HBV basic virology


Interferons Induce Degradation Of HBV CccDNA

Yuchen Xia1, Julie Lucifora1, Ke Zhang1, Xiaoming cheng1, Daniela Stadler1, Florian Reisinger1, Martin Feuerherd1, Zuzanna Makowska2, Daniel Hartmann3, Wolfgang E. Thasler4, Markus H. Heim2, Mathias Heikenwälder1, Ulrike Protzer1

1lnstitute of Virology, Technische Universität München / Helmholtz Zenfrum München, Munich, Germany; 2Department of Biomedicine, University Hospital Basel, Basel, Switzerland; 3Department of Surgery University Hospital rechts der Isar, Technische Universität München, Munich, Germany; 4Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany

Persistence of HBV cccDNA in infected hepatocytes is a major problem in chronic hepatitis B treatment. Noncytopathic viral clearance by interferon (IFN) has been described, but the mechanisms involved remain elusive. In our study, we investigated if IFNs can exert degradation of HBV cccDNA, the template of HBV transcription. In HBV infected primary human hepatocytes and HepaRG cells, treatment with IFN-α and IFN-۷ significantly reduced HBV cccDNA. HBV cccDNA specific 3D-PCR indicated sequence alterations. Sequence analysis showed Cto U transition of the HBV cccDNA minus strand after IFN-α and IFN-۷ treatment. A detailed analysis of the underlying mechanism after IFN-α treatment revealed upregulation of the APOBEc3 (A3) family cytidine deaminases A3A and A3G in both cell types and in IFN-α treated patient livers in a time and dose dependent manner. JAK-STAT signaling blockade or HIV-Vif expression proved that IFN-α induced cccDNA deamination by A3 lead to degradation. Subcellllular localization analysis and overexpression experiments demonstrated that A3A, which locates to the nucleus, was the active effector. Treatment of cccDNA with a DNA repair enzyme cocktail corrected all mutations indicating that uracil could be removed by uracil-DNA glycosylase inducing apurinic/apyrimidinic (AP) sites. AP endonuclease reduced cccDNA levels in IFN-a treated cells showing that the cccDNA can be further digested by this endonuclease. We did not observe any deamination of host genomic DNA upon IFN-a treatment by 3D-PCR analysis or deep sequencing. This suggested that A3A acts on and is directed specifically to viral DNA. Since A3A co-localized with HBV core protein (HBc) in confocal microscopy and interaction was confirmed by co-immunoprecipitation, we propose that A3A utilizes HBc to get access to cccDNA. Chromatin immunoprecipitation confirmed that both HBc and A3A were bound to the cccDNA minichromosome. In HBV(x-) infection, reduction of cccDNA by IFN-α depended on trans-complementation with HBx, which is required to activate cccDNA transcription and HBc expression. Since IFN-α needs to be applied at high doses to clear infection, we screened for other cytokines showing similar antiviral effects. Like IFN-α and IFN-γ, TNF-α and more importantly activation of the lymphotoxin-β receptor at therapeutic doses were able to trigger deamination and subsequent degradation of HBV cccDNA via base excision pathway in an NF-kB dependent fashion. Our studies for the first time show that HBV cccDNA can be degraded without affecting the host cell and thus open new options for the development of novel and safe treatments to eradicate HBV and cure chronic hepatitis B.


Ulrike Protzer - Consulting: GILEAD; Grant/Research Support: Janssen

The following people have nothing to disclose: Yuchen Xia, Julie Lucifora, Ke Zhang, Xiaoming Cheng, Daniela Stadler, Florian Reisinger, Martin Feuerherd, Zuzanna Makowska, Daniel Hartmann, Wolfgang E. Thasler, Markus H. Heim, Mathias Heikenwälder


Early and late changes in gene expression profiles following infection with hepatitis B or C virus in human hepatocyte chimeric mice

C.Nelson Hayes, Sakura Akamatsu, Masataka Tsuge, Daiki Miki, Nobuhiko Hiraga, Hiromi Abe, Michio Imomuro, Shoichi Tokohashi, Hidenori Ochi, Kazuaki Chayama

Hiroshima University, Hiroshima, Japan

Background and aim: Hepatitis B and し viruses (HBV and HCV) are both hepatotropic viruses that cause chronic necroinflammatory liver disease and lead to increased risk of cirrhosis and hepatocellular carcinoma. However, the natural history and pathogenesis of these viruses differ greatly, with important consequences for treatment and prognosis. Due to the lack of suitable animal models, it has been difficult to examine differences in gene expression in response to infection with HBV compared to HCV. In this study cDNA microarray analysis of human hepatocyte chimeric mice was performed to compare patterns of gene expression prior to HBV or HCV infection and at two time points after infection. Methods: 34 human hepatocyte chimeric mice were allocated into five experimental groups. 15 mice were infected with HBV, 13 were infected with HCV, and 6 were used as an uninfected control group. Mice were inoculated via the tail vein with human serum containing HBV or HCV genotype 1b particles. 5 HBV-infected mice and 5 HCVinfected mice were sacrificed 10 days after infection, whereas the remaining 18 mice were sacrificed 8 weeks after infection. Human hepatocytes were extracted from mouse livers and analyzed using Toray 3D-Gene Human Oigo chip 25k microarray. Results: Pairwise comparisons revealed a number of short and long-term differences in gene expression in response to HBV and HCV infection. Fuzzy c-means cluster analysis was used to identify patterns in gene expression among the experimental groups, and gene set enrichment analysis was used to characterize clusters based on enrichment of gene ontology terms and Reactome pathways. Response of interferon stimulated genes was faster in HCV infection than in HBV infection, whereas HBV infection resulted in stronger and more sustained induction of acute phase genes (CRP, SAA1, SAA2). Distinct patterns of gene expression were detected using fuzzy c-means clustering, and each cluster was significantly associated with one or more Reactome pathways involving, e. g., innate and adaptive immune responses, cytokine signaling, cholesterol biosynthesis, signal transduction, and cell cycle regulation. Conclusions: Analysis of early and late changes in gene expression following HBV versus HCV infection revealed diverging patterns of immune response. Better understanding of differences in the molecular pathogenesis of inflammation in HBV versus HCV infection may help to reduce immune-mediated liver damage and improve response to therapy.


Kazuaki Chayama - Consulting: Abbvie; Grant/Research Support: Dainippon Sumitomo, Chugai, Mitsubishi Tanabe, DaIICHI SANKYO, Toray, BMS, MSD; Speaking and Teaching: Chugai, Mitsubishi Tanabe, DAIICHI SANKYO, KYORIN, Nihon Medi-Physics, BMS, Dainippon Sumitomo, MSD, ASKA, Astellas, AstraZeneca, Eisai, Olympus, GlaxoSmithKline, ZERIA, Bayer, Minophagen,

JANSSEN, JIMRO, TSUMURA, Otsuka, Taiho, Nippon Kayaku, Nippon

Shinyaku, Takeda, AJINOMOTO, Meiji Seika, Toray

The following people have nothing to disclose: C. Nelson Hayes, Sakura Akamatsu, Masataka Tsuge, Daiki Miki, Nobuhiko Hiraga, Hiromi Abe, Michio Imamura, Shoichi Takahashi, Hidenori Ochi


Baseline liver gene expression profile associated with therapy response in chronic hepatitis B patients treated with peginterferon and adefovir

Louis Jansen1, 2, Annikki de Niet1, 2, Zuzanna Makowska3, Michael T. Dill3, Karel A. van Dort2, Bart Takkenberg1, Markus H. Heim3, Neeltje A. Kootstra2, Hendrik W. Reesink1

1Department of Gastroenterology ond Hepatology, Academic Medical Center (AMC), Amsterdam, Netherlands; 2Department of Experimental Immunology, Academic Medical Genfer (AMC), Amsterdam, Netherlands; 3Department of Biomedicine, University of Basel, Basel, Switzerland

Background and Aim: Expression levels of interferon stimulated genes negatively correlate with the rate of sustained virological response in chronic hepatitis C patients treated with peg-IFN and ribavirin. In this study we investigated whether gene expression profiles in pre-treatment liver biopsies of patients with chronic hepatitis B (CHB) were associated with treatment outcome. Patients and Methods: Pre-treatment frozen liver biopsies of 42 CHB patients treated with Peg-IFN-a and adefovir for 48 weeks were available for analysis. Primary clinical outcome was combined response (CR) at week 72, defined as HBeAgnegativity, HBV DNA levels < 2, 000 IU/mL and persistent normal ALT levels in both HBeAg-positive and -negative patients, and was compared to non-response (NR). Total RNA was extracted and gene expression profiling was performed in 8 HBeAg-positive and 7 HBeAg-negative patients (9 CR) using Affymetrix Human Gene 1.0 ST microarrays. Transcriptome data was analyzed using Bioconductor packages of R statistical software. Twenty-seven additional frozen liver biopsies were available for confirmation (12 HBeAg-positive, 8 CR), and gene expression values of selected genes from the microarray were determined by real-time qPCR in all biopsies. Mean expression values were tested by Student΄s T test, and classification was performed in all available biopsies using kappa nearest neighbor (KNN) analysis. Results: In the microarray analysis, 57 genes were differentially expressed at baseline between patients with CR and those with NR. Eight of these genes showed a more than 2-fold difference. Ten genes were selected for qPCR analysis in all available liver biopsies, based on significance or known immune function. In this analysis, expression of one gene was significantly higher in patients with combined response: IL17RB (p = 0.009), and in 2 genes expression was significantly higher in patients with nonresponse: PAI1(p = 0.013), and NR1D1(p = 0.026). KNN analysis using this 3-gene prediction set (n=39) correctly classified 11/14 (79%) of patients with CR and 19/25 (76%) with NR. Conclusion: We identified three candidate genes whose expression patterns in baseline liver biopsies correlated with CR and NR. Classification analysis with this 3-gene set could predict most responders and non-responders. Ultimately one could use this specific hepatic signature to determine the chance of response to peg-IFN based therapy in CHB patients. More research is needed to study the role of the identified genes in HBV treatment, and to confirm their predictive value in an independent cohort.


Hendrik W. Reesink - Consulting: Abbott, Gilead, Astex, Merck, Roche, JanssenCilag, GlaxoSmithKline, Tibotec/ JJ, PRA-International; Grant/Research Support: Vertex, Boehringer Ingelheim, Anadys, Phenomix, Chugai, Japan Tobacco, Santaris, SGS, Idenix, BMS

The following people have nothing to disclose: Louis Jansen, Annikki de Niet, Zuzanna Makowska, Michael T. Dill, Karel A. van Dort, Bart Takkenberg, Markus H. Heim, Neeltje A. Kootstra


The nuclear function of Hepatitis B capsid (HBc) protein is to inhibit IFN response very early after infection of hepatocytes

Marion Gruffaz1, 2, Barbara Testoni1, 2, Souphalone Luangsay1, 2, Florione Fusil3, 2, Ait-Goughoulte Malika1, Jimmy Mancip3, 2, Marie-Anne Petit1, 2, Hassan Javanbakht4, Francois-Loic Cosset4, Fabien Zoulim1, 5,David Duronfel1,2

1U1052/ INSERM, Lyon, France; 2University of Lyon (UCBL), Lyo, France; 3U1111, INSERM, Lyon, France; 4Hoffmann-La-Roche, Basel, Switzerland; 5Hospices Civils de Lyon (HCL), Lyon, France

Background & aims: HBV has evolved strategies to evade innate immunity, including the interferon (IFN) response. While trying to identify new mechanisms that could explain the precocity of this inhibition, and the “stealthy” character of HBV, we identified HBV core protein (HBc) as a master early negative regulator of the IFN response. Methods: Human hepatocytes and other liver cells were either infected/exposed to HBV or transfected with viral nucleocapsids or recombinant HBc. Engineered HepaRG lines, inducible-expressing HBV proteins were also used to analyze the role of individual proteins. Ligands of innate sensors (PRRs) were used to trigger innate signaling pathways and evaluate the inhibitory effect of HBV proteins. HBV replication was assessed with standard procedures, whereas the effect of viral proteins on various interferons, interferoninduced (ISG) and pro-inflammatory cytokines gene expression, was analyzed by RTqPCR, WB and ELISA. ChIP experiments were also performed to investigate the binding of HBc, as well as to determine the recruitment of epigenome-modifying enzymes to target promoters. Results: HBV is capable to inhibit dsRNA-mediated interferon responses within minutes/hours of infection/exposure in hepatocytes, LSEC, or Kupffer cells. This inhibition occurs also with UV-inactivated virus suggesting that neo-synthesis of HBV proteins is not necessary. Using engineered HepaRG lines, we demonstrate that HBc is the main viral component responsible for this very early inhibition. The transfection of nucleocapsid or recombinant HBc recapitulates the same inhibitory phenotype. Moreover, HBc nuclear localization is required to suppress the transcription of targeted genes (i. e. IFNs, ISG), as the blockade of its trafficking, with nocodazole or anti-capsid molecules, reverts the inhibitory phenotype. ChlP and interaction analyses revealed that HBc is capable to bind to targeted promoters and to recruit Ezh2 and G9a chromatin-modifying enzymes to establish negative transcriptional marks (H3K9- or H3K27me2/me3) on selected promoters. These results were also confirmed in vivo in liver-humanized mice chronically infected by HBV. Conclusion: HBc is a key and very early negative regulator of the IFN response in hepatocytes. The precocity of this inhibition, due to nuclear delivery of HBc from “incoming virions” that direct interference with transcriptional machinery and/or favor the recruitment of epigenetic enzymes leading to repressive marks on target promoters, is instrumental for the establishment of persistent infection in vitro and in vivo. Targeting HBc nuclear functions may therefore represent a novel immunotherapeutic option.


Fabien Zoulim - Advisory Committees or Review Panels: Gilead; Consulting: Roche; Grant/Research Support: Gilead, Scynexis, Roche; Speaking and Teaching: Novartis, Roche, Janssen, Bristol Myers Squibb, Gilead

David Durantel - Grant/Research Support: Hoffman-La-Roche

The following people have nothing to disclose: Marion Gruffaz, Barbara Testoni, Souphalone Luangsay, Floriane Fusil, Ait-Goughoulte Malika, Jimmy Mancip, Marie-Anne Petit, Hassan Javanbakht, Francois-Loic Cosset


Hepatitis B virus (HBV) core promoter (CP) mutations and AKT1(v-Akt murine thymona viral oncogene homolog 1)coactivation may be associated with hepatocellular carcinoma (HCC) prognosis

Yuehua Huang, Lin Gu, Xiaohui Huang

Laboratory of Liver Diseases, The Third Affliataed Hospital of Sun Yat-sen University, Gunagzhou, China

Background: Clinical studies have shown HBV CP mutant is an independent predictor for HCC prognosis. Mounting evidence indicates activation of AKT1 is a key oncogenic event in tumor progression. However, the molecular pathogenesis of HBV mutations accelerating HCC progression remains undetermined. Aim: Investigate the role of crosstalk between HBV mutaitons and AKT1 in HCC progression. Methods: 52 HBV associated HCC patients with better progression (HCCB, >3 years survival) and 7 3 with poor progression (HCCP, <3years survival) after partial liver resection were analysed, respectively. HBV CP mutations were detected in serum samples. Proliferation and apoptotic indices were determined by counting KI67-positive cells and apoptotic figures stained by using apoptosis kit, respectively, on 3000 hepatocytes in HCC tissues. The microvessel density (MVD) was assessed by using anti-PODXL1antibody. Expressions of cell cycle regulators (p21, p27, and p57) and AKT1 as well as its downstream gene S phase kinase associated protein 2 (SKP2) were examined in both human HCC tissues and HBV expressing Huh7 cells. Effects of coactivation of AKT1 and HBV mutations on cell cycle progression and celluar growth were also analysed. Results: When compared to patients with HCCB, KI67-positive cells and MVD were significantly higher, while apoptotic index was significantly lower in HCCP. Decreased levels of cell cycle regulators, and increased levels of AKT1 and SKP2 were more profound in HCCP than that in HCCB. Higher incidence of HBV CP mutations was significantly associated with HCCP when compared to HCCB (77.5% for HCCP and 27% for HCCB, respectively, p<0.05). The level of AKT1 expression correlated with enhanced proliferation and MVD, as well as the prevalence of CP mutations, and was inversely correlated with apoptosis and survival in HCC patients. HBV with CP mutations accelerated cell cycle regulators protein degradation while wild type HBV had no effect in Huh7 cells. These effects were accompanied by a profound increase in AKT1.Coexpression of AKT1 and HBV CP mutations resulted in a dramatic increase of SKP2 expression, which in turn accelerated cellular growth and cell cycle progression in hepatoma cells when compared with cells overexpresssing AKT1 or HBV CP mutant alone. Small interfering RNA knockdown of SKP2 abrogated the effect of CP mutations on levels of cell cycle regulators, decreased cell proliferation, and restored cell cycle arrest.Conclusion:. Our data demonstrate the crosstalk between HBV CP mutations and AKT1 in promoting liver tumor progression, and suggest that AKT1/SKP2 signals may serve as a potential target for treamtment of HBV associated HCC.


The following people have nothing to disclose: Yuehua Huang, Lin Gu, Xiaohui Huang


HAPs hepatitis B virus (HBV) capsid inhibitors block core protein interaction with the viral minichromosome and host cell genes and affect cccDNA transcription and stability

Laura Belloni1, 2, Lichun Li4, Gianna Aurora Palumbo1, 3, Srinivas Reddy Chirapu5, Ludovica Calvo1, 3, Mg Finn6, Uri Lopafin7, Adam Zlotnick4,7,Massimo Levrero1, 2

1Dept. Internal Medicine (DMISM), Sapienza University Rome, Rome, Italy; 2Life Nanosciences Laboratory, Sapienza University Rome, Rome, Italy; 3EAL Inserm U785, Sapienza University Rome, Rome,Italy; 4Department of Molecular &Cellular Biochemistry, Indiana University, Bloomington, IN 47405, IN; 5Department of Chemistry and Biochemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Lo Jolla, CA; 6Depf Chemistry, Georgia Institute of Technology, Atlanta' GA 30332, GA; 7Assembly Pharmaceuticals, Bloomington, IN 47401,IN

Background and aim: The development of novel therapies for HBV infection requires new antivirals that target viral life cycle functions other than the viral polymerase. HBV Core protein (Cp) represents an attractive new therapeutic target. Cp capsid assembly is critical for viral RNA packaging, reverse transcription and intracellular trafficking. Core proteins have been shown to bind the nuclear cccDNA, possibly contributing to the regulation of its function and stability. Hetero-aryl-dihydropyrimidines (HAPs), a new class of antivirals inhibiting HBV replication in vitro and in vivo, enhance the rate and the extent of Cp assembly and, at high concentration, stabilize preferentially non-capsid polymers of Cp. Here we investigated the impact of HAP12 on cccDNA formation, levels and transcription as part of its antiviral activity against HBV. Methods: Capsid-associated HBV-DNA (TaqMan real-time PCR), cccDNA (TaqMan realtime PCR) and pgRNA levels (quantitative real-time PCR with specific primers), were assessed in: a) HepG2 cells transfected with full length HBV genomes; b) the inducible HepAD38 stable HBV cell line, left untreated or treated with the hetero-aryl-dihydropyrimidine HAP12 at 1-5 microM. Recruitment of HBc and histone modifications on host genes and the viral minichromosome were assessed using standard ChIP and the cccDNA ChIP assay, respectively. Results: HAP12 treatment of cells transfected with wild type linear HBV genomes showed a complete suppression of HBV replication at 72 and 96 hrs with a peak >50% reduction of pgRNA transcription at 96 hours. The strong HAP12 inhibitory effect on pgRNA transcription and HBV replication was confirmed in the HepAD38 HBV inducible cell line. Following induction of HBV from an integrated transgene, HepAD38 cells have been show to accumulate cccDNA. A sharp, time-dependent reduction of steady state cccDNA levels in HepAD38 cells was observed with HAP12.Additionally, HAP12 treatment both inhibited HBc occupancy of cccDNA in induced HepAD38 cells and reduced cccDNA-bound H3 histone acetylation. Interestingly, HAP12 treatment also reduced H3 histone acetylation and HBc occupancy of the host c-Src oncogene promoter region. Conclusions: Targeting HBV Cp with HAPs results in inappropriate capsid assembly and function, presumably secondary to conformational changes in Cp oligomers. HAP12 treated cells demonstrate impaired functional capsid formation, reduced viral replication at both the DNA and pgRNA level, as well as altered Cp interaction with both host genes and the HBV cccDNA.


Uri Lopatin - Employment: Assembly Pharmaceuticals; Stock Shareholder: Gilead Sciences

Adam Zlotnick - Management Position: Assembly Pharmaceuticals

Massimo Levrero - Advisory Committees or Review Panels: BMS, Jansen, Gilead; Speaking and Teaching: MSD, Roche

The following people have nothing to disclose: Laura Belloni, Lichun Li, Gianna Aurora Palumbo, Srinivas Reddy Chirapu, Ludovica Calvo, Mg Finn