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1
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Zhang X, Yue L, Li C, Yuan Z. A Fluorescent In Situ Hybridization (FISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Cell Culture. Methods Mol Biol 2024; 2837:125-135. [PMID: 39044080 DOI: 10.1007/978-1-0716-4027-2_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/25/2024]
Abstract
Hepatitis B virus (HBV) is undoubtedly a master in exploiting host resources while evading host defense for its multiplication within a constrained genetic coding capacity. To further unravel these cunning strategies, a clear picture of virus-host interaction with key subcellular and molecular contexts is needed. Here, we describe a FISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in cell culture models (e.g., HepAD38, HepG2-NTCP). It can be coupled with immunofluorescence staining of viral or host proteins or other fluorescent tagging systems which could illuminate numerous aspects of virus-host interactions.
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Affiliation(s)
- Xiaonan Zhang
- Centre for Research in Therapeutic Solutions, Biomedical Sciences, Faculty of Science and Technology, University of Canberra, Canberra, ACT, Australia.
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.
| | - Lei Yue
- Key Laboratory of Medical Molecular Virology (MOE & NHC), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Chang Li
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology (MOE & NHC), Research Unit of Cure of Chronic Hepatitis B Virus Infection (CAMS), Shanghai Frontiers Science Center of Pathogenic Microorganisms and Infection, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China.
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2
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Li Y, Yang Y, Li T, Wang Z, Gao C, Deng R, Ma F, Li X, Ma L, Tian R, Li H, Zhu H, Zeng L, Gao Y, Lv G, Niu J, Crispe IN, Tu Z. Activation of AIM2 by hepatitis B virus results in antiviral immunity that suppresses hepatitis C virus during coinfection. J Virol 2023; 97:e0109023. [PMID: 37787533 PMCID: PMC10617567 DOI: 10.1128/jvi.01090-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Accepted: 08/28/2023] [Indexed: 10/04/2023] Open
Abstract
IMPORTANCE Clinical data suggest that Hepatitis C virus (HCV) levels are generally lower in Hepatitis B virus (HBV) co-infected patients, but the mechanism is unknown. Here, we show that HBV, but not HCV, activated absent in melanoma-2. This in turn results in inflammasome-mediated cleavage of pro-IL-18, leading to an innate immune activation cascade that results in increased interferon-γ, suppressing both viruses.
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Affiliation(s)
- Yongqi Li
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Yang Yang
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Tianyang Li
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Zhengmin Wang
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Chunfeng Gao
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Rilin Deng
- Hunan Provincial Key Laboratory of Medical Virology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Pathogen Biology and Immunology of College of Biology, Hunan University, Changsha, Hunan, China
| | - Faxiang Ma
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Xinyang Li
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Licong Ma
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Renyun Tian
- Hunan Provincial Key Laboratory of Medical Virology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Pathogen Biology and Immunology of College of Biology, Hunan University, Changsha, Hunan, China
| | - Huiyi Li
- Hunan Provincial Key Laboratory of Medical Virology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Pathogen Biology and Immunology of College of Biology, Hunan University, Changsha, Hunan, China
| | - Haizhen Zhu
- Hunan Provincial Key Laboratory of Medical Virology, State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Pathogen Biology and Immunology of College of Biology, Hunan University, Changsha, Hunan, China
| | - Lei Zeng
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
| | - Yanhang Gao
- Institute of Liver Diseases, The First Hospital of Jilin University, Changchun , Jilin, China
| | - Guoyue Lv
- Institute of Liver Diseases, The First Hospital of Jilin University, Changchun , Jilin, China
| | - Junqi Niu
- Institute of Liver Diseases, The First Hospital of Jilin University, Changchun , Jilin, China
| | - Ian Nicholas Crispe
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA
| | - Zhengkun Tu
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun, Jilin, China
- Institute of Liver Diseases, The First Hospital of Jilin University, Changchun , Jilin, China
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3
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Xiang J, Fan C, Dong H, Ma Y, Xu P. A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1. eLife 2023; 12:e85412. [PMID: 37702383 PMCID: PMC10522339 DOI: 10.7554/elife.85412] [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: 12/06/2022] [Accepted: 09/12/2023] [Indexed: 09/14/2023] Open
Abstract
The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an 'Escaping' effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for 'Escaping'. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.
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Affiliation(s)
- Juan Xiang
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Chaoyang Fan
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Hongchang Dong
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Yilei Ma
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
| | - Pei Xu
- The Centre for Infection and Immunity Studies, School of Medicine, Shenzhen Campus of Sun Yat-sen Univeristy, Sun Yat-sen UniversityShenzhenChina
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4
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Illuminating the Live-Cell Dynamics of Hepatitis B Virus Covalently Closed Circular DNA Using the CRISPR-Tag System. mBio 2023; 14:e0355022. [PMID: 36840581 PMCID: PMC10128046 DOI: 10.1128/mbio.03550-22] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023] Open
Abstract
The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major obstacle to curing chronic hepatitis B (CHB). Current cccDNA detection methods are mostly based on biochemical extraction and bulk measurements. They nevertheless generated a general sketch of its biological features. However, an understanding of the spatiotemporal features of cccDNA is still lacking. To achieve this, we established a system combining CRISPR-Tag and recombinant HBV minicircle technology to visualize cccDNA at single-cell level in real time. Using this system, we found that the observed recombinant cccDNA (rcccDNA) correlated quantitatively with its active transcripts when a low to medium number of foci (<20) are present, but this correlation was lost in cells harboring high copy numbers (≥20) of rcccDNA. The disruption of HBx expression seems to displace cccDNA from the dCas9-accessible region, while HBx complementation restored the number of observable cccDNA foci. This indicated regulation of cccDNA accessibility by HBx. Second, observable HBV and duck HBV (DHBV) cccDNA molecules are substantially lost during cell division, and the remaining ones were distributed randomly to daughter cells. In contrast, Kaposi's sarcoma-associated herpesvirus (KSHV)-derived episomes can be retained in a LANA (latency-associated nuclear antigen)-dependent manner. Last, the dynamics of rcccDNA episomes in nuclei displayed confined diffusion at short time scales, with directional transport over longer time scales. In conclusion, this system enables the study of physiological kinetics of cccDNA at the single-cell level. The differential accessibility of rcccDNA to dCas9 under various physiological conditions may be exploited to elucidate the complex transcriptional and epigenetic regulation of the HBV minichromosome. IMPORTANCE Understanding the formation and maintenance of HBV cccDNA has always been a central issue in the study of HBV pathobiology. However, little progress has been made due to the lack of robust assay systems and its resistance to genetic modification. Here, a live-cell imaging system by grafting CRISPR-Tag into the recombinant cccDNA was established to visualize its molecular behavior in real time. We found that the accessibility of rcccDNA to dCas9-based imaging is related to HBx-regulated mechanisms. We also confirmed the substantial loss of observable rcccDNA in one-round cell division and random distribution of the remaining molecules. Molecular dynamics analysis revealed the confined movement of the rcccDNA episome, suggesting its juxtaposition to chromatin domains. Overall, this novel system offers a unique platform to investigate the intranuclear dynamics of cccDNA within live cells.
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5
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Sun F, Xia W, Ouyang Y. Research progress on detection methods for hepatitis B virus covalently closed circular DNA. J Viral Hepat 2023; 30:366-373. [PMID: 36751941 DOI: 10.1111/jvh.13817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Accepted: 02/04/2023] [Indexed: 02/09/2023]
Abstract
Hepatitis B virus (HBV) infection remains a serious global public health problem, and HBV covalently closed circular DNA (cccDNA) in the nucleus of infected cells cannot be eliminated by current treatments and is a major factor in the persistence and recurrence of hepatitis B. Efficient and scientific detection methods are important for clinical monitoring of cccDNA and targeted drug development. Western blotting is the gold standard for the quantitative detection of cccDNA, but it is time-consuming and complex. In recent years, new detection technologies have been continuously updated. There are new developments and breakthroughs in both next-generation polymerase chain reaction (PCR) and non-PCR methods such as in situ hybridization. Some HBV-related markers (such as hepatitis B core-related antigen) have also been shown to be closely related to cccDNA, and they can be used as surrogate markers to indirectly reflect cccDNA content. In this paper, the main detection methods of cccDNA and their improvements are reviewed, the advantages and limitations of these methods are analysed and summarized, and future development directions are proposed.
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Affiliation(s)
- Fenglan Sun
- Department of Laboratory Medicine, Jingzhou Hospital Affiliated to Yangtze University, Jingzhou, China
| | - Wei Xia
- Department of Laboratory Medicine, Jingzhou Hospital Affiliated to Yangtze University, Jingzhou, China
| | - Yaoling Ouyang
- Department of Laboratory Medicine, Jingzhou Hospital Affiliated to Yangtze University, Jingzhou, China
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6
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Prange R. Hepatitis B virus movement through the hepatocyte: An update. Biol Cell 2022; 114:325-348. [PMID: 35984727 DOI: 10.1111/boc.202200060] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Revised: 07/26/2022] [Accepted: 08/12/2022] [Indexed: 11/29/2022]
Abstract
Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3 kb in size is one of the smallest viral genomes known. HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic shuttling transport, secretory and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture-based, HBV in vitro -replication and in vitro -infection systems. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Reinhild Prange
- Department of Virology, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz, Mainz, D-55131, Germany
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7
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Skrlec I, Talapko J. Hepatitis B and circadian rhythm of the liver. World J Gastroenterol 2022; 28:3282-3296. [PMID: 36158265 PMCID: PMC9346465 DOI: 10.3748/wjg.v28.i27.3282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 03/15/2022] [Accepted: 06/13/2022] [Indexed: 02/06/2023] Open
Abstract
The circadian rhythm in humans is determined by the central clock located in the hypothalamus's suprachiasmatic nucleus, and it synchronizes the peripheral clocks in other tissues. Circadian clock genes and clock-controlled genes exist in almost all cell types. They have an essential role in many physiological processes, including lipid metabolism in the liver, regulation of the immune system, and the severity of infections. In addition, circadian rhythm genes can stimulate the immune response of host cells to virus infection. Hepatitis B virus (HBV) infection is the leading cause of liver disease and liver cancer globally. HBV infection depends on the host cell, and hepatocyte circadian rhythm genes are associated with HBV replication, survival, and spread. The core circadian rhythm proteins, REV-ERB and brain and muscle ARNTL-like protein 1, have a crucial role in HBV replication in hepatocytes. In addition to influencing the virus's life cycle, the circadian rhythm also affects the pharmacokinetics and efficacy of antiviral vaccines. Therefore, it is vital to apply antiviral therapy at the appropriate time of day to reduce toxicity and improve the effectiveness of antiviral treatment. For these reasons, understanding the role of the circadian rhythm in the regulation of HBV infection and host responses to the virus provides us with a new perspective of the interplay of the circadian rhythm and anti-HBV therapy. Therefore, this review emphasizes the importance of the circadian rhythm in HBV infection and the optimization of antiviral treatment based on the circadian rhythm-dependent immune response.
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Affiliation(s)
- Ivana Skrlec
- Department of Biophysics, Biology, and Chemistry, Faculty of Dental Medicine and Health, J. J. Strossmayer University of Osijek, Osijek 31000, Croatia
| | - Jasminka Talapko
- Department of Anatomy Histology, Embryology, Pathology Anatomy and Pathology Histology, Faculty of Dental Medicine and Health, Osijek 31000, Croatia
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8
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Su PY, Yen SCB, Yang CC, Chang CH, Lin WC, Shih C. Hepatitis B virus virion secretion is a CRM1-spike-mediated late event. J Biomed Sci 2022; 29:44. [PMID: 35729569 PMCID: PMC9210616 DOI: 10.1186/s12929-022-00827-w] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Accepted: 06/16/2022] [Indexed: 11/10/2022] Open
Abstract
Background Hepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treatment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that nuclear export of HBc particles can be facilitated via two CRM1-specific nuclear export signals (NES) at the spike tip. Methods In this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibitors at a low concentration, or CRM1-specific shRNA knockdown, in HBV-producing cell culture, and measured the secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV particles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined by proximity ligation assay, immunofluorescence microscopy, and nocodazole treatment. Results We report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in a CRM1-dependent manner. At the conformationally flexible spike tips of HBc particles, NES motifs overlap extensively with motifs important for secretion of HBV virions and naked capsids. Conclusions We provided experimental evidence that virions and naked capsids can egress via two distinct, yet overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubule-dependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the first report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for chronic HBV patients in clinical medicine. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-022-00827-w.
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Affiliation(s)
- Pei-Yi Su
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, No.100, Shih-Chuan 1st Road, Sanmin, 80708, Kaohsiung, Taiwan.,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Shin-Chwen Bruce Yen
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, No.100, Shih-Chuan 1st Road, Sanmin, 80708, Kaohsiung, Taiwan.,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Ching-Chun Yang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Chih-Hsu Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Wen-Chang Lin
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Chiaho Shih
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, No.100, Shih-Chuan 1st Road, Sanmin, 80708, Kaohsiung, Taiwan. .,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
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9
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Bustamante-Jaramillo LF, Fingal J, Blondot ML, Rydell GE, Kann M. Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges. Viruses 2022; 14:v14030557. [PMID: 35336964 PMCID: PMC8950347 DOI: 10.3390/v14030557] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Revised: 01/30/2022] [Accepted: 03/01/2022] [Indexed: 11/16/2022] Open
Abstract
Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 1010 virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research.
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Affiliation(s)
- Luisa F. Bustamante-Jaramillo
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden; (L.F.B.-J.); (J.F.); (G.E.R.)
| | - Joshua Fingal
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden; (L.F.B.-J.); (J.F.); (G.E.R.)
| | - Marie-Lise Blondot
- Microbiologie Fondamentale et Pathogénicité (MFP), CNRS UMR 5234, University of Bordeaux, 33076 Bordeaux, France;
| | - Gustaf E. Rydell
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden; (L.F.B.-J.); (J.F.); (G.E.R.)
| | - Michael Kann
- Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 405 30 Gothenburg, Sweden; (L.F.B.-J.); (J.F.); (G.E.R.)
- Region Västra Götaland, Department of Clinical Microbiology, Sahlgrenska University Hospital, 405 30 Gothenburg, Sweden
- Correspondence:
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10
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Zai W, Hu K, Ye J, Ding J, Huang C, Li Y, Fang Z, Wu M, Wang C, Chen J, Yuan Z. Long-Term Hepatitis B Virus Infection Induces Cytopathic Effects in Primary Human Hepatocytes, and Can Be Partially Reversed by Antiviral Therapy. Microbiol Spectr 2022; 10:e0132821. [PMID: 35171034 PMCID: PMC8849052 DOI: 10.1128/spectrum.01328-21] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 01/21/2022] [Indexed: 01/05/2023] Open
Abstract
Chronic infection of hepatitis B virus (HBV) remains a major health burden worldwide. While the immune response has been recognized to play crucial roles in HBV pathogenesis, the direct cytopathic effects of HBV infection and replication on host hepatocytes and the HBV-host interactions are only partially defined due to limited culture systems. Here, based on our recently developed 5 chemical-cultured primary human hepatocytes (5C-PHHs) model that supports long-term HBV infection, we performed multiplexed quantitative analysis of temporal changes of host proteome and transcriptome on PHHs infected by HBV for up to 4 weeks. We showed that metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels during long-term HBV infection, which led to cytopathic effects and could be partially rescued by early, rather than late, nucleot(s)ide analog (NA) administration and could be significantly relieved by blocking viral antigens with RNA interference (RNAi). Overexpression screening of the dysregulated proteins identified a series of host factors that may contribute to pro- or anti-HBV responses of the infected hepatocytes. In conclusion, our results suggest that long-term HBV infection in primary human hepatocytes leads to cytopathic effects through remodeling the proteome and transcriptome and early antiviral treatment may reduce the extent of such effects, indicating a role of virological factors in HBV pathogenesis and a potential benefit of early administration of antiviral treatment. IMPORTANCE Global temporal quantitative proteomic and transcriptomic analysis using long-term hepatitis B virus (HBV)-infected primary human hepatocytes uncovered extensive remodeling of the host proteome and transcriptome and revealed cytopathic effects of long-term viral replication. Metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels, which could be partially rescued by early, rather than late, NA therapy and could be relieved by blocking viral antigens with RNAi. Overexpression screening identified a series of pro- or anti-HBV host factors. These data have deepened the understanding of the mechanisms of viral pathogenesis and HBV-host interactions in hepatocytes, with implications for therapeutic intervention.
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Affiliation(s)
- Wenjing Zai
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Kongying Hu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jianyu Ye
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Jiahui Ding
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Chao Huang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Yaming Li
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
| | - Zhong Fang
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Min Wu
- Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Cong Wang
- Shanghai Public Health Clinical Center, 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
| | - Zhenghong Yuan
- 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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