Browsing by Author "Zhang, Hu"

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    Biogenesis and molecular characteristics of serum hepatitis B virus RNA
    (Public Library of Science, 2020-10-20) Shen, Sheng; Xie, Zhanglian; Cai, Dawei; Yu, Xiaoyang; Zhang, Hu; Kim, Elena S.; Zhou, Bin; Hou, Jinlin; Zhang, Xiaoyong; Huang, Qi; Sun, Jian; Guo, Haitao; Medicine, School of Medicine
    HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
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    Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA
    (American Society for Microbiology, 2020-12-09) Cai, Dawei; Yan, Ran; Xu, Jerry Z.; Zhang, Hu; Shen, Sheng; Mitra, Bidisha; Marchetti, Alexander; Kim, Elena S.; Guo, Haitao; Microbiology and Immunology, School of Medicine
    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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    Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion
    (NAS, 2020-12) Zang, Ruochen; Case, James Brett; Yutuc, Eylan; Ma, Xiucui; Shen, Sheng; Gomez Castro, Maria Florencia; Liu, Zhuoming; Zeng, Qiru; Zhao, Haiyan; Son, Juhee; Rothlauf, Paul W.; Kreutzberger, Alex J. B.; Hou, Gaopeng; Zhang, Hu; Bose, Sayantan; Wang, Xin; Vahey, Michael D.; Mani, Kartik; Griffiths, William J.; Kirchhausen, Tom; Fremont, Daved H.; Guo, Haitao; Diwan, Abhinav; Wang, Yuqin; Diamond, Michael S.; Whelan, Sean P. J.; Ding, Siyuan; Microbiology and Immunology, School of Medicine
    Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
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    Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA
    (PLOS, 2022-06-09) Kim, Elena S.; Zhou, Jun; Zhang, Hu; Marchetti, Alexander; van de Klundert, Maarten; Cai, Dawei; Yu, Xiaoyang; Mitra, Bidisha; Liu, Yuanjie; Wang, Mu; Protzer, Ulrike; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.
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    Naturally Occurring Core Protein Mutations Compensate for the Reduced Replication Fitness of a Lamivudine-Resistant HBV Isolate
    (Elsevier Masson, 2019-05) Zhang, Yongmei; Zhang, Hu; Zhang, Junjie; Zhang, Jiming; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of an RNA intermediate. The lack of proofreading capacity of the viral DNA polymerase results in a high mutation rate of HBV genome. Under the selective pressure created by the nucleos(t)ide analogue (NA) antiviral drugs, viruses with resistance mutations are selected. However, the replication fitness of NA-resistant mutants is markedly reduced compared to wild-type. Compensatory mutations in HBV polymerase, which restore the viral replication capacity, have been reported to arise under continuous treatment with lamivudine (LMV). We have previously identified a highly replicative LMV-resistant HBV isolate from a chronic hepatitis B patient experiencing acute disease exacerbation. Besides the common YMDD drug-resistant mutations, this isolate possesses multiple additional mutations in polymerase and core regions. The transcomplementation assay demonstrated that the enhanced viral replication is due to the mutations of core protein. Further mutagenesis study revealed that the P5T mutation of core protein plays an important role in the enhanced viral replication through increasing the levels of capsid formation and pregenomic RNA encapsidation. However, the LMV-resistant virus harboring compensatory core mutations remains sensitive to capsid assembly modulators (CpAMs). Taken together, our study suggests that the enhanced HBV nucleocapsid formation resulting from core mutations represents an important viral strategy to surmount the antiviral drug pressure and contribute to viral pathogenesis, and CpAMs hold promise for developing the combinational antiviral therapy for hepatitis B.
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    NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner
    (Public Library of Science, 2024-09-11) Shen, Sheng; Cai, Dawei; Liang, Hongyan; Zeng, Ge; Liu, Wendong; Yan, Ran; Yu, Xiaoyang; Zhang, Hu; Liu, Shi; Li, Wanying; Deng, Rui; Lu, Xingyu; Liu, Yuanjie; Sun, Jian; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.
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    Proteomic Analysis of Nuclear Hepatitis B Virus Relaxed Circular DNA-Associated Proteins Identifies UV-Damaged DNA Binding Protein as a Host Factor Involved in Covalently Closed Circular DNA Formation
    (American Society for Microbiology, 2022) Marchetti, Alexander L.; Zhang, Hu; Kim, Elena S.; Yu, Xiaoyang; Jang, Sunbok; Wang, Mu; Guo, Haitao; Microbiology and Immunology, School of Medicine
    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 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 coprecipitated proteins by mass spectrometry. DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported short hairpin RNA (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 the 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. Transcomplementation 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 several host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNA interference (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, 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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    RNA Helicase DDX17 Inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation
    (American Society for Microbiology, 2021) Mao, Richeng; Dong, Minhui; Shen, Zhongliang; Zhang, Hu; Liu, Yuanjie; Cai, Dawei; Mitra, Bidisha; Zhang, Jiming; Guo, Haitao; Microbiology and Immunology, School of Medicine
    DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.
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    The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation
    (Public Library of Science, 2017-12-29) Long, Quanxin; Yan, Ran; Hu, Jieli; Cai, Dawei; Mitra, Bidisha; Kim, Elena S.; Marchetti, Alexander; Zhang, Hu; Wang, Soujuan; Liu, Yuanjie; Huang, Ailong; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.
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    Screening of an epigenetic compound library identifies BRD4 as a potential antiviral target for hepatitis B virus covalently closed circular DNA transcription
    (Elsevier, 2023) Yu, Xiaoyang; Long, Quanxin; Shen, Sheng; Liu, Zhentao; Chandran, Jithin; Zhang, Junjie; Ding, Hao; Zhang, Hu; Cai, Dawei; Kim, Elena S.; Huang, Yufei; Guo, Haitao; Microbiology and Immunology, School of Medicine
    HBV cccDNA is the persistent form of viral genome, which exists in host cell nucleus as an episomal minichromosome decorated with histone and non-histone proteins. cccDNA is the authentic viral transcription template and resistant to current antivirals. Growing evidence shows that the transcriptional activity of cccDNA minichromosome undergoes epigenetic regulations, suggesting a new perspective for anti-cccDNA drug development through targeting histone modifications. In this study, we screened an epigenetic compound library in the cccDNA reporter cell line HepBHAe82, which produces the HA-tagged HBeAg in a cccDNA-dependent manner. Among the obtained hits, a bromodomain-containing protein 4 (BRD4) inhibitor MS436 exhibited marked inhibition of cccDNA transcription in both HBV stable cell line HepAD38 and HepG2-NTCP or primary human hepatocyte infection system under noncytotoxic concentrations. Chromatin immunoprecipitation (ChIP) assay demonstrated that MS436 dramatically reduced the enrichment of H3K27ac, an activating histone modification pattern, on cccDNA minichromosome. RNAseq differential analysis showed that MS436 does not drastically change host transcriptome or induce any known anti-HBV factors/pathways, indicating a direct antiviral effect of MS436 on cccDNA minichromosome. Interestingly, the MS436-mediated inhibition of cccDNA transcription is accompanied by cccDNA destabilization in HBV infection and a recombinant cccDNA system, indicating that BRD4 activity may also play a role in cccDNA maintenance. Furthermore, depletion of BRD4 by siRNA knockdown or PROTAC degrader resulted in cccDNA inhibition in HBV-infected HepG2-NTCP cells, further validating BRD4 as an antiviral target. Taken together, our study has demonstrated the practicality of HepBHAe82-based anti-HBV drug screening system and provided a proof-of-concept for targeting HBV cccDNA with epigenetic compounds.