therapeutic targets to develop drugs for the treatment of chronically-infected HBV patients.
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Affiliation(s)
| | - Daniel Garrido
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile
| | - Manuel Varas-Godoy
- Centro de Biología Celular y Biomedicina (CEBICEM), Facultad de Medicina y Ciencia, Universidad, San Sebastián, Santiago, 7510157, Chile
| | - José Leonardo Gutiérrez
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Barrio Universitario s/n, Concepción, 4070043, Chile
| | - Rodrigo A Villanueva
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile.
| | - Alejandra Loyola
- Fundación Ciencia & Vida, Avenida Zañartu 1482, Ñuñoa, 7780272, Santiago, Chile; Universidad San Sebastián, Santiago, 7510157, Chile.
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25
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Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV. Cell Discov 2021; 7:105. [PMID: 34725333 PMCID: PMC8560872 DOI: 10.1038/s41421-021-00337-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2021] [Accepted: 09/22/2021] [Indexed: 12/13/2022] Open
Abstract
Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.
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26
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Moon IY, Kim JW. Methylation profile of hepatitis B virus is not influenced by interferon α in human liver cancer cells. Mol Med Rep 2021; 24:715. [PMID: 34396432 PMCID: PMC8383030 DOI: 10.3892/mmr.2021.12354] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2020] [Accepted: 07/12/2021] [Indexed: 12/17/2022] Open
Abstract
Interferon (IFN) α is used for the treatment of chronic hepatitis B virus (HBV) infection, but the molecular mechanisms underlying its antiviral effect have not been fully elucidated. Epigenetic modifications regulate the transcriptional activity of covalently closed circular DNA (cccDNA) in cells with chronic HBV infection. IFN‑α has been shown to modify cccDNA‑bound histones, but it is not known whether the anti‑HBV effect of IFN‑α involves methylation of cccDNA. The present study aimed to determine whether IFN‑α induced methylation of HBV cccDNA in a cell‑based model in which HepG2 cells were directly infected with wild‑type HBV virions. Methylation status of HBV cccDNA was assessed using global DNA methylation ELISA assay, methylation‑specific PCR and bisulfite sequencing. IFN‑α suppressed HBV DNA and RNA transcripts, but methylation profiles were similar between the control and IFN‑α treated groups. Chromatin immunoprecipitation results revealed binding of DNA methyltransferases (DNMT) 3A and DNMT3B to HBV cccDNA and treatment with IFN‑α suppressed the recruitment of DNMT3B to cccDNA. Taken together, these results suggest that IFN‑α does not induce methylation of HBV cccDNA. Therefore, it was concluded that methylation is unlikely to contribute to the anti‑HBV effect of IFN‑α in HepG2 cells, and that alternative mechanisms need to be sought to enhance cccDNA methylation as a novel therapy against HBV.
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Affiliation(s)
- In Young Moon
- Department of Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi 13620, Republic of Korea
| | - Jin-Wook Kim
- Department of Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi 13620, Republic of Korea
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
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27
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Akbar SMF, Al Mahtab M, Cesar Aguilar J, Uddin MH, Khan MSI, Yoshida O, Penton E, Gerardo GN, Hiasa Y. Exploring evidence-based innovative therapy for the treatment of chronic HBV infection: experimental and clinical. EXPLORATION OF MEDICINE 2021. [DOI: 10.37349/emed.2021.00058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 08/03/2021] [Indexed: 01/02/2025] Open
Abstract
With the advent of various vaccines and antimicrobial agents during the 20th century, the control and containment of infectious diseases appeared to be a matter of time. However, studies unveiled the diverse natures of microbes, their lifestyle, and pathogenetic potentials. Since the ground-breaking discovery of the hepatitis B virus (HBV) by Baruch Blumberg and the subsequent development of a vaccine in the early 1980s, the main task of the scientific community has been to develop a proper management strategy for HBV-induced chronic liver diseases. In the early 1980’s, standard interferon (IFN) induced a reduction of HBV DNA levels, followed by the normalization of serum transaminases (alanine aminotransferase, ALT), in some chronic hepatitis B (CHB) patients. However, in the course of time, the limitations of standard IFN became evident, and the search for an alternative began. In the late 1980’s, nucleoside analogs entered the arena of CHB treatment as oral drugs with potent antiviral capacities. At the beginning of the 21st century, insights were developed into the scope and limitations of standard IFN, pegylated-IFN as well as nucleoside analogs for treating CHB. Considering the non-cytopathic nature of the HBV, the presence of covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocytes and HBV-induced immune-mediated liver damages, a new field of CHB management was initiated by modulating the hosts’ immune system through immune therapy. This review will discuss the nature and design of innovative immune therapy for CHB.
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Affiliation(s)
- Sheikh Mohammad Fazle Akbar
- Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Ehime 7910295, Japan
| | - Mamun Al Mahtab
- Department of Hepatology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka 1000, Bangladesh
| | - Julio Cesar Aguilar
- Center for Genetic Engineering and Biotechnology, Havana, Havana 10600, Cuba
| | | | - Md. Sakirul Islam Khan
- Department of Anatomy and Embryology, Ehime University Graduate School of Medicine, Ehime 7910295, Japan
| | - Osamu Yoshida
- Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Ehime 7910295, Japan
| | - Eduardo Penton
- Center for Genetic Engineering and Biotechnology, Havana, Havana 10600, Cuba
| | | | - Yoichi Hiasa
- Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Ehime 7910295, Japan
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28
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Lv X, Xiang X, Wu Y, Liu Y, Xu R, Xiang Q, Lai G. GATA binding protein 4 promotes the expression and transcription of hepatitis B virus by facilitating hepatocyte nuclear factor 4 alpha in vitro. Virol J 2021; 18:196. [PMID: 34583732 PMCID: PMC8479913 DOI: 10.1186/s12985-021-01668-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Accepted: 09/21/2021] [Indexed: 12/24/2022] Open
Abstract
Background GATA binding protein 4 (GATA4) has been reported as a potential target of gene therapy for hepatocellular carcinoma (HCC). It is well known that the main cause of HCC is the chronic infection of hepatitis B virus (HBV). However, whether the effect of GATA4 on HBV has not yet been reported. Methods In this study, the regulation of GATA4 on HBV was analyzed in vitro. In turn, the effect of HBV on GATA4 was also observed in vitro, in vivo, and clinical HCC patients. Subsequently, we analyzed whether the effect of GATA4 on HBV was related to hepatocyte nuclear factor 4 alpha (HNF4α) in vitro. Results The results showed that GATA4 significantly promoted the secretion of HBV surface antigen (HBsAg) and HBV e antigen in the cell culture medium, improved the replication of HBV genomic DNA, and increased the level of HBV 3.5 kb pre-genomic RNA and HBV total RNA (P < 0.05). Moreover, it was showed that HBV had no significant effect on GATA4 in vitro and in vivo (P > 0.05). At the same time, GATA4 expression was decreased in 78.9% (15/19) of HCC patients regardless of the HBV and HBsAg status. Among them, there were 76.9% (10/13) in HBV-associated patients with HCC (HBV-HCC), and 83.3% (5/6) in non-HBV-HCC patients. In addition, the expression of HNF4α was also up-regulated or down-regulated accordingly when stimulating or interfering with the expression of GATA4. Furthermore, stimulating the expression of HNF4α could only alleviate the HBsAg level and HBV transcription levels, but had no significant effect on GATA4. Conclusions In summary, this study found that GATA4 has a positive effect on HBV, and the potential pathway may be related to another transcription factor HNF4α that regulates HBV.
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Affiliation(s)
- Xiaoqin Lv
- Laboratory Animal Center of Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong District, Chongqing, 400016, China
| | - Xia Xiang
- Laboratory Animal Center of Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong District, Chongqing, 400016, China
| | - Yue Wu
- The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 40010, China
| | - Yang Liu
- LuXian No. 2 High School, Sichuan, 646100, China
| | - Ruqing Xu
- Laboratory Animal Center of Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong District, Chongqing, 400016, China
| | - Qin Xiang
- Molecular Oncology and Epigenetics Laboratory of the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China
| | - Guoqi Lai
- Laboratory Animal Center of Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong District, Chongqing, 400016, China.
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29
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Ye J, Chen J. Interferon and Hepatitis B: Current and Future Perspectives. Front Immunol 2021; 12:733364. [PMID: 34557195 PMCID: PMC8452902 DOI: 10.3389/fimmu.2021.733364] [Citation(s) in RCA: 107] [Impact Index Per Article: 26.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Accepted: 08/17/2021] [Indexed: 12/12/2022] Open
Abstract
Chronic hepatitis B virus (HBV) infection remains a major health burden worldwide for which there is still no effective curative treatment. Interferon (IFN) consists of a group of cytokines with antiviral activity and immunoregulatory and antitumor effects, that play crucial roles in both innate and adaptive immune responses. IFN-α and its pegylated form have been used for over thirty years to treat chronic hepatitis B (CHB) with advantages of finite treatment duration and sustained virologic response, however, the efficacy is limited and side effects are common. Here, we summarize the status and unique advantages of IFN therapy against CHB, review the mechanisms of IFN-α action and factors affecting IFN response, and discuss the possible improvement of IFN-based therapy and the rationale of combinations with other antiviral agents in seeking an HBV cure.
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Affiliation(s)
- Jianyu Ye
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jieliang Chen
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.,Research Unit of Cure of Chronic Hepatitis B Virus Infection, Chinese Academy of Medical Sciences, Shanghai, China
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30
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Ligat G, Verrier ER, Nassal M, Baumert TF. Hepatitis B virus-host interactions and novel targets for viral cure. Curr Opin Virol 2021; 49:41-51. [PMID: 34029994 PMCID: PMC7613419 DOI: 10.1016/j.coviro.2021.04.009] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 02/06/2023]
Abstract
Chronic infection with HBV is a major cause of advanced liver disease and hepatocellular carcinoma. Nucleos(t)ide analogues effectively control HBV replication but viral cure is rare. Hence treatment has often to be administered for an indefinite duration, increasing the risk for selection of drug resistant virus variants. PEG-interferon-α-based therapies can sometimes cure infection but suffer from a low response rate and severe side-effects. CHB is characterized by the persistence of a nuclear covalently closed circular DNA (cccDNA), which is not targeted by approved drugs. Targeting host factors which contribute to the viral life cycle provides new opportunities for the development of innovative therapeutic strategies aiming at HBV cure. An improved understanding of the host immune system has resulted in new potentially curative candidate approaches. Here, we review the recent advances in understanding HBV-host interactions and highlight how this knowledge contributes to exploiting host-targeting strategies for a viral cure.
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Affiliation(s)
- Gaëtan Ligat
- Université de Strasbourg, F-67000 Strasbourg, France; Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMRS 1110, F-67000 Strasbourg, France.
| | - Eloi R Verrier
- Université de Strasbourg, F-67000 Strasbourg, France; Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMRS 1110, F-67000 Strasbourg, France.
| | - Michael Nassal
- University Hospital Freiburg, Dept. of Internal Medicine 2/Molecular Biology, D79106 Freiburg, Germany.
| | - Thomas F Baumert
- Université de Strasbourg, F-67000 Strasbourg, France; Inserm, Institut de Recherche sur les Maladies Virales et Hépatiques UMRS 1110, F-67000 Strasbourg, France; Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Nouvel Hôpital Civil, 67000 Strasbourg, France.
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Nishio A, Bolte FJ, Takeda K, Park N, Yu ZX, Park H, Valdez K, Ghany MG, Rehermann B. Clearance of pegylated interferon by Kupffer cells limits NK cell activation and therapy response of patients with HBV infection. Sci Transl Med 2021; 13:13/587/eaba6322. [PMID: 33790025 DOI: 10.1126/scitranslmed.aba6322] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Accepted: 02/24/2021] [Indexed: 12/12/2022]
Abstract
Pegylated interferon-α (PEG-IFN-α), where IFN-α is attached to polyethylene glycol (PEG), is an approved treatment for chronic hepatitis B virus (HBV) infection, a disease that causes liver-related morbidity and mortality in 257 million people worldwide. It is unknown why only a minority of patients respond to PEG-IFN-α. Using sequential blood samples and liver biopsies of patients with chronic HBV infection before, during, and after PEG-IFN-α treatment, we find that patients with early natural killer (NK) cell activation after PEG-IFN-α injection experienced greater liver inflammation, lysis of HBV-infected hepatocytes, and hepatitis B surface antigen (HBsAg) decline than those without. NK cell activation was associated with induction of interferon-stimulated genes and determined by PEG-IFN-α pharmacokinetics. Patients with delayed increases in PEG-IFN-α concentrations had greater amounts of PEG-specific immunoglobulin M (IgM) immune complexes in the blood and more PEG and IgM detected in the liver than patients with rapid increase in PEG-IFN-α concentration. This was associated with reduced NK cell activation. These results indicate that the immunomodulatory functions of PEG-IFN-α, particularly activation of NK cells, play a pivotal role in the response to treatment and further demonstrate that these functions are affected by PEG-IFN-α pharmacokinetics. Accelerated clearance of antibody-complexed pegylated drugs by Kupffer cells may be important beyond the field of HBV therapeutics. Thus, these findings may contribute to improving the efficacy of pegylated drugs that are now being developed for other chronic diseases and cancer.
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Affiliation(s)
- Akira Nishio
- Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Fabian J Bolte
- Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Kazuyo Takeda
- Pathology Core, National Heart, Lung and Blood Institute, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Nana Park
- Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Zu-Xi Yu
- Pathology Core, National Heart, Lung and Blood Institute, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Heiyoung Park
- Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Kristin Valdez
- Clinical Research Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Marc G Ghany
- Clinical Research Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
| | - Barbara Rehermann
- Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA.
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Qu B, Brown RJP. Strategies to Inhibit Hepatitis B Virus at the Transcript Level. Viruses 2021; 13:v13071327. [PMID: 34372533 PMCID: PMC8310268 DOI: 10.3390/v13071327] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 07/02/2021] [Accepted: 07/06/2021] [Indexed: 12/11/2022] Open
Abstract
Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.
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Affiliation(s)
- Bingqian Qu
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- European Virus Bioinformatics Center, 07743 Jena, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
| | - Richard J. P. Brown
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
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Singh P, Kairuz D, Arbuthnot P, Bloom K. Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach. World J Gastroenterol 2021; 27:3182-3207. [PMID: 34163105 PMCID: PMC8218364 DOI: 10.3748/wjg.v27.i23.3182] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 03/23/2021] [Accepted: 05/07/2021] [Indexed: 02/06/2023] Open
Abstract
Global prophylactic vaccination programmes have helped to curb new hepatitis B virus (HBV) infections. However, it is estimated that nearly 300 million people are chronically infected and have a high risk of developing hepatocellular carcinoma. As such, HBV remains a serious health priority and the development of novel curative therapeutics is urgently needed. Chronic HBV infection has been attributed to the persistence of the covalently closed circular DNA (cccDNA) which establishes itself as a minichromosome in the nucleus of hepatocytes. As the viral transcription intermediate, the cccDNA is responsible for producing new virions and perpetuating infection. HBV is dependent on various host factors for cccDNA formation and the minichromosome is amenable to epigenetic modifications. Two HBV proteins, X (HBx) and core (HBc) promote viral replication by modulating the cccDNA epigenome and regulating host cell responses. This includes viral and host gene expression, chromatin remodeling, DNA methylation, the antiviral immune response, apoptosis, and ubiquitination. Elimination of the cccDNA minichromosome would result in a sterilizing cure; however, this may be difficult to achieve. Epigenetic therapies could permanently silence the cccDNA minichromosome and promote a functional cure. This review explores the cccDNA epigenome, how host and viral factors influence transcription, and the recent epigenetic therapies and epigenome engineering approaches that have been described.
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Affiliation(s)
- Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Dylan Kairuz
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
| | - Kristie Bloom
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand, Johannesburg 2050, Gauteng, South Africa
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Goh ZY, Ren EC, Ko HL. Intracellular interferon signalling pathways as potential regulators of covalently closed circular DNA in the treatment of chronic hepatitis B. World J Gastroenterol 2021; 27:1369-1391. [PMID: 33911462 PMCID: PMC8047536 DOI: 10.3748/wjg.v27.i14.1369] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Revised: 02/23/2021] [Accepted: 03/17/2021] [Indexed: 02/06/2023] Open
Abstract
Infection with the hepatitis B virus (HBV) is still a major global health threat as 250 million people worldwide continue to be chronically infected with the virus. While patients may be treated with nucleoside/nucleotide analogues, this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA (cccDNA) genome. As a result, HBV infection cannot be cured, and the virus reactivates when conditions are favorable. Interferons (IFNs) are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells. They have been shown to induce cccDNA clearance, but their use in the treatment of HBV infection is limited as HBV-targeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling. Thus, to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV, instead of direct IFN administration, novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed. This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions. These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications. In addition, the mechanisms that HBV employs to disrupt IFN signalling will be discussed. Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections. Together, these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.
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Affiliation(s)
- Zhi Yi Goh
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Integrative Sciences and Engineering Programme, NUS Graduate School, National University of Singapore, Singapore 119077, Singapore
| | - Ee Chee Ren
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119260, Singapore
| | - Hui Ling Ko
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore 138648, Singapore
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A putative amphipathic alpha helix in hepatitis B virus small envelope protein plays a critical role in the morphogenesis of subviral particles. J Virol 2021; 95:JVI.02399-20. [PMID: 33536177 PMCID: PMC8103704 DOI: 10.1128/jvi.02399-20] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization, but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent, but ubiquitination-independent non-classic ER-associated degradation (ERAD) pathway. Taken together, the results reported herein favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC compartment, whereas the failure of budding process may result in S protein degradation by 20S proteasome in an ubiquitination-independent manner.Importance Subviral particles are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed the virion particles by 10,000 to 100,000-fold in the blood of HBV infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induces immune tolerance and contributes to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by appearance of anti-HBs antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is, therefore, considered to be essential for the restoration of host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.
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Fatehi F, Bingham RJ, Dykeman EC, Patel N, Stockley PG, Twarock R. An Intracellular Model of Hepatitis B Viral Infection: An In Silico Platform for Comparing Therapeutic Strategies. Viruses 2020; 13:v13010011. [PMID: 33374798 PMCID: PMC7823939 DOI: 10.3390/v13010011] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2020] [Revised: 12/17/2020] [Accepted: 12/19/2020] [Indexed: 12/23/2022] Open
Abstract
Hepatitis B virus (HBV) is a major focus of antiviral research worldwide. The International Coalition to Eliminate HBV, together with the World Health Organisation (WHO), have prioritised the search for a cure, with the goal of eliminating deaths from viral hepatitis by 2030. We present here a comprehensive model of intracellular HBV infection dynamics that includes all molecular processes currently targeted by drugs and agrees well with the observed outcomes of several clinical studies. The model reveals previously unsuspected kinetic behaviour in the formation of sub-viral particles, which could lead to a better understanding of the immune responses to infection. It also enables rapid comparative assessment of the impact of different treatment options and their potential synergies as combination therapies. A comparison of available and currently developed treatment options reveals that combinations of multiple capsid assembly inhibitors perform best.
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Affiliation(s)
- Farzad Fatehi
- York Cross-Disciplinary Centre for Systems Analysis, University of York, York YO10 5GE, UK; (F.F.); (R.J.B.); (E.C.D.)
- Department of Mathematics, University of York, York YO10 5DD, UK
| | - Richard J. Bingham
- York Cross-Disciplinary Centre for Systems Analysis, University of York, York YO10 5GE, UK; (F.F.); (R.J.B.); (E.C.D.)
- Department of Mathematics, University of York, York YO10 5DD, UK
- Department of Biology, University of York, York YO10 5NG, UK
| | - Eric C. Dykeman
- York Cross-Disciplinary Centre for Systems Analysis, University of York, York YO10 5GE, UK; (F.F.); (R.J.B.); (E.C.D.)
- Department of Mathematics, University of York, York YO10 5DD, UK
| | - Nikesh Patel
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT UK;
| | - Peter G. Stockley
- Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT UK;
- Correspondence: (P.G.S.); (R.T.)
| | - Reidun Twarock
- York Cross-Disciplinary Centre for Systems Analysis, University of York, York YO10 5GE, UK; (F.F.); (R.J.B.); (E.C.D.)
- Department of Mathematics, University of York, York YO10 5DD, UK
- Department of Biology, University of York, York YO10 5NG, UK
- Correspondence: (P.G.S.); (R.T.)
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Li MH, Chen QQ, Zhang L, Lu HH, Sun FF, Zeng Z, Lu Y, Yi W, Xie Y. Association of cytokines with hepatitis B virus and its antigen. J Med Virol 2020; 92:3426-3435. [PMID: 32662892 DOI: 10.1002/jmv.26301] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Revised: 06/02/2020] [Accepted: 07/09/2020] [Indexed: 12/14/2022]
Abstract
To investigate the characteristics of cytokines in patients with different HBV infection status and their correlation with HBV DNA, HBsAg, and HBeAg levels. Peripheral blood samples were collected from patients with chronic HBV infection in immune tolerance phase (IT), HBeAg-positive chronic hepatitis B (CHB), and acute hepatitis B (AHB) groups, and levels of cytokines were detected by Luminex technique, and analyzed by FLEXMAP 3D analyzer. The correlation between cytokines and HBV DNA load, HBsAg, HBeAg, and alanine aminotransferase (ALT) level in patients with chronic HBV infection was analyzed. In total 312 subjects (184 males and 128 females) were enrolled in the study. There were significant differences among IT, CHB, and AHB groups in Flt-3L value (P = .003; H = 12.312), IFN-γ (P = .001; H = 11.723), IL-10 (P = .001; H = 18.736), IL-17A ((P = .001; H = 12.735), and TGF-β1 (P = .001; Z = 48.571). IFN-α2 levels in CHB group were significantly higher than those in IT and AHB groups (15.24 vs 35.78 pg/mL, P = .000; Z = 3.727; 13.88 vs 35.78 pg/mL, P = .024; Z = -2.258. In CHB group, the levels of HBsAg and ALT were positively correlated with the levels of IL-10 (r = .173; P = .006; r = 0.176; P = .006, respectively), while HBeAg level was positively correlated with the IFN-α2 level (r = .153; P = .016). In AHB group, the HBsAg level was positively correlated with Flt-3L, IFN-α2, IL-10, and IL-6 (r = .402; P = .023; r = .436; P = .016; r = .524, P = .002; r = .405; P = .022, respectively). HBeAg level was positively correlated with IFN-γ and IL-17A levels (r = .400; P = .023; r = .373; P = .036, respectively), and ALT level was positively correlated with IL-6 levels (r = .367; P = .039). In either AHB or CHB group, HBV DNA load was only related to TGF-β level (r = .493; P = .004; r = -.218, P = 0.009 respectively). The correlation between Flt-3L and HBsAg (F = 7.422; P = .007); IL-17, IL-6, and HBeAg (F = 5.757; P = .017; F = 6.156; P = .014) were statistically significant. There was significant correlation between TGF-β2 and HBV DNA (F = 11.795; P = .001), and between ALT and HBsAg, HBV DNA (F = 26.089; P = .000; F = 4.724; P = .031). HBsAg, HBeAg, and HBV DNA were correlated with cytokines and ALT in patients with HBV infection. The level of IFN-α2 was significantly higher in patients with CHB. HBV DNA load was only correlated with the level of TGF-β in acute or CHB.
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Affiliation(s)
- Ming-Hui Li
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
| | - Qi-Qi Chen
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
| | - Lu Zhang
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Hui-Hui Lu
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Fang-Fang Sun
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Zhan Zeng
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
| | - Yao Lu
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Wei Yi
- Department of Gynecology and Obstetrics, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Yao Xie
- Department of Hepatology Division 2, Beijing Ditan Hospital, Capital Medical University, Beijing, China
- Department of Hepatology Division 2, Peking University Ditan Teaching Hospital, Beijing, China
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Lau KC, Burak KW, Coffin CS. Impact of Hepatitis B Virus Genetic Variation, Integration, and Lymphotropism in Antiviral Treatment and Oncogenesis. Microorganisms 2020; 8:E1470. [PMID: 32987867 PMCID: PMC7599633 DOI: 10.3390/microorganisms8101470] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/16/2020] [Accepted: 09/22/2020] [Indexed: 12/14/2022] Open
Abstract
Chronic Hepatitis B Virus (HBV) infection poses a significant global health burden. Although, effective treatment and vaccinations against HBV are available, challenges still exist, particularly in the development of curative therapies. The dynamic nature and unique features of HBV such as viral variants, integration of HBV DNA into host chromosomes, and extrahepatic reservoirs are considerations towards understanding the virus biology and developing improved anti-HBV treatments. In this review, we highlight the importance of these viral characteristics in the context of treatment and oncogenesis. Viral genotype and genetic variants can serve as important predictive factors for therapeutic response and outcomes in addition to oncogenic risk. HBV integration, particularly in coding genes, is implicated in the development of hepatocellular carcinoma. Furthermore, we will discuss emerging research that has identified various HBV nucleic acids and infection markers within extrahepatic sites (lymphoid cells). Intriguingly, the presence of hepatocellular carcinoma (HCC)-associated HBV variants and viral integration within the lymphoid cells may contribute towards the development of extrahepatic malignancies. Improved understanding of these HBV characteristics will enhance the development of a cure for chronic HBV infection.
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Affiliation(s)
- Keith C.K. Lau
- Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;
- Calgary Liver Unit, Division of Gastroenterology and Hepatology, Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;
| | - Kelly W. Burak
- Calgary Liver Unit, Division of Gastroenterology and Hepatology, Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;
| | - Carla S. Coffin
- Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;
- Calgary Liver Unit, Division of Gastroenterology and Hepatology, Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada;
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Tsukuda S, Watashi K. Hepatitis B virus biology and life cycle. Antiviral Res 2020; 182:104925. [PMID: 32866519 DOI: 10.1016/j.antiviral.2020.104925] [Citation(s) in RCA: 214] [Impact Index Per Article: 42.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Revised: 07/24/2020] [Accepted: 08/26/2020] [Indexed: 12/12/2022]
Abstract
Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. The HBV life cycle is unique in that the genomic DNA (relaxed-circular partially double-stranded DNA: rcDNA) is converted to a molecular template DNA (covalently closed circular DNA: cccDNA) to amplify a viral RNA intermediate, which is then reverse-transcribed back to viral DNA. The highly stable characteristics of cccDNA result in chronic infection and a poor rate of cure. This complex life cycle of HBV offers a variety of targets to develop antiviral agents. We provide here an update on the current knowledge of HBV biology and its life cycle, which may help to identify new antiviral targets.
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Affiliation(s)
- Senko Tsukuda
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Department of Applied Biological Science, Tokyo University of Science, Noda, Japan; Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; MIRAI, JST, Saitama, Japan.
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40
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Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription. J Virol 2020; 94:JVI.00442-20. [PMID: 32581092 DOI: 10.1128/jvi.00442-20] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2020] [Accepted: 06/15/2020] [Indexed: 12/12/2022] Open
Abstract
Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.
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Wang Z, Wang W, Wang L. Epigenetic regulation of covalently closed circular DNA minichromosome in hepatitis B virus infection. BIOPHYSICS REPORTS 2020. [DOI: 10.1007/s41048-020-00112-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
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Stadelmayer B, Diederichs A, Chapus F, Rivoire M, Neveu G, Alam A, Fraisse L, Carter K, Testoni B, Zoulim F. Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum. J Hepatol 2020; 73:40-51. [PMID: 32087349 DOI: 10.1016/j.jhep.2020.01.028] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Revised: 01/10/2020] [Accepted: 01/15/2020] [Indexed: 12/22/2022]
Abstract
BACKGROUND & AIMS Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, i.e. preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. METHODS We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. RESULTS In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-β treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. CONCLUSIONS This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA. LAY SUMMARY Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.
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Affiliation(s)
- Bernd Stadelmayer
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude-Bernard (UCBL), 69008 Lyon, France.
| | - Audrey Diederichs
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude-Bernard (UCBL), 69008 Lyon, France
| | - Fleur Chapus
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude-Bernard (UCBL), 69008 Lyon, France
| | - Michel Rivoire
- INSERM U1032, Centre Léon Bérard (CLB), 69008 Lyon, France
| | - Gregory Neveu
- Evotec, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France
| | - Antoine Alam
- Evotec, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France
| | - Laurent Fraisse
- Evotec, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France
| | - Kara Carter
- Evotec, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France
| | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude-Bernard (UCBL), 69008 Lyon, France
| | - Fabien Zoulim
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, 69008, France; University of Lyon, Université Claude-Bernard (UCBL), 69008 Lyon, France; Hospices Civils de Lyon (HCL), 69002 Lyon, France.
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Menne S, Wildum S, Steiner G, Suresh M, Korolowicz K, Balarezo M, Yon C, Murreddu M, Hong X, Kallakury BV, Tucker R, Yang S, Young JAT, Javanbakht H. Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus. Hepatol Commun 2020; 4:916-931. [PMID: 32490326 PMCID: PMC7262289 DOI: 10.1002/hep4.1502] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 02/13/2020] [Accepted: 02/18/2020] [Indexed: 12/16/2022] Open
Abstract
RG7834 is a small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon‐α (wIFN‐α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN‐α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN‐α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off‐treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV‐specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.
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Affiliation(s)
- Stephan Menne
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Steffen Wildum
- Roche Pharma Research and Early Development Roche Innovation Center Basel Basel Switzerland
| | - Guido Steiner
- Roche Pharma Research and Early Development Roche Innovation Center Basel Basel Switzerland
| | - Manasa Suresh
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Kyle Korolowicz
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Maria Balarezo
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Changsuek Yon
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Marta Murreddu
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | - Xupeng Hong
- Department of Microbiology and Immunology Georgetown University Medical Center Washington DC
| | | | - Robin Tucker
- Department of Pharmacology Georgetown University Medical Center Washington DC
| | - Song Yang
- Roche Pharma Research and Early Development Roche Innovation Center Shanghai Shanghai China
| | - John A T Young
- Roche Pharma Research and Early Development Roche Innovation Center Basel Basel Switzerland
| | - Hassan Javanbakht
- Roche Pharma Research and Early Development Roche Innovation Center Basel Basel Switzerland
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Imam H, Kim GW, Mir SA, Khan M, Siddiqui A. Interferon-stimulated gene 20 (ISG20) selectively degrades N6-methyladenosine modified Hepatitis B Virus transcripts. PLoS Pathog 2020; 16:e1008338. [PMID: 32059034 PMCID: PMC7046284 DOI: 10.1371/journal.ppat.1008338] [Citation(s) in RCA: 93] [Impact Index Per Article: 18.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2019] [Revised: 02/27/2020] [Accepted: 01/20/2020] [Indexed: 12/24/2022] Open
Abstract
Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation. Hepatitis B Virus (HBV) is a DNA virus but replicates through a transitional pregenomic RNA (pgRNA). Interferon stimulated antiviral RNase, ISG20 selectively binds to the lower epsilon stem loop of HBV RNA and causes their degradation. Surprisingly this ISG20 binding site is chemically modified by N6-methyladenosine addition to A1907 residue, which resides in the lower region of the epsilon stem loop. This single m6A site occurs twice due to terminal redundancy of sequences in the pgRNA. We demonstrated herein that IFN-α-induced ISG20 can selectively degrade m6A modified HBV RNA. Using a combined strategy of silencing cellular methyltransferases, m6A binding protein YTHDF2 and the m6A sites mutants, we show that HBV transcripts are resistant to either IFN-α treatment or ectopically introduced ISG20 mediated degradation. YTHDF2 is an m6A binding protein which makes the HBV RNAs less stable. YTHDF2 protein forms a complex with IFN-α stimulated ISG20 and executes the nuclease digestion of the recruited m6A modified transcripts. Absence of cellular m6A machinery (methyltransferases or m6A reader proteins) makes the HBV RNA unresponsive to ISG20 mediated decay. This study provides molecular explanation of IFN-α mediated degradation of m6A modified HBV RNAs.
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Affiliation(s)
- Hasan Imam
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, United States of America
| | - Geon-Woo Kim
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, United States of America
| | - Saiful Anam Mir
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, United States of America
| | - Mohsin Khan
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, United States of America
- * E-mail: (MK); (AS)
| | - Aleem Siddiqui
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, California, United States of America
- * E-mail: (MK); (AS)
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Ostankova YV, Semenov AV, Totolian AA. [The quantitative determination method of covalently closed circular DNA HBV in puncture biopsy specimens of the liver.]. Klin Lab Diagn 2019; 64:565-570. [PMID: 31610110 DOI: 10.18821/0869-2084-2019-64-9-565-570] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2019] [Accepted: 06/25/2019] [Indexed: 12/16/2022]
Abstract
To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the β-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage» t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.
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Affiliation(s)
- Yu V Ostankova
- Saint-Petersburg Pasteur Institute, 197191, Saint Petersburg, Russia
| | - A V Semenov
- Saint-Petersburg Pasteur Institute, 197191, Saint Petersburg, Russia.,Saint-Petersburg State Medical University n.a. acad. I.P. Pavlov, 197022, Saint Petersburg, Russia.,North-West State Medical University n.a. I.I. Mechnikov, 191015, Saint Petersburg, Russia
| | - A A Totolian
- Saint-Petersburg Pasteur Institute, 197191, Saint Petersburg, Russia.,Saint-Petersburg State Medical University n.a. acad. I.P. Pavlov, 197022, Saint Petersburg, Russia
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Cao WH, Li MH, Pan CQ, Lu Y, Zhang L, Ran CP, Wu SL, Hua WH, Liu SA, Shen G, Chang M, Liu RY, Hao HX, Hu LP, Xie Y. Quantitation of Plasmacytoid Dendritic Cells in Chronic Hepatitis B Patients with HBeAg Positivity During PEG-IFN and Entecavir Therapy. J Interferon Cytokine Res 2019; 38:197-205. [PMID: 29791282 DOI: 10.1089/jir.2018.0014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Plasmacytoid dendritic cells (pDCs) are crucial for control of chronic hepatitis B (CHB) virus infection. In this study, we evaluated the frequencies of pDCs and expression of functional molecules on pDCs in patients treated with PEG-IFN-α-2a or entecavir (ETV) and investigated changes during treatment. The mean fluorescence intensity of CD86 (CD86MFI) on the surface of pDCs and frequencies of pDCs and CD86+ pDCs in peripheral blood were measured. Compared with baseline, CD86+ pDC% and CD86MFI increased obviously after PEG-IFN-α-2a treatment for 12 and 24 weeks. For patients treated with ETV, only pDC% increased observably after treatment weeks 12 and 24 (P < 0.001) compared with baseline. Hepatitis B surface antigen (HBsAg) decline was significantly associated with elevated CD86+ pDC% (r = 0.348, P = 0.015) during PEG-IFN-α-2a treatment. In the HBsAg response group, CD86+ pDC% and CD86MFI (P < 0.001) increased observably after PEG-IFN-α-2a therapy, whereas only CD86MFI had a statistically significant difference after therapy compared with baseline (12 weeks versus 0 weeks, P = 0.022; 24 weeks versus 0 weeks, P = 0.015) in the HBsAg nonresponse group. CD86+ pDC% between the 2 groups had statistically significant differences at baseline (P = 0.001) and at the treatment time points of 12 and 24 weeks (P < 0.001), respectively. For patients receiving ETV therapy, pDC% increased observably, but CD86+ pDC% decreased significantly (P < 0.001) in the HBV DNA nonresponse group during early treatment with ETV. In CHB patients, HBsAg response in PEG-IFN-α-2a therapy correlated with the increase of CD86+ pDC% and HBV DNA nonresponse in ETV treatment correlated with the decrease of CD86+ pDC%.
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Affiliation(s)
- Wei-Hua Cao
- 1 Department of Hepatology Division 2, Liver Diseases Center, Peking University Ditan Teaching Hospital , Beijing, China
| | - Ming-Hui Li
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Calvin Q Pan
- 3 Division of Gastroenterology and Hepatology, Department of Medicine, NYU Langone Health, New York University School of Medicine , New York, New York
| | - Yao Lu
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Lu Zhang
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Chong-Ping Ran
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Shu-Ling Wu
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Wen-Hao Hua
- 4 Clinical Test Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Shun-Ai Liu
- 5 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Ge Shen
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Min Chang
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Ru-Yu Liu
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Hong-Xiao Hao
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Lei-Ping Hu
- 2 Department of Hepatology Division 2, Liver Diseases Center, Beijing Ditan Hospital, Capital Medical University , Beijing, China
| | - Yao Xie
- 1 Department of Hepatology Division 2, Liver Diseases Center, Peking University Ditan Teaching Hospital , Beijing, China
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Zhu A, Liao X, Li S, Zhao H, Chen L, Xu M, Duan X. HBV cccDNA and Its Potential as a Therapeutic Target. J Clin Transl Hepatol 2019; 7:258-262. [PMID: 31608218 PMCID: PMC6783673 DOI: 10.14218/jcth.2018.00054] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 04/02/2019] [Accepted: 07/10/2019] [Indexed: 12/13/2022] Open
Abstract
Chronic hepatitis B virus infection continues to be a major health burden worldwide. It can cause various degrees of liver damage and is strongly associated with the development of liver cirrhosis and hepatocellular carcinoma. Covalently closed circular DNA in the nucleus of infected cells cannot be disabled by present therapies which may lead to HBV persistence and relapse. In this review, we summarized the current knowledge on hepatitis B virus covalently closed circular DNA and its potential role as a therapeutic target.
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Affiliation(s)
- Anjing Zhu
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Xinzhong Liao
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Shuang Li
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Hang Zhao
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Limin Chen
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Min Xu
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
| | - Xiaoqiong Duan
- Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College, Chengdu, China
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Abstract
Chronic hepatitis B remains a significant cause of morbidity and mortality worldwide. Most hepatitis B virus (HBV)-infected individuals are neither diagnosed nor treated. In those treated, nucleos(t)ide polymerase inhibitors persistently suppress viremia to the limits of quantitation; however, few achieve a "functional cure," defined as sustained off-treatment loss of detectable serum HBV DNA with or without loss of hepatitis B surface antigen. The low cure rate has been attributed to an inability to eliminate the viral reservoir of covalently closed circular DNA from hepatocytes. This review focuses on the diverse therapeutic approaches currently under development that may contribute to the goal of HBV cure.
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Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA. J Virol 2019; 93:JVI.02230-18. [PMID: 30867306 DOI: 10.1128/jvi.02230-18] [Citation(s) in RCA: 53] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Accepted: 03/01/2019] [Indexed: 12/18/2022] Open
Abstract
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
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Zhu W, Liu H, Zhang X. Toward Curative Immunomodulation Strategies for Chronic Hepatitis B Virus Infection. ACS Infect Dis 2019; 5:703-712. [PMID: 30907080 DOI: 10.1021/acsinfecdis.8b00297] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Chronic hepatitis B virus (HBV) infection remains a major cause of morbidity and mortality worldwide. HBV surface antigen loss is considered a functional cure and is an ideal goal for antiviral therapy. However, current treatment regimens, including nucleos(t)ide analogues or interferons monotherapy and combination therapy, rarely achieve this goal in chronic hepatitis B patients. Nucleos(t)ide analogues (NAs), as well as many direct antiviral drugs in ongoing development, are able to inhibit HBV replication and gene expression, but it is hard to achieve immune control and prevent recurrence after therapy cessation. Host immunity, especially HBV-specific T cell response, is proven to play a critical role in control or clearance of HBV infection. Considering HBV chronically infected patients display varying degrees of dysfunction regarding their immune system, novel approaches to enhancing antiviral immune responses are necessary in order to combine with current antiviral agents. In this Review, we focus on the role of innate and adaptive immune responses in HBV immunopathogenesis and discuss attractive strategies or drugs that aim to activate or rebuild antiviral immunity to achieve the goal of an HBV functional cure.
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Affiliation(s)
- Wei Zhu
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838, North Guangzhou Avenue, Guangzhou, Guangdong 510515, China
| | - Hongyan Liu
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838, North Guangzhou Avenue, Guangzhou, Guangdong 510515, China
| | - Xiaoyong Zhang
- State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838, North Guangzhou Avenue, Guangzhou, Guangdong 510515, China
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