Browsing by Subject "cccDNA"

Now showing 1 - 6 of 6
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    Biological Functions of Intracellular Hepatitis B e Antigen
    (2019-09) Mitra, Bidisha; Guo, Haitao; Androphy, Elliot J.; Kaplan, Mark; Yu, Andy; Lu, Tao
    The function(s) of the intracellular form of HBeAg, previously reported as the preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we propose to elucidate the translocation of p22 during its formation from endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have identified that a portion of p22, after the cleavage of its signal peptide in ER, is released back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except for the extended 10 amino acid precore region at the N-terminus, we observed that p22 wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not the secreted HBeAg significantly reduced interferon stimulated response element (ISRE) activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN- α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1, indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus evade host innate immune response and cause resistance to IFN therapy in patients. Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results indicate that there is a novel ER sorting mechanism for the distribution of the intracellular and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism.
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    Establishment of an Inducible HBV Stable Cell Line that Expresses cccDNA-dependent Epitope-tagged HBeAg for Screening of cccDNA Modulators
    (Elsevier, 2016-08) Cai, Dawei; Wang, Xiaohe; Yan, Ran; Mao, Richeng; Liu, Yuanjie; Ji, Changhua; Cuconati, Andrea; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.
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    Identification of Hydrolyzable Tannins (Punicalagin, Punicalin and Geraniin) as Novel inhibitors of Hepatitis B Virus Covalently Closed Circular DNA
    (Elsevier, 2016-10) Liu, Chunlan; Cai, Dawei; Zhang, Lin; Tang, Wei; Yan, Ran; Guo, Haitao; Chen, Xulin; Department of Microbiology and Immunology, IU School of Medicine
    The development of new agents to target HBV cccDNA is urgently needed because of the limitations of current available drugs for treatment of hepatitis B. By using a cell-based assay in which the production of HBeAg is in a cccDNA-dependent manner, we screened a compound library derived from Chinese herbal remedies for inhibitors against HBV cccDNA. Three hydrolyzable tannins, specifically punicalagin, punicalin and geraniin, emerged as novel anti-HBV agents. These compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner in our assay, without dramatic alteration of viral DNA replication. Furthermore, punicalagin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. By employing the cell-based cccDNA accumulation and stability assay, we found that these tannins significantly inhibited the establishment of cccDNA and modestly facilitated the degradation of preexisting cccDNA. Collectively, our results suggest that hydrolyzable tannins inhibit HBV cccDNA production via a dual mechanism through preventing the formation of cccDNA and promoting cccDNA decay, although the latter effect is rather minor. These hydrolyzable tannins may serve as lead compounds for the development of new agents to cure HBV infection.
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    Metabolism and function of hepatitis B virus cccDNA: Implications for the development of cccDNA-targeting antiviral therapeutics.
    (Elsevier, 2015-10) Guo, Ju-Tao; Guo, Haitao; Department of Microbiology and Immunology, IU School of Medicine
    Persistent hepatitis B virus (HBV) infection relies on the stable maintenance and proper functioning of a nuclear episomal form of the viral genome called covalently closed circular (ccc) DNA. One of the major reasons for the failure of currently available antiviral therapeutics to achieve a cure of chronic HBV infection is their inability to eradicate or inactivate cccDNA. In this review article, we summarize our current understanding of cccDNA metabolism in hepatocytes and the modulation of cccDNA by host pathophysiological and immunological cues. Perspectives on the future investigation of cccDNA biology, as well as strategies and progress in therapeutic elimination and/or transcriptional silencing of cccDNA through rational design and phenotypic screenings, are also discussed. 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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    Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation
    (2022-01) Marchetti, Alexander Lloyd; Guo, Haitao; Yu, Andy; Androphy, Elliot J.; Robinson, Christopher
    Despite the lifecycle of the hepatitis B virus (HBV) being extensively investigated and described, there remains a significant gap in our knowledge of arguably one of the most crucial steps in the HBV lifecycle, the formation and maintenance of a covalently closed circular DNA (cccDNA) reservoir. Advancements in our understanding of host factors and pathways involved in cccDNA formation have been made through hypothesis driven studies and shRNA/siRNA screenings. We sought to create a targeted-unbiased assay to directly observe host factor-rcDNA interactions. This was achieved through an rcDNA Co-Immunoprecipitation paired Mass Spectrometry (rcDNA-CoIP/MS) assay. We created a DNA oligo complimentary to the open portion of the HBV rcDNA, labeled with biotin, to facilitate easy precipitation of nuclear rcDNA and complexed proteins. Proteins precipitated were analyzed through liquid chromatography paired mass spectrometry (LC/MS). Along with previously reported host factors, several factors of DNA damage repair pathways/complexes were also identified. A component of the UV-DDB complex, DDB1, surfaced as a hit. UV-DDB/rcDNA binding was confirmed through ChIP-qPCR. DDB2, the DNA damage binding component of the UV-DDB complex was knocked out in HepG2-NTCP and HepAD38 cells. This resulted in a significant decrease in the formation of cccDNA in DDB2 knockout cell lines following infection or induction. The subsequent reduction of downstream indicators of cccDNA formation such as viral RNA and proteins, HBcAg and HBeAg, showed a consistent decrease with cccDNA levels. Ectopic expression of DDB2 in the knockout cell lines rescued HBV phenotypes of cccDNA levels and its downstream indicators. Inactive mutant DDB2 plasmids were also transfected into the DDB2 K/O cell lines and failed to rescue cccDNA indicators. We therefore showed through a novel assay that we can discover novel viral rcDNA-host interactions, such as the UV-DDB complex recruiting DNA repair pathways to “repair” rcDNA to cccDNA.
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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.