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1
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Hsieh CL, Chang LY, Chen PJ, Yeh SH. HBV polymerase recruits the phosphatase PP1 to dephosphorylate HBc-Ser170 to complete encapsidation. PLoS Pathog 2025; 21:e1012905. [PMID: 39932960 PMCID: PMC11813143 DOI: 10.1371/journal.ppat.1012905] [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: 06/18/2024] [Accepted: 01/14/2025] [Indexed: 02/13/2025] Open
Abstract
The HBV core (HBc) protein contains an N-terminal domain (NTD) for capsid assembly and an arginine-rich C-terminal domain (CTD) for pregenomic RNA (pgRNA) encapsidation. Phosphorylation of the HBc CTD, especially at Ser162 and Ser170, is essential for nucleation with the polymerase (Pol) to initiate pgRNA encapsidation. As capsids mature, the HBc CTD undergoes dephosphorylation, suggesting the involvement of a phosphatase in the late stage of encapsidation, which remains to be determined. Using a C-S170 antibody specific for non-phosphorylated HBc-Ser170, we observed a transition from a phosphorylated to a dephosphorylated state during pgRNA packaging. The Pol-dependent dephosphorylation of HBc-Ser170 was confirmed by the substitution of one single amino acid at Val782 in the RNase H domain, which abolished the dephosphorylation of HBc-Ser170. Immunoprecipitation, mass spectrometry analyses, and the protein structural analyses showed that the recruitment of the host phosphatase PP1 is dependent on the Pol-Val782 domain. This recruitment does not require HBc but does require Pol via epsilon RNA signal, suggesting that the Pol-pgRNA complex plays a key role in PP1 recruitment. Pol-pgRNA-PP1-mediated dephosphorylation of HBc-Ser170 is essential for the completion of pgRNA encapsidation and appears to be associated with late endosomes/multivesicular bodies (MVBs). Therefore, HBV Pol may play a dual role by initially bringing pgRNA to phosphorylated HBc and recruiting PP1 for later completion of RNA packaging into the capsids. These findings not only decipher the mechanism by which Pol-mediated dephosphorylation of HBc regulates pgRNA encapsulation, but also reveal the possibility of PP1 as a potential target for antiviral development.
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Affiliation(s)
- Chi-Ling Hsieh
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Li-Yang Chang
- Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Pei-Jer Chen
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
- Center of Precision Medicine, National Taiwan University, Taipei, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Shiou-Hwei Yeh
- Graduate Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan
- Center of Precision Medicine, National Taiwan University, Taipei, Taiwan
- Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan
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2
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Morita C, Wada M, Ohsaki E, Kimura-Ohba S, Ueda K. Generation of Replication-Competent Hepatitis B Virus Harboring Tagged Polymerase for Visualization and Quantification of the Infection. Microbiol Immunol 2025; 69:43-58. [PMID: 39620377 DOI: 10.1111/1348-0421.13183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2024] [Revised: 11/03/2024] [Accepted: 11/04/2024] [Indexed: 01/07/2025]
Abstract
Hepatitis B virus (HBV) infection is a serious global health problem causing acute and chronic hepatitis and related diseases. Approximately, 296 million patients have been chronically infected with the virus, leading to cirrhosis and hepatocellular carcinoma. Although HBV polymerase (HBVpol, pol) plays a pivotal role in HBV replication and must be a definite therapeutic target. The problems are that the detailed functions and intracellular dynamics of HBVpol remain unclear. Here, we constructed two kinds of tagged HBVpol, PA-tagged and HiBiT-tagged pol, and the HBV-producing vectors. Each PA tag and HiBiT tag were inserted into N-terminus of spacer region on HBVpol open reading frame. Transfection of the plasmids into HepG2 cells led to production of HBV. These tagged HBVpol were detectable in HBV replicating cells and pol-HiBiT enabled quantitative analysis. Furthermore, these recombinant HBV were infectious to primary human hepatocytes. Thus, we successfully designed infectious and replication-competent recombinant HBV harboring detectable tagged HBVpol. Such infectious recombinant HBV will provide a novel tool to study HBVpol dynamics and develop new therapeutics against HBV.
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Grants
- This research was supported by Grants from the Japan Agency for Medical Research and Development (AMED) Grants (16fk0310504h0005, 17fk0310105h0001, 18fk0310105h0002, 19fk0310105h0003, 20fk0310105h0004, 21fk0310105h005, 22fk0310505h0001, 23fk0310505h0002, and 24fk0310505h003) to K.U. and from JST SPRING, Grant Number JPMJSP2138, to C.M. and from the Osaka University Transdisciplinary Program for Biomedical Entrepreneurship and Innovation (WISE program) to C.M.
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Affiliation(s)
- Chiharu Morita
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Masami Wada
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
- Center for Infectious Disease Education and Research (CiDER), Osaka, Japan
| | - Shihoko Kimura-Ohba
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan
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3
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Yasutake Y, Hattori SI, Kumamoto H, Tamura N, Maeda K, Mitsuya H. Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations. Sci Rep 2024; 14:15742. [PMID: 38977798 PMCID: PMC11231328 DOI: 10.1038/s41598-024-66505-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Accepted: 07/02/2024] [Indexed: 07/10/2024] Open
Abstract
While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4'-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT's dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4'-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.
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Affiliation(s)
- Yoshiaki Yasutake
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, 062-8517, Japan.
- Computational Bio Big-Data Open Innovation Laboratory (CBBD-OIL), AIST, Tokyo, 169-8555, Japan.
| | - Shin-Ichiro Hattori
- National Center for Global Health and Medicine (NCGM) Research Institute, Tokyo, 162-8655, Japan
| | - Hiroki Kumamoto
- Department of Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama, 362-0806, Japan
| | - Noriko Tamura
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, 062-8517, Japan
| | - Kenji Maeda
- National Center for Global Health and Medicine (NCGM) Research Institute, Tokyo, 162-8655, Japan
- Division of Antiviral Therapy, Joint Research Center for Human Retrovirus Infection, Kagoshima University, Kagoshima, 890-8544, Japan
| | - Hiroaki Mitsuya
- National Center for Global Health and Medicine (NCGM) Research Institute, Tokyo, 162-8655, Japan.
- Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
- Department of Clinical Sciences, Kumamoto University Hospital, Kumamoto, 860-8556, Japan.
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4
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Su PY(A, Chang CH, Yen SCB, Wu HY, Tung WJ, Hu YP, Chen YYI, Lin MH, Shih C, Chen PJ, Tsai K. Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production. Proc Natl Acad Sci U S A 2024; 121:e2400378121. [PMID: 38830096 PMCID: PMC11181118 DOI: 10.1073/pnas.2400378121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 04/20/2024] [Indexed: 06/05/2024] Open
Abstract
Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.
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Affiliation(s)
- Pei-Yi (Alma) Su
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Chih-Hsu Chang
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Shin-Chwen Bruce Yen
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
- Taiwan International Graduate Program, National Yang-Ming Chiao-Tung University and Academia Sinica, Taipei115, Taiwan
| | - Hsiu-Yi Wu
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Wan-Ju Tung
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
| | - Yu-Pei Hu
- Institute of Biomedical Sciences Summer Undergraduate Internship Program, Academia Sinica, Taipei115, Taiwan
| | - Yen-Yu Ian Chen
- Institute of Biomedical Sciences Summer Undergraduate Internship Program, Academia Sinica, Taipei115, Taiwan
| | - Miao-Hsia Lin
- Department of Microbiology, National Taiwan University College of Medicine, Taipei100, Taiwan
| | - Chiaho Shih
- Graduate Institute of Cell Biology, College of Life Sciences, China Medical University, Taichung404, Taiwan
| | - Pei-Jer Chen
- National Taiwan University Center for Genomic Medicine, National Taiwan University, Taipei100, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei100, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital, Taipei100, Taiwan
| | - Kevin Tsai
- Institute of Biomedical Sciences, Academia Sinica, Taipei115, Taiwan
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5
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Deng W, Chen F, Zhao Y, Zhou M, Guo M. Anti-hepatitis B virus activities of natural products and their antiviral mechanisms. Chin J Nat Med 2023; 21:803-811. [PMID: 38035936 DOI: 10.1016/s1875-5364(23)60505-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Indexed: 12/02/2023]
Abstract
Chronic hepatitis B (CHB) infections caused by the hepatitis B virus (HBV) continue to pose a significant global public health challenge. Currently, the approved treatments for CHB are limited to interferon and nucleos(t)ide analogs, both of which have their limitations, and achieving a complete cure remains an elusive goal. Therefore, the identification of new therapeutic targets and the development of novel antiviral strategies are of utmost importance. Natural products (NPs) constitute a class of substances known for their diverse chemical structures, wide-ranging biological activities, and low toxicity profiles. They have shown promise as potential candidates for combating various diseases, with a substantial number demonstrating anti-HBV properties. This comprehensive review focuses on the current applications of NPs in the fight against HBV and provides a summary of their antiviral mechanisms, considering their impact on the viral life cycle and host hepatocytes. By offering insights into the world of anti-HBV NPs, this review aims to furnish valuable information to support the future development of antiviral drugs.
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Affiliation(s)
- Wanyu Deng
- College of Life Science, Shangrao Normal University, Shangrao 334001, China
| | - Fu Chen
- College of Life Science, Shangrao Normal University, Shangrao 334001, China
| | - Yue Zhao
- State Key Laboratory of Natural Medicines, School of Life Science&Technology, China Pharmaceutical University, Nanjing 211198, China
| | - Ming Zhou
- BGI-Shenzhen, Shenzhen 518000, China; Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518110, China; Liver-biotechnology (Shenzhen) Co., Ltd., Shenzhen 518110, China.
| | - Min Guo
- State Key Laboratory of Natural Medicines, School of Life Science&Technology, China Pharmaceutical University, Nanjing 211198, China.
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6
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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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7
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Ranga A, Gupta A, Yadav L, Kumar S, Jain P. Advancing beyond reverse transcriptase inhibitors: The new era of hepatitis B polymerase inhibitors. Eur J Med Chem 2023; 257:115455. [PMID: 37216809 DOI: 10.1016/j.ejmech.2023.115455] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2023] [Revised: 05/03/2023] [Accepted: 05/03/2023] [Indexed: 05/24/2023]
Abstract
Hepatitis B virus (HBV) is a genetically diverse blood-borne virus responsible for chronic hepatitis B. The HBV polymerase plays a key role in viral genome replication within the human body and has been identified as a potential drug target for chronic hepatitis B therapeutics. However, available nucleotide reverse transcriptase inhibitors only target the reverse transcriptase domain of the HBV polymerase; they also pose resistance issues and require lifelong treatment that can burden patients financially. In this study, various chemical classes are reviewed that have been developed to target different domains of the HBV polymerase: Terminal protein, which plays a vital role in the formation of the viral DNA; Reverse transcriptase, which is responsible for the synthesis of the viral DNA from RNA, and; Ribonuclease H, which is responsible for degrading the RNA strand in the RNA-DNA duplex formed during the reverse transcription process. Host factors that interact with the HBV polymerase to achieve HBV replication are also reviewed; these host factors can be targeted by inhibitors to indirectly inhibit polymerase functionality. A detailed analysis of the scope and limitations of these inhibitors from a medicinal chemistry perspective is provided. The structure-activity relationship of these inhibitors and the factors that may affect their potency and selectivity are also examined. This analysis will be useful in supporting the further development of these inhibitors and in designing new inhibitors that can inhibit HBV replication more efficiently.
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Affiliation(s)
- Abhishek Ranga
- Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research, Delhi Pharmaceutical Sciences and Research University, Pushp Vihar, MB Road, New Delhi, 110017, India
| | - Aarti Gupta
- Department of Pharmaceutical Biotechnology, Delhi Pharmaceutical Sciences and Research University, Pushp Vihar, MB Road, New Delhi, 110017, India
| | - Laxmi Yadav
- Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research, Delhi Pharmaceutical Sciences and Research University, Pushp Vihar, MB Road, New Delhi, 110017, India
| | - Sachin Kumar
- Department of Pharmacology, Delhi Institute of Pharmaceutical Sciences and Research, Delhi Pharmaceutical Sciences and Research University, Pushp Vihar, MB Road, New Delhi, 110017, India.
| | - Priti Jain
- Department of Pharmaceutical Chemistry, Delhi Pharmaceutical Sciences and Research University, Pushp Vihar, MB Road, New Delhi, 110017, India.
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8
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Olenginski LT, Kasprzak WK, Attionu SK, Shapiro BA, Dayie TK. Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target. Molecules 2023; 28:1803. [PMID: 36838792 PMCID: PMC9963113 DOI: 10.3390/molecules28041803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/07/2023] [Accepted: 02/11/2023] [Indexed: 02/17/2023] Open
Abstract
The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.
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Affiliation(s)
- Lukasz T. Olenginski
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Wojciech K. Kasprzak
- Bioinformatics and Computational Science Program, Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
| | - Solomon K. Attionu
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
| | - Bruce A. Shapiro
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA
| | - Theodore K. Dayie
- Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA
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9
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Kar A, Samanta A, Mukherjee S, Barik S, Biswas A. The HBV web: An insight into molecular interactomes between the hepatitis B virus and its host en route to hepatocellular carcinoma. J Med Virol 2023; 95:e28436. [PMID: 36573429 DOI: 10.1002/jmv.28436] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2022] [Revised: 11/26/2022] [Accepted: 12/23/2022] [Indexed: 12/28/2022]
Abstract
Hepatitis B virus (HBV) is a major aetiology associated with the development and progression of hepatocellular carcinoma (HCC), the most common primary liver malignancy. Over the past few decades, direct and indirect mechanisms have been identified in the pathogenesis of HBV-associated HCC which include altered signaling pathways, genome integration, mutation-induced genomic instability, chromosomal deletions and rearrangements. Intertwining of the HBV counterparts with the host cellular factors, though well established, needs to be systemized to understand the dynamics of host-HBV crosstalk and its consequences on HCC progression. Existence of a vast array of protein-protein and protein-nucleic acid interaction databases has led to the uncoiling of the compendia of genes/gene products associated with these interactions. This review covers the existing knowledge about the HBV-host interplay and brings it down under one canopy emphasizing on the HBV-host interactomics; and thereby highlights new strategies for therapeutic advancements against HBV-induced HCC.
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Affiliation(s)
- Arpita Kar
- Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
| | - Abhisekh Samanta
- Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
| | - Soumyadeep Mukherjee
- Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
| | - Subhasis Barik
- Department of In Vitro Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
| | - Avik Biswas
- Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
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10
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Olenginski LT, Kasprzak WK, Bergonzo C, Shapiro BA, Dayie TK. Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication. J Mol Biol 2022; 434:167633. [PMID: 35595167 DOI: 10.1016/j.jmb.2022.167633] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Revised: 04/28/2022] [Accepted: 05/07/2022] [Indexed: 12/30/2022]
Abstract
Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5'-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and μs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.
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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
| | - Wojciech K Kasprzak
- Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
| | - Christina Bergonzo
- Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and University of Maryland, Rockville, MD 20850, USA
| | - Bruce A Shapiro
- RNA Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA
| | - Theodore K Dayie
- Center for Biomolecular Structure and Organization, Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
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11
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Dörnbrack K, Beck J, Nassal M. Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro. PLoS Pathog 2022; 18:e1010362. [PMID: 35259189 PMCID: PMC8903280 DOI: 10.1371/journal.ppat.1010362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Accepted: 02/10/2022] [Indexed: 11/18/2022] Open
Abstract
Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P—ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process. Chronic hepatitis B virus (HBV) infection puts >250 million people at an increased risk for severe liver disease. Current treatments can control but rarely cure infection. HBV features a 3,200 bp DNA genome, generated by reverse transcription of a pregenomic (pg) RNA. To initiate DNA synthesis the viral polymerase, P protein, employs a stem-loop on pgRNA, ε, to covalently link a defined first nucleotide to its Terminal Protein (TP) domain. This protein-priming is highly virus-specific yet poorly understood. More is known for duck HBV (DHBV) where, different from HBV, protein-priming was successfully reconstituted in vitro years ago. One insight was that gaining priming-competence involves opening of the apical stem in DHBV ε RNA (Dε); in HBV ε the more extensive basepairing might restrict such dynamics. Here we relaxed these constraints by identifying functional but less stably folded, including split, HBV ε variants. Several such variants supported in vitro priming, including in a simple two-component-system employing a shortened recombinant P protein. Amongst other data the new cell-free systems yielded a first view on a major conformational change in HBV ε RNA bound to P protein, highlighting the importance of RNA dynamics for the human virus. Beyond furthering basic understanding our data should facilitate screening for protein-priming inhibitors as new anti-HBV agents.
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Affiliation(s)
- Katharina Dörnbrack
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
| | - Jürgen Beck
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
- * E-mail: (JB); , (MN)
| | - Michael Nassal
- Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Freiburg, Germany
- * E-mail: (JB); , (MN)
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12
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Abstract
Hepatitis B virus (HBV) is a hepatotropic, partially double-stranded DNA virus that replicates by reverse transcription and is a major cause of chronic liver disease and hepatocellular carcinoma. Reverse transcription is catalyzed by the four-domain multifunctional HBV polymerase (P) protein that has protein-priming, RNA- and DNA-dependent DNA synthesis (i.e., reverse transcriptase), and ribonuclease H activities. P also likely promotes the three strand transfers that occur during reverse transcription, and it may participate in immune evasion by HBV. Reverse transcription is primed by a tyrosine residue in the amino-terminal domain of P, and P remains covalently attached to the product DNA throughout reverse transcription. The reverse transcriptase activity of P is the target for the nucleos(t)ide analog drugs that dominate HBV treatment, and P is the target of ongoing efforts to develop new drugs against both the reverse transcriptase and ribonuclease H activities. Despite the unusual reverse transcription pathway catalyzed by P and the importance of P to HBV therapy, understanding the enzymology and structure of HBV P severely lags that of the retroviral reverse transcriptases due to substantial technical challenges to studying the enzyme. Obtaining a better understanding of P will broaden our appreciation of the diversity among reverse transcribing elements in nature, and will help improve treatment for people chronically infected with HBV.
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Affiliation(s)
- Daniel N Clark
- Department of Microbiology, Weber State University, Ogden, UT, United States
| | - Razia Tajwar
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, United States
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA, United States
| | - John E Tavis
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, United States.
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13
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Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation. J Virol 2021; 96:e0136021. [PMID: 34705558 DOI: 10.1128/jvi.01360-21] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify said host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the co-precipitated proteins by mass spectrometry. The DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported shRNA screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Trans-complementation of wild type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA-binding, DDB1-binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified a handful of host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNAi screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay we discovered a novel relationship between the UV-DDB complex and cccDNA formation, hence, providing a proof-of-concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.
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Wei L, Ploss A. Mechanism of Hepatitis B Virus cccDNA Formation. Viruses 2021; 13:v13081463. [PMID: 34452329 PMCID: PMC8402782 DOI: 10.3390/v13081463] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Revised: 07/14/2021] [Accepted: 07/21/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.
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15
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Identification of hepatitis B virus core protein residues critical for capsid assembly, pgRNA encapsidation and resistance to capsid assembly modulators. Antiviral Res 2021; 191:105080. [PMID: 33933516 DOI: 10.1016/j.antiviral.2021.105080] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 04/23/2021] [Accepted: 04/25/2021] [Indexed: 02/06/2023]
Abstract
Assembly of hepatitis B virus (HBV) capsids is driven by the hydrophobic interaction of core protein (Cp) at dimer-dimer interface. Binding of core protein allosteric modulators (CpAMs) to a hydrophobic "HAP" pocket formed between the inter-dimer interface strengths the dimer-dimer interaction and misdirects the assembly of Cp dimers into non-capsid Cp polymers or morphologically normal capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In this study, we performed a systematic mutagenesis analysis to identify Cp amino acid residues at Cp dimer-dimer interface that are critical for capsid assembly, pgRNA encapsidation and resistance to CpAMs. By analyzing 70 mutant Cp with a single amino acid substitution of 25 amino acid residues around the HAP pocket, our study revealed that residue W102 and Y132 are critical for capsid assembly. However, substitution of many other residues did not significantly alter the amount of capsids, but reduced the amount of encapsidated pgRNA, suggesting their critical roles in pgRNA packaging. Interestingly, several mutant Cp with a single amino acid substitution of residue P25, T33 or I105 supported high levels of DNA replication, but conferred strong resistance to multiple chemotypes of CpAMs. In addition, we also found that WT Cp, but not the assembly incompetent Cp, such as Y132A Cp, interacted with HBV DNA polymerase (Pol). This later finding implies that encapsidation of viral DNA polymerase may depend on the interaction of Pol with a capsid assembly intermediate, but not free Cp dimers. Taking together, our findings reported herein shed new light on the mechanism of HBV nucleocapsid assembly and mode of CpAM action.
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Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription. Proc Natl Acad Sci U S A 2021; 118:2022373118. [PMID: 33753499 DOI: 10.1073/pnas.2022373118] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.
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17
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Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA. J Virol 2020; 95:JVI.00922-20. [PMID: 33055252 DOI: 10.1128/jvi.00922-20] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2020] [Accepted: 10/05/2020] [Indexed: 12/13/2022] Open
Abstract
The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand [(-)strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand [(+)strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.IMPORTANCE The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
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18
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Marchetti AL, Guo H. New Insights on Molecular Mechanism of Hepatitis B Virus Covalently Closed Circular DNA Formation. Cells 2020; 9:cells9112430. [PMID: 33172220 PMCID: PMC7694973 DOI: 10.3390/cells9112430] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Revised: 11/03/2020] [Accepted: 11/04/2020] [Indexed: 12/15/2022] Open
Abstract
The chronic factor of the Hepatitis B Virus (HBV), specifically the covalently closed circular DNA (cccDNA), is a highly stable and active viral episomal genome established in the livers of chronic hepatitis B patients as a constant source of disease. Being able to target and eliminate cccDNA is the end goal for a genuine cure for HBV. Yet how HBV cccDNA is formed from the viral genomic relaxed circular DNA (rcDNA) and by what host factors had been long-standing research questions. It is generally acknowledged that HBV hijacks cellular functions to turn the open circular DNA conformation of rcDNA into cccDNA through DNA repair mechanisms. With great efforts from the HBV research community, there have been several recent leaps in our understanding of cccDNA formation. It is our goal in this review to analyze the recent reports showing evidence of cellular factor's involvement in the molecular pathway of cccDNA biosynthesis.
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Affiliation(s)
- Alexander L. Marchetti
- Department of Microbiology and Immunology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA;
- Cancer Virology Program, Hillman Cancer Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Haitao Guo
- Cancer Virology Program, Hillman Cancer Center, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA
- Correspondence:
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19
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Bak E, Miller JT, Noronha A, Tavis J, Gallicchio E, Murelli RP, Le Grice SFJ. 3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis. Molecules 2020; 25:molecules25194434. [PMID: 32992516 PMCID: PMC7583054 DOI: 10.3390/molecules25194434] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/15/2020] [Accepted: 09/19/2020] [Indexed: 02/07/2023] Open
Abstract
Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5' terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).
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Affiliation(s)
- Ellen Bak
- Basic Research Laboratory National Cancer Institute, Frederick, MD 21702, USA; (E.B.); (J.T.M.); (A.N.)
| | - Jennifer T. Miller
- Basic Research Laboratory National Cancer Institute, Frederick, MD 21702, USA; (E.B.); (J.T.M.); (A.N.)
| | - Andrea Noronha
- Basic Research Laboratory National Cancer Institute, Frederick, MD 21702, USA; (E.B.); (J.T.M.); (A.N.)
| | - John Tavis
- Department of Molecular Microbiology and Immunology, St. Louis University, St. Louis, MO 63104, USA;
| | - Emilio Gallicchio
- Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY 11210, USA; (E.G.); (R.P.M.)
- PhD Program in Chemistry, The Graduate Center of The City University of New York, New York, NY 10016, USA
- PhD Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY 10016, USA
| | - Ryan P. Murelli
- Department of Chemistry, Brooklyn College, The City University of New York, Brooklyn, NY 11210, USA; (E.G.); (R.P.M.)
- PhD Program in Chemistry, The Graduate Center of The City University of New York, New York, NY 10016, USA
- PhD Program in Biochemistry, The Graduate Center of The City University of New York, New York, NY 10016, USA
| | - Stuart F. J. Le Grice
- Basic Research Laboratory National Cancer Institute, Frederick, MD 21702, USA; (E.B.); (J.T.M.); (A.N.)
- Correspondence:
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20
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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: 206] [Impact Index Per Article: 41.2] [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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21
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Abstract
Hepatitis B virus (HBV), which was discovered in 1965, is a threat to global public health. HBV infects human hepatocytes and leads to acute and chronic liver diseases, and there is no cure. In cells infected by HBV, viral DNA can be integrated into the cellular genome. HBV DNA integration is a complicated process during the HBV life cycle. Although HBV integration normally results in replication-incompetent transcripts, it can still act as a template for viral protein expression. Of note, it is a primary driver of hepatocellular carcinoma (HCC). Recently, with the development of detection methods and research models, the molecular biology and the pathogenicity of HBV DNA integration have been better revealed. Here, we review the advances in the research of HBV DNA integration, including molecular mechanisms, detection methods, research models, the effects on host and viral gene expression, the role of HBV integrations in the pathogenesis of HCC, and potential treatment strategies. Finally, we discuss possible future research prospects of HBV DNA integration.
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Affiliation(s)
- Kaitao Zhao
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
| | - Andrew Liu
- Laboratory of Molecular Cardiology, National Heart Lung Blood Institute, National Institutes of Health, Bethesda, MD 20814, USA
| | - Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China
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22
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Xia Y, Guo H. Hepatitis B virus cccDNA: Formation, regulation and therapeutic potential. Antiviral Res 2020; 180:104824. [PMID: 32450266 PMCID: PMC7387223 DOI: 10.1016/j.antiviral.2020.104824] [Citation(s) in RCA: 119] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 05/03/2020] [Accepted: 05/18/2020] [Indexed: 02/06/2023]
Abstract
Hepatitis B virus (HBV) infection remains a major public health concern worldwide with about 257 million individuals chronically infected. Current therapies can effectively control HBV replication and slow down disease progress, but cannot cure HBV infection. Upon infection, HBV establishes a pool of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. The cccDNA exists as a minichromosome and resists to antivirals, thus a therapeutic eradication of cccDNA from the infected cells remains unattainable. In this review, we summarize the state of knowledge on the mechanisms underlying cccDNA formation and regulation, and discuss the possible strategies that may contribute to the eradication of HBV through targeting cccDNA.
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Affiliation(s)
- Yuchen Xia
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, China.
| | - Haitao Guo
- UPMC Hillman Cancer Center, Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA.
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23
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Liu XQ, Ohsaki E, Ueda K. Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase. Genes Cells 2020; 25:523-537. [PMID: 32415897 PMCID: PMC7496097 DOI: 10.1111/gtc.12778] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Revised: 04/14/2020] [Accepted: 05/08/2020] [Indexed: 12/18/2022]
Abstract
Although several nucleo(s)tide analogs are available for treatment of HBV infection, long‐term treatment with these drugs can lead to the emergence of drug‐resistant viruses. Recent HIV‐1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non‐nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI‐resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)‐reverse transcriptase (RT) (TP‐RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP‐RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA‐binding assay system. Then, we used TP‐RT in cell‐free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6‐hydroxy‐DL‐DOPA and N‐oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell‐based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP‐RT domain to treat HBV infection.
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Affiliation(s)
- Xiao-Quan Liu
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Eriko Ohsaki
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Keiji Ueda
- Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Japan
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24
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Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target. Viruses 2020; 12:v12050570. [PMID: 32455999 PMCID: PMC7291194 DOI: 10.3390/v12050570] [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: 04/15/2020] [Revised: 05/17/2020] [Accepted: 05/19/2020] [Indexed: 12/15/2022] Open
Abstract
Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure.
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Toyoda T, Wang Y, Wen Y, Tanaka Y. Fluorescence-based biochemical analysis of human hepatitis B virus reverse transcriptase activity. Anal Biochem 2020; 597:113642. [PMID: 32171777 DOI: 10.1016/j.ab.2020.113642] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 02/04/2020] [Accepted: 02/18/2020] [Indexed: 12/14/2022]
Abstract
Although the unique mechanism by which hepatitis B virus (HBV) polymerase primes reverse transcription is now well-characterized, the subsequent elongation process remains poorly understood. Reverse transcriptase (RT)-RNase H sequences from polymerase amino acid 304 (the C-terminal part of spacer domain) to 843 were expressed in Escherichia coli and purified partially. RT elongation activity was investigated using the fluorescent-tagged primer and homopolymeric RNA templates. RT elongation activity depended on both Mg2+ and Mn2+, and had low affinity for purine deoxynucleotides, which may be related with the success of adefovir, tenofovir, and entecavir. However, the polymerization rate was lower than that of human immunodeficiency virus RT. All HBV genotypes displayed similar RT activity, except for genotype B, which demonstrated increased elongation activity.
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Affiliation(s)
- Tetsuya Toyoda
- Choju Medical Institute, Fukushimura Hospital, 19-14 Azayamanaka, Noyori-Cho, Toyohashi, Aichi, 441-8124, Japan.
| | - Yongxiang Wang
- Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China
| | - Yumei Wen
- Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China
| | - Yasuhito Tanaka
- Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay. J Gastroenterol 2020; 55:441-452. [PMID: 31768802 DOI: 10.1007/s00535-019-01643-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 11/12/2019] [Indexed: 02/04/2023]
Abstract
BACKGROUND Hepatitis B virus (HBV) polymerase is the only virus-encoded enzyme essential for producing the HBV genome and is regarded as an attractive drug target. However, the difficulty of synthesizing and purifying recombinant HBV polymerase protein has hampered the development of new drugs targeting this enzyme, especially compounds unrelated to the nucleoside structure. We recently have developed a technique for the synthesis and purification of recombinant HBV polymerase containing the reverse transcriptase (RT) domain that carried DNA elongation activity in vitro. METHODS We used the overproduced protein to establish an in vitro high-throughput screening system to identify compounds that inhibit the elongation activity of HBV polymerase. RESULTS We screened 1120 compounds and identified a stilbene derivative, piceatannol, as a potential anti-HBV agent. Derivative analysis identified another stilbene derivative, PDM2, that was able to inhibit HBV replication with an IC50 of 14.4 ± 7.7 μM. An infection experiment suggested that the compounds inhibit the replication of HBV rather than the entry process, as expected. Surface plasmon resonance analysis demonstrated a specific interaction between PDM2 and the RT domain. Importantly, PDM2 showed similar inhibitory activity against the replication of both wild-type HBV and a lamivudine/entecavir-resistant HBV variant. Furthermore, PDM2 showed an additive effect in combination with clinically used nucleos(t)ide analogs. CONCLUSIONS We report the development of a screening system that is useful for identifying non-nucleos(t)ide RT inhibitors.
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Abstract
Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth. The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5′ truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production.
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Wang J, Huang H, Liu Y, Chen R, Yan Y, Shi S, Xi J, Zou J, Yu G, Feng X, Lu F. HBV Genome and Life Cycle. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1179:17-37. [PMID: 31741332 DOI: 10.1007/978-981-13-9151-4_2] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.
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Affiliation(s)
- Jie Wang
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Hongxin Huang
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Yongzhen Liu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Ran Chen
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Ying Yan
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Shu Shi
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Jingyuan Xi
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Jun Zou
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Guangxin Yu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Xiaoyu Feng
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China
| | - Fengmin Lu
- Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, P.R. China.
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Tramontano E, Corona A, Menéndez-Arias L. Ribonuclease H, an unexploited target for antiviral intervention against HIV and hepatitis B virus. Antiviral Res 2019; 171:104613. [PMID: 31550450 DOI: 10.1016/j.antiviral.2019.104613] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Revised: 09/18/2019] [Accepted: 09/19/2019] [Indexed: 12/18/2022]
Abstract
Ribonucleases H (RNases H) are endonucleolytic enzymes, evolutionarily related to retroviral integrases, DNA transposases, resolvases and numerous nucleases. RNases H cleave RNA in RNA/DNA hybrids and their activity plays an important role in the replication of prokaryotic and eukaryotic genomes, as well as in the replication of reverse-transcribing viruses. During reverse transcription, the RNase H activity of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) degrades the viral genomic RNA to facilitate the synthesis of viral double-stranded DNA. HIV and HBV reverse transcriptases contain DNA polymerase and RNase H domains that act in a coordinated manner to produce double-stranded viral DNA. Although RNase H inhibitors have not been developed into licensed drugs, recent progress has led to the identification of a number of small molecules with inhibitory activity at low micromolar or even nanomolar concentrations. These compounds can be classified into metal-chelating active site inhibitors and allosteric inhibitors. Among them, α-hydroxytropolones, N-hydroxyisoquinolinediones and N-hydroxypyridinediones represent chemotypes active against both HIV and HBV RNases H. In this review we summarize recent developments in the field including the identification of novel RNase H inhibitors, compounds with dual inhibitory activity, broad specificity and efforts to decrease their toxicity.
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Affiliation(s)
- Enzo Tramontano
- Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.
| | - Angela Corona
- Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy
| | - Luis Menéndez-Arias
- Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid), Madrid, Spain.
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Abstract
With a yearly death toll of 880,000, hepatitis B virus (HBV) remains a major health problem worldwide, despite an effective prophylactic vaccine and well-tolerated, effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long-term antiviral therapy, and its integration, though no longer able to support viral replication, destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome, which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products, and thus, it is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.
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RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element. J Virol 2019; 93:JVI.02161-18. [PMID: 30626666 DOI: 10.1128/jvi.02161-18] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2018] [Accepted: 12/18/2018] [Indexed: 12/14/2022] Open
Abstract
Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.
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Anikhindi SA, Kumar A, Sharma P, Singla V, Bansal N, Arora A. Ideal Cure for Hepatitis B Infection: The Target is in Sight. J Clin Exp Hepatol 2018; 8:188-194. [PMID: 29892183 PMCID: PMC5992304 DOI: 10.1016/j.jceh.2017.10.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Accepted: 10/30/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) is one of the most common causes of liver cirrhosis and hepatocellular carcinoma. Despite recent strides in pharmacotherapy, complete cure of HBV infection still remains an enigma. The biggest obstacle in HBV therapy is clearance of covalently closed circular deoxyribonucleic acid (cccDNA). We discuss about the role of cccDNA in HBV life cycle, efficacy and shortcomings of currently available antivirals as well as promising novel targets to achieve ideal HBV cure.
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Affiliation(s)
- Shrihari A. Anikhindi
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
| | - Ashish Kumar
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
| | - Praveen Sharma
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
| | - Vikas Singla
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
| | - Naresh Bansal
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
| | - Anil Arora
- Department of Gastroenterology, Sir Ganga Ram Hospital, New Delhi, India
- Department of Pathology, Sir Ganga Ram Hospital, New Delhi, India
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Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection. J Virol 2017. [PMID: 28637752 DOI: 10.1128/jvi.00539-17] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3' exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved.IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC DNA to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved.
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Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming. Sci Rep 2017; 7:7120. [PMID: 28769080 PMCID: PMC5541001 DOI: 10.1038/s41598-017-07657-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Accepted: 06/28/2017] [Indexed: 12/12/2022] Open
Abstract
Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.
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35
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Ko C, Michler T, Protzer U. Novel viral and host targets to cure hepatitis B. Curr Opin Virol 2017; 24:38-45. [DOI: 10.1016/j.coviro.2017.03.019] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Revised: 03/06/2017] [Accepted: 03/30/2017] [Indexed: 02/07/2023]
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Affiliation(s)
- Amanda L. Garner
- Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan USA
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37
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Menéndez-Arias L, Sebastián-Martín A, Álvarez M. Viral reverse transcriptases. Virus Res 2017; 234:153-176. [PMID: 28043823 DOI: 10.1016/j.virusres.2016.12.019] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2016] [Revised: 12/19/2016] [Accepted: 12/24/2016] [Indexed: 12/11/2022]
Abstract
Reverse transcriptases (RTs) play a major role in the replication of Retroviridae, Metaviridae, Pseudoviridae, Hepadnaviridae and Caulimoviridae. RTs are enzymes that are able to synthesize DNA using RNA or DNA as templates (DNA polymerase activity), and degrade RNA when forming RNA/DNA hybrids (ribonuclease H activity). In retroviruses and LTR retrotransposons (Metaviridae and Pseudoviridae), the coordinated action of both enzymatic activities converts single-stranded RNA into a double-stranded DNA that is flanked by identical sequences known as long terminal repeats (LTRs). RTs of retroviruses and LTR retrotransposons are active as monomers (e.g. murine leukemia virus RT), homodimers (e.g. Ty3 RT) or heterodimers (e.g. human immunodeficiency virus type 1 (HIV-1) RT). RTs lack proofreading activity and display high intrinsic error rates. Besides, high recombination rates observed in retroviruses are promoted by poor processivity that causes template switching, a hallmark of reverse transcription. HIV-1 RT inhibitors acting on its polymerase activity constitute the backbone of current antiretroviral therapies, although novel drugs, including ribonuclease H inhibitors, are still necessary to fight HIV infections. In Hepadnaviridae and Caulimoviridae, reverse transcription leads to the formation of nicked circular DNAs that will be converted into episomal DNA in the host cell nucleus. Structural and biochemical information on their polymerases is limited, although several drugs inhibiting HIV-1 RT are known to be effective against the human hepatitis B virus polymerase. In this review, we summarize current knowledge on reverse transcription in the five virus families and discuss available biochemical and structural information on RTs, including their biosynthesis, enzymatic activities, and potential inhibition.
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Affiliation(s)
- Luis Menéndez-Arias
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain.
| | - Alba Sebastián-Martín
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain
| | - Mar Álvarez
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Nicolás Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain
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Liu Y, Nie H, Mao R, Mitra B, Cai D, Yan R, Guo JT, Block TM, Mechti N, Guo H. Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA. PLoS Pathog 2017; 13:e1006296. [PMID: 28399146 PMCID: PMC5388505 DOI: 10.1371/journal.ppat.1006296] [Citation(s) in RCA: 103] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 03/15/2017] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general. HBV is a DNA virus but replicates its DNA via retrotranscription of a viral RNA pregenome. ISG20, an antiviral RNase induced by interferons, inhibits the replication of many RNA viruses but the underlying molecular antiviral mechanism remains elusive. Since all the known viruses, except for prions, have RNA products in their life cycles, ISG20 can be a broad spectrum antiviral protein; but in order to distinguish viral RNA from host RNA, ISG20 may have evolved to recognize virus-specific signals as its antiviral target. We demonstrated herein that ISG20 selectively binds to a unique stem-loop structure called epsilon (ε) in all HBV RNA species and degrades viral RNA to inhibit HBV replication. Because ε is the HBV pregenomic RNA packaging signal and reverse transcription priming site, the binding of ISG20 to ε, even in the absence of ribonuclease activity, results in antiviral effect to prevent DNA replication due to preventing viral polymerase binding to pgRNA. We also determined the structure and sequence requirements of ε RNA and ISG20 protein for ISG20-ε binding and antiviral activity. Such information will aid the function study of ISG20 against viral pathogens in host innate defense, and ISG20 has potentials to be developed into a therapeutic agent for viral diseases including hepatitis B.
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Affiliation(s)
- Yuanjie Liu
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Hui Nie
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America
| | - Richeng Mao
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Bidisha Mitra
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Dawei Cai
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Ran Yan
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Timothy M. Block
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Nadir Mechti
- CNRS, UMR5235, DIMNP, University of Montpellier 2, Montpellier, France
| | - Haitao Guo
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America
- * E-mail:
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Chakraborty D, Ghosh S. The epsilon motif of hepatitis B virusRNAexhibits a potassium‐dependent ribonucleolytic activity. FEBS J 2017; 284:1184-1203. [DOI: 10.1111/febs.14050] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2016] [Revised: 01/21/2017] [Accepted: 02/22/2017] [Indexed: 12/01/2022]
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Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase. J Virol 2017; 91:JVI.01785-16. [PMID: 27852858 DOI: 10.1128/jvi.01785-16] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Accepted: 11/12/2016] [Indexed: 01/08/2023] Open
Abstract
Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain. IMPORTANCE HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and high-resolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.
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Clark DN, Jones SA, Hu J. In Vitro Assays for RNA Binding and Protein Priming of Hepatitis B Virus Polymerase. Methods Mol Biol 2017; 1540:157-177. [PMID: 27975315 DOI: 10.1007/978-1-4939-6700-1_13] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
The hepatitis B virus (HBV) polymerase synthesizes the viral DNA genome from the pre-genomic RNA (pgRNA) template through reverse transcription. Initiation of viral DNA synthesis is accomplished via a novel protein priming mechanism, so named because the polymerase itself acts as a primer, whereby the initiating nucleotide becomes covalently linked to a tyrosine residue on the viral polymerase. Protein priming, in turn, depends on specific recognition of the packaging signal on pgRNA called epsilon. These early events in viral DNA synthesis can now be dissected in vitro as described here.The polymerase is expressed in mammalian cells and purified by immunoprecipitation. The purified protein is associated with host cell factors, is enzymatically active, and its priming activity is epsilon dependent. A minimal epsilon RNA construct from pgRNA is co-expressed with the polymerase in cells. This RNA binds to and co-immunoprecipitates with the polymerase. Modifications can be made to either the epsilon RNA or the polymerase protein by manipulating the expression plasmids. Also, the priming reaction itself can be modified to assay for the initiation or subsequent DNA synthesis during protein priming, the susceptibility of the polymerase to chemical inhibitors, and the precise identification of the DNA products upon their release from the polymerase. The identity of associated host factors can also be evaluated. This protocol closely mirrors our current understanding of the RNA binding and protein priming steps of the HBV replication cycle, and it is amenable to modification. It should therefore facilitate both basic research and drug discovery.
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Affiliation(s)
- Daniel N Clark
- Department of Microbiology and Immunology-H107, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA, 17033, USA.
| | - Scott A Jones
- Department of Microbiology and Immunology-H107, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA, 17033, USA
- Nevada Division of Public and Behavioral Health, Primary Care Office, 4150 Technology Way, Suite 104, Carson City, NV, 89706, USA
| | - Jianming Hu
- Department of Microbiology and Immunology-H107, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA, 17033, USA
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Qi Y, Gao Z, Xu G, Peng B, Liu C, Yan H, Yao Q, Sun G, Liu Y, Tang D, Song Z, He W, Sun Y, Guo JT, Li W. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus. PLoS Pathog 2016; 12:e1005893. [PMID: 27783675 PMCID: PMC5081172 DOI: 10.1371/journal.ppat.1005893] [Citation(s) in RCA: 155] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2016] [Accepted: 08/24/2016] [Indexed: 12/11/2022] Open
Abstract
Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. HBV chronically infects 240 million people worldwide. Persistent HBV infection relies on stable maintenance of the nuclear form of viral genome, the covalently closed circular (ccc) DNA. However, the molecular mechanism underlying the conversion of HBV genomic relaxed circular (rc) DNA into cccDNA remains elusive. Our studies reported herein provide unambiguous evidence suggesting that host DNA polymerase κ (POLK) is required for repairing the gap of rcDNA and formation of cccDNA in a de novo HBV infection. POLK is thus a potential therapeutic target for treatment of chronic hepatitis B.
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Affiliation(s)
- Yonghe Qi
- National Institute of Biological Sciences, Beijing, China
| | - Zhenchao Gao
- National Institute of Biological Sciences, Beijing, China
- Graduate program in School of Life Sciences, Peking University, Beijing, China
| | - Guangwei Xu
- National Institute of Biological Sciences, Beijing, China
| | - Bo Peng
- National Institute of Biological Sciences, Beijing, China
- Graduate program in School of Life Sciences, Peking University, Beijing, China
| | - Chenxuan Liu
- National Institute of Biological Sciences, Beijing, China
- College of Life Sciences Beijing Normal University, Beijing, China
| | - Huan Yan
- National Institute of Biological Sciences, Beijing, China
| | - Qiyan Yao
- National Institute of Biological Sciences, Beijing, China
| | - Guoliang Sun
- National Institute of Biological Sciences, Beijing, China
| | - Yang Liu
- National Institute of Biological Sciences, Beijing, China
- School of Life Science, Tsinghua University, Beijing, China
| | - Dingbin Tang
- National Institute of Biological Sciences, Beijing, China
- Graduate program in School of Life Sciences, Peking University, Beijing, China
| | - Zilin Song
- National Institute of Biological Sciences, Beijing, China
| | - Wenhui He
- National Institute of Biological Sciences, Beijing, China
| | - Yinyan Sun
- National Institute of Biological Sciences, Beijing, China
| | - Ju-Tao Guo
- Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America
| | - Wenhui Li
- National Institute of Biological Sciences, Beijing, China
- * E-mail: (WL)
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Hermann T. Small molecules targeting viral RNA. WILEY INTERDISCIPLINARY REVIEWS-RNA 2016; 7:726-743. [PMID: 27307213 PMCID: PMC7169885 DOI: 10.1002/wrna.1373] [Citation(s) in RCA: 112] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Revised: 04/29/2016] [Accepted: 05/23/2016] [Indexed: 02/06/2023]
Abstract
Highly conserved noncoding RNA (ncRNA) elements in viral genomes and transcripts offer new opportunities to expand the repertoire of drug targets for the development of antiinfective therapy. Ligands binding to ncRNA architectures are able to affect interactions, structural stability or conformational changes and thereby block processes essential for viral replication. Proof of concept for targeting functional RNA by small molecule inhibitors has been demonstrated for multiple viruses with RNA genomes. Strategies to identify antiviral compounds as inhibitors of ncRNA are increasingly emphasizing consideration of drug‐like properties of candidate molecules emerging from screening and ligand design. Recent efforts of antiviral lead discovery for RNA targets have provided drug‐like small molecules that inhibit viral replication and include inhibitors of human immunodeficiency virus (HIV), hepatitis C virus (HCV), severe respiratory syndrome coronavirus (SARS CoV), and influenza A virus. While target selectivity remains a challenge for the discovery of useful RNA‐binding compounds, a better understanding is emerging of properties that define RNA targets amenable for inhibition by small molecule ligands. Insight from successful approaches of targeting viral ncRNA in HIV, HCV, SARS CoV, and influenza A will provide a basis for the future exploration of RNA targets for therapeutic intervention in other viral pathogens which create urgent, unmet medical needs. Viruses for which targeting ncRNA components in the genome or transcripts may be promising include insect‐borne flaviviruses (Dengue, Zika, and West Nile) and filoviruses (Ebola and Marburg). WIREs RNA 2016, 7:726–743. doi: 10.1002/wrna.1373 This article is categorized under:
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
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Affiliation(s)
- Thomas Hermann
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. .,Center for Drug Discovery Innovation, University of California, San Diego, La Jolla, CA, USA.
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Huang YH, Tseng YH, Lin WR, Hung G, Chen TC, Wang TH, Lee WC, Yeh CT. HBV polymerase overexpression due to large core gene deletion enhances hepatoma cell growth by binding inhibition of microRNA-100. Oncotarget 2016; 7:9448-9461. [PMID: 26824500 PMCID: PMC4891051 DOI: 10.18632/oncotarget.7021] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2015] [Accepted: 01/17/2016] [Indexed: 01/04/2023] Open
Abstract
Different types of hepatitis B virus (HBV) core gene deletion mutants were identified in chronic hepatitis B patients. However, their clinical roles in different stages of natural chronic HBV infection remained unclear. To address this issue, HBV core genes were sequenced in three gender- and age-matched patient groups diagnosed as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), respectively. Functional analysis of the identified mutants was performed. A novel type of large-fragment core gene deletion (LFCD) was identified exclusively in HCC patients and significantly associated with unfavorable postoperative survival. The presence of LFCDs resulted in generation of precore-polymerase fusion protein or brought the polymerase reading frame under direct control of HBV precore/core promoter, leading to its over-expression. Enhanced cell proliferation and increased tumorigenicity in nude mice were found in hepatoma cells expressing LFCDs. Because of the epsilon-binding ability of HBV polymerase, we hypothesized that the over-expressed polymerase carrying aberrant amino-terminal sequence could bind to cellular microRNAs. Screening of a panel of microRNAs revealed physical association of a precore-polymerase fusion protein with microRNA-100. A binding inhibition effect on microRNA-100 by the precore-polymerase fusion protein with up-regulation of its target, polo-like kinase 1 (PLK1), was discovered. The binding inhibition and growth promoting effects could be reversed by overexpressing microRNA-100. Together, HCC patients carrying hepatitis B large-fragment core gene deletion mutants had an unfavorable postoperative prognosis. The growth promoting effect was partly due to polymerase overexpression, leading to binding inhibition of microRNA-100 and up-regulation of PLK1.
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MESH Headings
- Animals
- Apoptosis/genetics
- Base Sequence
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/virology
- Cell Cycle Proteins/metabolism
- Cell Line, Tumor
- Cell Proliferation/genetics
- Cell Transformation, Neoplastic/genetics
- DNA, Viral/genetics
- Female
- Gene Deletion
- Gene Products, pol/biosynthesis
- Gene Products, pol/genetics
- Hep G2 Cells
- Hepatitis B virus/enzymology
- Hepatitis B virus/genetics
- Hepatitis B, Chronic/virology
- Humans
- Liver Cirrhosis/virology
- Liver Neoplasms/pathology
- Liver Neoplasms/virology
- Male
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- MicroRNAs/antagonists & inhibitors
- MicroRNAs/genetics
- Middle Aged
- Neoplasm Transplantation
- Prognosis
- Protein Binding/genetics
- Protein Serine-Threonine Kinases/metabolism
- Proto-Oncogene Proteins/metabolism
- Sequence Analysis, DNA
- Transplantation, Heterologous
- Polo-Like Kinase 1
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Affiliation(s)
- Ya-Hui Huang
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
| | - Ying-Hsin Tseng
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
| | - Wey-Ran Lin
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
- Department of Hepato-Gastroenterology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - George Hung
- Department of Molecular Biology, Princeton University, NJ, USA
| | - Tse-Ching Chen
- Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Tong-Hong Wang
- Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Wei-Chen Lee
- Division of Liver and Transplantation Surgery, Department of General Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Chau-Ting Yeh
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
- Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
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Clark DN, Hu J. Hepatitis B virus reverse transcriptase - Target of current antiviral therapy and future drug development. Antiviral Res 2015; 123:132-7. [PMID: 26408354 DOI: 10.1016/j.antiviral.2015.09.011] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2015] [Revised: 09/09/2015] [Accepted: 09/21/2015] [Indexed: 02/07/2023]
Abstract
Hepatitis B virus (HBV) infections rely on the proper functioning of the viral polymerase enzyme, a specialized reverse transcriptase (RT) with multiple activities. All currently approved antiviral drugs for the treatment of chronic HBV infection, except for interferon, target the RT and belong to the same chemical class - they are all nucleoside analogs. Viral DNA synthesis is carried out by the RT enzyme in several different steps, each with distinct RT conformational requirements. In principle, each stage may be targeted by distinct antiviral drugs. In particular, the HBV RT has the unique ability to initiate viral DNA synthesis using itself as a protein primer in a novel protein priming reaction. In order to help identify RT inhibitors and study their mechanisms of action, a number of experimental systems have been developed, each varying in its ability to dissect the protein priming stage and subsequent stages of viral DNA synthesis at the molecular level. Two of the most effective drugs to date, entecavir and tenofovir, can inhibit both the protein priming and the subsequent DNA elongation stages of HBV DNA synthesis. Interestingly, clevudine, a thymidine analog, can inhibit both protein priming and DNA elongation in a non-competitive manner and without being incorporated into the viral DNA. Thus, a nucleoside RT inhibitor (NRTI) can functionally mimic a non-NRTI (NNRTI) in its inhibition of the HBV RT. Therefore, novel NRTIs as well as NNRTIs may be developed to inhibit the DNA synthesis activity of the HBV RT. Furthermore, additional activities of the RT that are also essential to HBV replication, including specific recognition of the viral RNA and its packaging into viral nucleocapsids, may be exploited for antiviral development. To achieve a more potent inhibition of viral replication and ultimately cure chronic HBV infection, the next generation of anti-HBV therapies will likely need to include NRTIs, NNRTIs, and other agents that target the viral RT as well as other viral and host factors in various combinations. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."
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Affiliation(s)
- Daniel N Clark
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States.
| | - Jianming Hu
- Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States
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Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation. J Virol 2015. [PMID: 26202253 DOI: 10.1128/jvi.01481-15] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. IMPORTANCE Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.
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Hu J, Seeger C. Hepadnavirus Genome Replication and Persistence. Cold Spring Harb Perspect Med 2015; 5:a021386. [PMID: 26134841 DOI: 10.1101/cshperspect.a021386] [Citation(s) in RCA: 104] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Hallmarks of the hepadnavirus replication cycle are the formation of covalently closed circular DNA (cccDNA) and the reverse transcription of a pregenomic RNA (pgRNA) in core particles leading to synthesis of the relaxed circular DNA (rcDNA) genome. cccDNA, the template for viral RNA transcription, is the basis for the persistence of these viruses in infected hepatocytes. In this review, we summarize the current state of knowledge on the mechanisms of hepadnavirus reverse transcription and the biochemical and structural properties of the viral reverse transcriptase (RT). We highlight important gaps in knowledge regarding cccDNA biosynthesis and stability. In addition, we discuss the impact of current antiviral therapies on viral persistence, particularly on cccDNA.
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Affiliation(s)
- Jianming Hu
- Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, Pennsylvania 17033
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48
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Chen J, Wu M, Liu K, Zhang W, Li Y, Zhou X, Bai L, Yuan Z. New insights into hepatitis B virus biology and implications for novel antiviral strategies. Natl Sci Rev 2015. [DOI: 10.1093/nsr/nwv044] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Abstract
Hepatitis B virus (HBV), a small DNA virus with a unique replication mode, can cause chronic hepatitis (CHB), which is characterized by the persistence of the viral covalently closed circular DNA that serves as the template for HBV replication and the production of large amounts of secreted HBV surface antigen (HBsAg) that is present in excess of the levels of infectious virus. Despite the success of currently approved antiviral treatments for CHB patients, including interferon and nucleotide analogs, which suppress HBV replication and reduce the risk of CHB-related liver diseases, these therapies fail to eradicate the virus in most of the patients. With the development of the cell and animal models for HBV study, a better understanding of the HBV life cycle has been achieved and a series of novel antiviral strategies that target different stages of HBV replication have been designed to overcome the viral factors that contribute to HBV persistence. Such basic HBV research advancements and therapeutic developments are the subject of this review.
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Affiliation(s)
- Jieliang Chen
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
- Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Min Wu
- Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Kuancheng Liu
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
- Institutes of Medical Microbiology and Biomedical Sciences, Fudan University, Shanghai 200032, China
| | - Wen Zhang
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
- Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Yaming Li
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Xiaohui Zhou
- Research Unit, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
| | - Lu Bai
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Zhenghong Yuan
- Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
- Institutes of Medical Microbiology and Biomedical Sciences, Fudan University, Shanghai 200032, China
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49
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Cui X, McAllister R, Boregowda R, Sohn JA, Ledesma FC, Caldecott KW, Seeger C, Hu J. Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair? PLoS One 2015; 10:e0128401. [PMID: 26079492 PMCID: PMC4469307 DOI: 10.1371/journal.pone.0128401] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Accepted: 04/28/2015] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.
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Affiliation(s)
- Xiuji Cui
- Department of Microbiology and Immunology, Hershey, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States of America
| | - Rebecca McAllister
- Department of Microbiology and Immunology, Hershey, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States of America
| | - Rajeev Boregowda
- Department of Microbiology and Immunology, Hershey, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States of America
| | - Ji A. Sohn
- Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America
| | - Felipe Cortes Ledesma
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)—CSIC, Av. Américo Vespucio s/n, 41092 Sevilla, Spain
| | - Keith W. Caldecott
- Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton, Sussex BN1 9RQ, United Kingdom
| | - Christoph Seeger
- Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America
| | - Jianming Hu
- Department of Microbiology and Immunology, Hershey, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States of America
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50
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Abstract
Infection with HBV is common worldwide, with over 350 million chronic carriers. Chronic HBV infection is associated with cirrhosis and hepatocellular carcinoma. All currently available oral antivirals are directed against the HBV polymerase enzyme, a reverse transcriptase. HBV polymerase contains several important domains and motifs which define its functions and reveal ways to further target it. This enzyme executes many functions required for the HBV replication cycle, including viral RNA binding, RNA packaging, protein priming, template switching, DNA synthesis and RNA degradation. In addition, HBV polymerase must interact with host proteins for its functions. Future therapeutics may inhibit not only the DNA synthesis steps which are carried out by the reverse transcriptase domain (as all current antivirals do) but other domains, functions and interactions which are essential to the HBV replication cycle.
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Affiliation(s)
- Daniel N Clark
- The Pennsylvania State University College of Medicine, Milton S Hershey Medical Center, PA 17033, USA
| | - Jianming Hu
- The Pennsylvania State University College of Medicine, Milton S Hershey Medical Center, PA 17033, USA
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