F Sureau

P Zoulim

David Deny

Durantel

Dulce Alfaiate

Julie Lucifora
email: julie.lucifora@inserm.fr

Natali Abeywickrama-Samarakoon

Maud Michelet

Barbara Testoni

Jean-Claude Cortay

Camille Sureau

Fabien Zoulim

Paul Dény
email: paul.deny@inserm.fr

David Durantel
email: david.durantel@inserm.fr

HDV RNA replication is associated with HBV repression and interferonstimulated genes induction in super-infected hepatocytes

Keywords: cccDNA, circular covalently closed DNA, CHD, chronic hepatitis delta, dHepaRG, differentiated HepaRG, HBV, hepatitis B virus, HDV, hepatitis D virus, HDAg, Hepatitis delta antigen, HIV, human immunodeficiency virus, hNTCP, human sodium taurocholate cotransporting polypeptide, IFN, interferon, ISG, interferon stimulated genes, LR-β lymphotoxin receptor-, MOI, multiplicity significant, PAMP, pathogen-associated molecular pattern, pgRNA, pregenomic RNA, PHH, primary human hepatocytes, p.i., post-infection, PRR, pathogen recognition receptor, PEG, polyethylene glycol, SRB, sulforhodamine B, TLR ,toll-like receptor, VGE, virus genome equivalent, Hepatitis D virus, hepatitis B virus, viral interference, IFN response, Interferon stimulated genes

HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes

HIGHLIGHTS

A model of super-infection with HDV on HBV-infected hepatocytes was established; HDV infection induces a strong IFN response in these immune-competent hepatocytes;

In this model, HDV infection is associated with HBV inhibition, thus access to recapitulating in vivo viral interference; This super infection model is also suitable for the evaluation of novel drugs/antivirals, including immune-modulators.

Introduction

Chronic hepatitis delta (CHD) affects 15-20 million people worldwide (5-10% of the hepatitis B virus (HBV)-infected patients) [START_REF] Hughes | Hepatitis delta virus[END_REF]. It is considered to be the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis and cirrhosis and an increased risk of liver disease decompensation, hepatocellular carcinoma and premature death [START_REF] Fattovich | Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep)[END_REF]. Pegylated-alpha interferon (Peg-IFN) remains the sole therapeutic option for these patients, leading to a low virological response rate (< 30%) at 24 weeks post-treatment and high rate (>50%) of late relapse [START_REF] Heidrich | Late HDV RNA relapse after peginterferon alpha-based therapy of chronic hepatitis delta[END_REF]. The overall long-term sustained virologic response (SVR) rate is therefore very low in the clinical trial setting, and is even lower in the "real-life" clinical management [START_REF] Yurdaydin | Treatment of chronic delta hepatitis[END_REF]. HBV-reverse transcriptase inhibitors have no effect on hepatitis D virus (HDV) replication. The pipeline of investigational drugs against HDV infection remains limited due to the fact that i) HDV does not encode enzymatic activities and uses cell DNA-dependent RNA polymerases (particularly RNA pol II) for its replication, ii) there are remaining gaps in the knowledge of the viral life-cycle, and iiii) no appropriate in vitro model of this satellite co-or superinfection exists to screen antiviral drugs. Amongst few others, Myrcludex (a viral entry inhibitors) and farnesyl transferase (i.e. Lonafarnib) inhibitors are in early clinical trial evaluation [START_REF] Koh | Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-ofconcept randomised, double-blind, placebo-controlled phase 2A trial[END_REF][START_REF] Bogomolov | Interim results of a Phase Ib/IIa study of the entry inhibitor myrcludex B in chronic hepatitis D infected patients[END_REF].

HDV is a subviral agent satellite of HBV, and its genome, the smallest known among mammalian viruses, has similarities to plant viroids. To ensure propagation, HDV relies on HBV, as HDV ribonucleoproteins are surrounded by HBV envelope-embedded glycoproteins. Furthermore, HDV entry into human hepatocytes is mediated through the large HBV envelope protein (L-HBsAg) interaction with the recently discovered cell surface HBV receptor, i.e. the human sodium taurocholate cotransporting polypeptide (hNTCP) [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF].

HDV genome is a single-stranded circular RNA of ~1680 bp, with high intra-molecular base pairing, allowing a rod-like structure folding. Its complementary 'antigenomic' strand encompasses the SHD gene that codes a single protein, the small, 24 kDa, HD protein (S-HDAg), which is essential for HDV RNA replication. At a later phase of the HDV replication cycle, SHD stop codon editing, catalyzed by Adenosine Deaminase acting on RNA-1 (ADAR-1), leads to the synthesis of a 19-20 amino-acid (aa) carboxy-terminal extended isoform of HDAg; this large, 27kDa, protein (L-HDAg), thwarts HDV RNA replication and, in its farnesylated form, is involved in particle assembly [START_REF] Lai | RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus[END_REF][START_REF] Taylor | Virology of hepatitis D virus[END_REF]. Both clinical and experimental data support the existence of viral interference between HDV and HBV. In the clinical setting, most patients infected with both HBV and HDV feature a pattern of HDV dominance, with a significant decrease in HBV-DNA viral load, when compared to mono-infected patients [START_REF] Schaper | Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D[END_REF][START_REF] Krogsgaard | Delta-infection and suppression of hepatitis B virus replication in chronic HBsAg carriers[END_REF][START_REF] Genesca | Hepatitis B virus replication in acute hepatitis B, acute hepatitis B virushepatitis delta virus coinfection and acute hepatitis delta superinfection[END_REF]. Moreover, studies on liver biopsies from chronically HDV-infected patients have shown a decreased level of HBV replicative intermediates in the liver [START_REF] Pollicino | Replicative and transcriptional activities of hepatitis B virus in patients coinfected with hepatitis B and hepatitis delta viruses[END_REF]. Finally, this negative interference has been confirmed in vivo, in super-infection conditions, using HBV-infected chimpanzees, woodchuck hepatitis virus (WHV)-infected woodchucks, and more recently HBV-infected humanized mice [START_REF] Rizzetto | Transmission of the hepatitis B virus-associated delta antigen to chimpanzees[END_REF][START_REF] Lütgehetmann | Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation[END_REF][START_REF] Negro | Hepatitis delta virus (HDV) and woodchuck hepatitis virus (WHV) nucleic acids in tissues of HDV-infected chronic WHV carrier woodchucks[END_REF][START_REF] Sureau | Cloned hepatitis delta virus cDNA is infectious in the chimpanzee[END_REF].

To understand the molecular basis of HDV interference on HBV, relevant infection-based in vitro models are essential. Viral interference has been observed in Huh7 cells by transfection of DNA vectors expressing HBV and HDV (or either HDAg isoforms) [START_REF] Wu | Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line[END_REF]. Direct inhibition of HBV enhancer-1 and activation of MxA gene, an interferon-stimulated gene (ISG) known to suppress HBV replication, have been documented in the same cell line [START_REF] Williams | Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene[END_REF]. However, transfection models with cDNAs expressing HDV genome have limitations and protein overexpression may lead to inaccurate assumptions. To explore HBV/HDV interference, the access to a cell culture model featuring both cccDNA formation and a competent innate immunity would be instrumental. Until recently, the knowledge on innate immune response related to HDV infection remained scarce. After in vitro studies suggesting a modulation of the IFN response [START_REF] Mcnair | Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA[END_REF][START_REF] Pugnale | Hepatitis delta virus inhibits alpha interferon signaling[END_REF], recent data from mouse models (both the humanized uPA-SCID and the hNTCP transgenic mice) revealed a strong induction of the intra-hepatocyte ISG expression [START_REF] Giersch | Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection[END_REF][START_REF] He | Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide[END_REF]. Further knowledge on the interactions between HDV and the innate immune system could be invaluable to get insights on the interplay between HDV and its helper, as well as to identify novel therapeutic strategies. The aim of this study was to establish a novel cellular model of HDV super-infection, and characterize HBV/HDV interactions via direct viral interference mechanisms or through hepatocyte innate immune response to infection. This model could furthermore allow an evaluation of novel drugs on HDV replication. Using the differentiated HepaRG (dHepaRG) cells, which are immune-competent [START_REF] Luangsay | Expression and Functionality of Toll-and RIG-like receptors in HepaRG Cells[END_REF], we confirmed an efficient suppression of HBV replication (i.e. inhibition of intracellular HBV RNA and DNA accumulation, as well as HBeAg secretion), with no detectable effect on cccDNA nor HBsAg expression, and showed that HDV infection is associated with induction of ISGs, but not with induction of NF-kappaB regulated genes. Finally, we demonstrate the usefulness of this model with respect to antiviral discovery, by studying the antiviral activity of interferon alpha, specific anti-HBV and investigational specific anti-HDV drugs.

Material and Methods

Production of HBV and HDV virions

High-titer HBV particles were retrieved from HepG2.2.15 cells supernatant as previously described [START_REF] Luangsay | Early Inhibition of Hepatocyte Innate Responses by Hepatitis B Virus[END_REF]. HDV particles were produced by Huh7 cotransfection of a trimer HDV-1 prototype replication-competent plasmid (pSVLD3) and an HBsAg-encoding plasmid (pT7HB2.7) according to Sureau et al. [START_REF] Sureau | The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model[END_REF] (see also Sup. Fig. 1). Both HBV and HDV supernatants were concentrated with 8% PEG 8000 (Sigma-Aldrich). All virus preparations were tested for the absence of endotoxin (Lonza).

Cell culture and infection

The human liver progenitor HepaRG cells were cultured, differentiated using DMSO and infected using PEG4% overnight with either HBV or HDV as previously described [START_REF] Luangsay | Expression and Functionality of Toll-and RIG-like receptors in HepaRG Cells[END_REF][START_REF] Luangsay | Early Inhibition of Hepatocyte Innate Responses by Hepatitis B Virus[END_REF][START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF]. For super-infection experiments, cells already infected with HBV for 6 days (100 viral genome equivalents [vge]/cell, unless otherwise indicated) were exposed to HDV overnight (100 vge/cell, unless otherwise specified). Primary human hepatocytes (PHH) were isolated and infected as previously reported [START_REF] Luangsay | Early Inhibition of Hepatocyte Innate Responses by Hepatitis B Virus[END_REF][START_REF] Sureau | The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model[END_REF]. HepG2-NTCP were kindly provided by Dr Stephan Urban (Univ. Heidelberg, Germany); there were cultivated in 10%-FCS supplemented DMEM (4.5 g/L glucose) without DMSO until confluency and with 2% DMSO after and infected as for HepaRG.

Nucleic acid quantification: qPCR, RT-qPCR and Northern Blot

For HBV titration, DNA was extracted with the QiAmp Ultrasens Virus kit (Qiagen) and submitted to qPCR. HDV was titrated by qRT-PCR after RNA extraction with the NucleoSpin RNA Virus kit (Macherey-Nagel) and digestion with DNAse I (Life Technologies) 1h at 37°C, followed by 20 min at 70ºC, to eliminate residual plasmid DNA. Supernatants from infected-dHepaRG cells were used for viral particle RNA and DNA quantification. In order to remove free nucleic acid, clarified supernatants were submitted to DNAse and RNAse digestion (Roche), followed by overnight precipitation with 8% PEG 8000 (Sigma-Aldrich). After centrifugation, pellets were suspended in PBS 1X and nucleic acids were extracted with the Nucleospin 96 Virus kit (Macherey Nagel). The resultant nucleic acids were quantified using qPCR and RT-qPCR for HBV and HDV, respectively.

For HBV intracellular total DNA quantification, DNA extraction was performed using the Master Pure Complete DNA and RNA extraction kit (Epicentre), or the Nucleospin 96 tissue kit (Macherey Nagel). Intracellular total RNA was extracted with the NucleoSpin RNA kit (Macherey Nagel), which includes a DNAse digestion step. All primers and probes are listed in Supplementary Table 1. HDV quantification was performed by one-step RT-qPCR (Express One-Step SYBR Greener, Life Technologies) using the primers described by Scholtès & colleagues [START_REF] Scholtes | Standardized one-step real-time reverse transcription-PCR assay for universal detection and quantification of hepatitis delta virus from clinical samples in the presence of a heterologous internal-control RNA[END_REF], and the following cycling conditions: 50°C for 20 min (retro-transcription -RT), 95°C for 5 min and then 40 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 20 s. PCR was run in the Roche LightCycler 480. Serial dilution of quantified full length HDV-1 RNA (obtained from in vitro transcription of a pCDNA-3-derived plasmid containing a monomeric full length HDV-1 cDNA insert) was used as a quantification standard (Sup. Fig. 2).

HBV DNA/RNA and innate immune gene expression were performed as previously described [START_REF] Luangsay | Early Inhibition of Hepatocyte Innate Responses by Hepatitis B Virus[END_REF]. For cccDNA quantification, total DNA was submitted to digestion with plasmid-safe DNAse (Epicentre) for 4hours at 37ºC, followed by 30 minutes of heat inactivation. Quantification was performed by FRET-based qPCR as previously described [START_REF] Werle-Lapostolle | Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[END_REF]. Beta globin was used as a house-keeping gene. For all intracellular gene expression analysis, the comparative cycle threshold (Ct) method was applied and results displayed as a ratio, to a control sample (described for each experiment) [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF]. Northern blot for HDV and HBV RNA detection was essentially performed as previously described [START_REF] Sureau | The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model[END_REF][START_REF] Lucifora | Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector[END_REF]. Briefly, purified RNA was denatured at 50°C for one hour with glyoxal (Life Technologies), subjected to electrophoresis through a phosphate 1.2% agarose gel and transferred to a nylon membrane (Amersham N+, GE). Membrane-bound RNA was hybridized to 32P-labeled full HDV genome or DIG-labeled HBV-specific probes. Quantitative analysis of HDV RNA was achieved by phosphorimager scanning (Typhoon Fla 9500, GE); 18S and/or 28S rRNA quantification was used as loading control. Quantitative analysis of HBV RNAs was achieved using "ImageLab software" (Bio-Rad).

Elisa, immunofluorescence and western blotting

Commercial immunoassay kits (Autobio Diagnostics Co., China) were used for HBsAg and HBeAg quantification in the cell culture supernatant. Results are presented as a ratio to a control sample, described for each experiment. Cut-offs for these ELISA were 1 NCU/mL (i.e. 1 NCU ≈ 13 ng) for HBeAg and 2.5 ng/mL for HBsAg. Human IP-10 cytokines were detected in the supernatants using the DuoSet® ELISA kit according to the manufacturer (R&D Systems). Analysis of Secreted Type I Interferon was performed as described previously [START_REF] Luangsay | Expression and Functionality of Toll-and RIG-like receptors in HepaRG Cells[END_REF].

To perform immunofluorescence, cells were fixated with paraformaldehyde 4% and permeabilized by Triton 0.3%. Labeling was done using the following antibodies: HBcAg -monoclonal mouse antibody from Abcam (Ab-8637 -

Results

In a mono-infection setting, dHepaRG cells support a strong, yet transient, HDV replication, associated with a strong expression of ISGs

To assess the conditions of HDV inoculation, dHepaRG cells were either mock-infected or infected with HDV at multiplicities of infection (MOI) ranging from 1 to 500 vge/cell. At day-6 post-infection (p.i.), intracellular HDV RNA could be detected by RT-qPCR from the lowest MOI tested (1 vge/cell), with a linear increase up to 50 vge/cell, reaching a plateau for higher MOIs, up to 500 vge/cell (Fig. 1A). Northern blot analysis confirmed RT-qPCR findings and, using a genomic sense probe, indicated de novo formation of HDV replicative antigenomic RNA through the initiation of a replicative cycle (Fig 1B). This was further confirmed in western blot and IF analyses, showing a plateau of HDV protein expression for MOIs higher than 100 vge/cell and a number of infected cells of no more than 5% of the monolayer (Fig. 1C andS3). Importantly, the level of HDV replication in dHepaRG was as high as that observed in HepG2-NTCP cells [START_REF] Ni | Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[END_REF], but 10x lower than that obtained in infected PHH (Fig. S4). For the experiments performed later, HDV MOIs of 10 or 100 vge/cell were mainly used for an optimal viral stock management.

To get insights on infection kinetics, dHepaRG cells were infected with HDV and total RNA and proteins were collected sequentially. As a negative control, cells were treated with the entry inhibitor Myrcludex®, from 2 hours before infection up to the end of viral inoculation, as shown in Fig. 2A. A steep rise in HDV intracellular RNA accumulation was detected from day 2 p.i. by RT-qPCR, reaching a peak at day 6 and a subsequent decrease (Fig. 2B and2C). At later time point p.i., HDV RNA remained detectable (Fig. 2A and2B), indicating a residual accumulation of replicative intermediates and/or a low replication persistence. As expected, no significant increase in HDV intracellular RNA occurred in the Myrcludex®-treated control (Fig. 2), highly suggesting that the replication detected in the assay occurred after hNTCP receptor-mediated specific entry process. Increase in HDV RNA levels was associated with an increase in the expression of HDAg (Fig. 2C andS3), with both forms (S-HDAg and L-HDAg) being clearly detectable from day 3 p.i. (Fig. 2C). The pattern of expression was slightly delayed as compared to HDV RNA, but followed the same bell-shaped curve. At later time points both HDAg isoform signals decreased but remained detectable (Fig. 2C andS3). A very slight impairment of hepatocyte viability was associated with HDV infection, as documented by levels of secreted ApoB throughout time, neutral-red staining, and sulforhodamine assays (Fig.

S6A, S6B and S6C

). However this weakly measurable toxicity did not parallel the kinetic of replication of HDV, as it was constant over time. Moreover, the use of an apoptosis inhibitor (i.e. QVD-OPH) did not modify the level of HDV replication, therefore confirming that HDV do not induce a specific death of infected dHepaRG cells (Fig. S6D andS6E).

In contrast to hepatoma cells, dHepaRG cells express functional innate immune sensors, namely pathogen recognition receptors (PRRs), and, therefore may be relevant to study antiviral response in hepatocytes [START_REF] Luangsay | Expression and Functionality of Toll-and RIG-like receptors in HepaRG Cells[END_REF]. We aimed to decipher IFN response to HDV infection in this model. Upon HDV mono-infection, increased expression of several representative ISGs could be detected. Interestingly, ISGs expression peaked at day-6 p.i. and correlated with HDV RNA replication kinetics (Fig. 3A). No ISGs' induction occurred in the presence of Myrcludex®, excluding a non-specific stimulation by the viral inoculum. Furthermore, no induction was detected during the first 3 days p.i., which may suggest that the IFN response matched HDV RNA replication and HDV RNA neosynthesis, rather than the incoming viral RNA material. In comparison to non-infected cells, highest expressions were detected for RSAD2 (i.e. VIPERIN; mean fold change 289) and IFI78 (i.e. MXA; mean fold change 143,2). Other evaluated genes included ISG15 (mean fold change 36,2), OAS1 (mean fold change 21,2), DDX58 (i.e. RIGI; mean fold change 20,7), MDA5 (mean fold change 9) and IFN-(mean fold change 4,4). For all studied time points, non-significant and less than 2 fold differences in expression were found respectively for IFN-and IL-6 between HDV infected cells and mock or Myrcludex® treated controls. In addition, we observed an increased secretion of IP-10 and type-I IFN that paralleled the increase of HDV RNA observed with the different amounts of HDV particles used for infection (Fig. 3B).

During super-infection, secretion of infectious HDV particles demonstrates the existence of HBV/HDV co-infected cells.

To set up the super-infection model, dHepaRG cells were first inoculated with HBV (100 or 500 vge/cell), and, at the plateau of HBV replication (i.e. day-6) (32), cells were inoculated with HDV. In this setting, three infected cell populations could be identified, upon labeling with anti-HBcAg and anti-HDAg antibodies: HBcAg positive/HDAg negative cells, HBcAg negative/HDAg positive cells and HBcAg positive/HDAg positive cells (Fig. 4A). This suggested that cells were either mono-infected by either HBV or HDV, or by both viruses, respectively. The same observation was obtained when HBsAg immunostaining was used instead of HBcAg labeling (data not shown). The proportion of infected cells expressing either antigen remained below 5% for either HBV or HDV markers, whereas co-labeling occurred in approximately 1-2% of the total dHepaRG cells.

Interestingly, despite the low number of detectable co-labeled cells, quantification of HDV RNA reached 1.3x10 7 vge/mL in the supernatant of HBV-HDV super-infected cells for the best condition. This result was obtained without detectable cell toxicity (data not shown), suggesting the secretion of viral particles. As expected, HDV intracellular RNA levels increased significantly with HDV MOI, but non-significantly with HBV MOI (Fig. 4B), whereas HDV secretion was proportional to both HDV and HBV MOIs (Fig. 4C). Of note, infections of HepG2-NTCP cells with concentrated HDV particles secreted from our super-infection experiments (called HDV-2P, for second passage) were as efficient as primary HDV inoculum (using equivalent MOI). This was demonstrated both by intracellular HDV RNA quantification and HDAg IF staining (Fig. 4D). Despite the low number of HBcAg/HDAg-positive cells, and likely due to the high efficiency of HDVreplication per cell [START_REF] Chen | Structure and replication of the genome of the hepatitis delta virus[END_REF], in the dHepaRG HBV/HDV super-infection setting, supernatant RNA associated from infectious HDV particles could be easily quantified, demonstrating that some dHepaRG cells can be infected by both viruses and are hence a suitable model for the evaluation of HDV-HBV interactions and the selection of drug resistant HDV variants.

In HBV-infected cells, HDV super-infection is associated with a MOI-dependent induction of ISGs and decreased HBV replication

The expression of innate immune related genes was evaluated at day-15 post-HBV infection (day-9 post-HDV infection). As previously determined [START_REF] Luangsay | Early Inhibition of Hepatocyte Innate Responses by Hepatitis B Virus[END_REF], HBV alone did not induce any innate gene expression at this time point (Fig. 5). In contrast, HDV infection was clearly associated with a strong induction of all studied ISGs, which was HDV MOIdependent but HBV-independent. Consistent with HDV mono-infection, super-infection, induced preferentially RSAD2 and MXA, with a respective 83.5 and a 48.6 fold increased gene expression (at HBV 100 vge/mL and HDV 100 vge/mL). Finally, no induction of NF-B induced genes was identified in HDV infections, as exemplified for IL-6 (Fig. 5), IL-8 and IL-1 (data not shown). Collectively, these data indicate that in dHepaRG cells, HDV infection induces a strong IFN response at the peak of RNA replication, independently of both HBV infection and NF-B pathway.

To investigate viral interference in the same setting, replication parameters were analyzed at day-15 post-HBV inoculation (9 days post-HDV super-infection). A significant decrease in both HBeAg secretion and intracellular HBV DNA accumulation was observed upon HDV super-infection of HBV-infected cells and the decrease was more pronounced with increasing HDV MOIs (Fig. 6A and6E). Concomitantly, HBV-DNA level decreased in the supernatant when cells were super-infected by HDV (Fig. 6D). In such conditions, no reduction of HBsAg was observed (Fig. 6B), and no variation of cccDNA levels occurred (as measured by specific qPCR; Fig. 6C). Considering HBV MOI condition of 100 vge/cell, the decrease of HBV pgRNA correlated with the HDV MOI (25% reduction for HDV MOI 100 vs HBV mono-infection; p<0.05) (Fig. 6G and6H). In contrast, no variation in the amount of total HBV RNA was observed even at high HDV MOI (Fig. 6F and6H).

To further characterize this viral interference, HBeAg and HBsAg secretion were followed throughout time in dHepaRG inoculated, or not, with HBV (100 vge/cells) and superinfected, or not, with HDV (Fig. 7A and7B). Compared to HBV-mono-infected cells, HDV-super-infected cells displayed a significant decrease of HBeAg secretion (34% decrease, p<0.01) (Fig. 7A). Increasing HDV MOI further inhibited HBeAg secretion (Fig. 7B and7C) and decreased the number of HBcAg positive cells (Fig. 7C) suggesting that viral interference was dependent of HDV-MOI. In contrast, there was no significant difference in HBsAg secretion levels between HBV-mono-infected and HDVsuper-infected cells (Fig. 7A and7B).

Study of various drugs for their anti-HDV efficiency

In order to further validate this HBV-HDV super-infection model, we aimed to explore the inhibitory effect of different molecules that could interfere with different steps of HBV and HDV life cycles in infected dHepaRG cells. Besides approved compounds such as IFN and tenofovir di-fumarate (TDF), we also verified the potential action of the entry inhibitor Myrcludex® and lonafarnib, a farnesyl transferase inhibitor (FTI), that have recently entered in phase II clinical trials for chronic HBV/HDV liver disease indication. Doses and treatment schedules were selected based on previously published data [START_REF] Lucifora | Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA[END_REF][START_REF] Bordier | A prenylation inhibitor prevents production of infectious hepatitis delta virus particles[END_REF][START_REF] Ni | Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[END_REF][START_REF] Isorce | Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus[END_REF].

As expected, upon treatment with the HBV-polymerase inhibitor tenofovir, a significant decrease was observed in the amount of secreted HBV DNA (70%; p<0,0001), but not in secreted antigens or intracellular RNA levels (Fig. S7A). No effect was documented on HDV replication or viral secretion (Fig. 8A). IFN treatment led to an important reduction of both HBV and HDV replicative parameters (Fig 8B andS7B). Unlike the other drugs, IFN treatment was associated with a significant decrease in Apolipoprotein B secretion (67%; p<0,0001). As no increased LDH release was observed, such finding may be related to hepatocyte de-differentiation rather than cytotoxicity (Fig. S8).

Prenylation inhibitors have been shown both in vitro and in vivo to impact HDV envelopment and secretion without having a direct effect on viral replication. By treating HBV/HDV super-infected cells with FTI-277, we could observe a modest, albeit nonsignificant, reduction of HDV secretion into the supernatant (40%, p=0,16), which, interestingly, was associated with an increase of intracellular HDV RNA levels (2 fold increase; p<0,05) (Fig. 8C). As expected, FTI-277 treatment had no effect on HBV replicative markers (Fig. S7C). Treatment with combinations of IFN and tenofovir or FTI-277 and IFN did not evidence a further decrease of neither HBV nor HDV parameters compared to single drug treatments (Fig. 8D, 8E, S7D andS7E). In this model, we could also confirm a suppression of HDV entry by Myrcludex® treatment previous and during HDV inoculation, while excluding a post-entry effect on both HBV and HDV replications (Fig. 8F andS7F). Overall, these results validate this model for the evaluation of both immune modulatory and direct-acting antiviral compounds acting on both HBV and HDV and at different steps of the viral life cycles.

Discussion

Despite leading to the most severe form of chronic viral hepatitis and infecting 15 to 20 million of HBV-positive people worldwide, HDV remains a neglected pathogen. Getting more fundamental knowledge on HBV/HDV co-infections and viral interference may ultimately translate into the development of much needed new therapeutic strategies against HDV. One aim of this work was to implement a relevant cell culture model to study this viral interplay, taking into account a subcellular innate immunity component. PHH are considered as the gold standard to perform in vitro studies on HBV and by extension on HDV. However, the low accessibility of fresh human liver resections, as well as the quality and variability of individual preparations limit their use. Interestingly, similarly to PHH, and in contrast to widely-used HepG2 and Huh7 cells, HepaRG cells functionally express most of innate immunity sensors [START_REF] Luangsay | Expression and Functionality of Toll-and RIG-like receptors in HepaRG Cells[END_REF] and are therefore considered as immunecompetent [START_REF] Maire | Characterization of the double-stranded RNA responses in human liver progenitor cells[END_REF]. Despite their lower susceptibility to HBV and HDV infection, dHepaRG are the best alternative to PHH cultures to study HBV infection, as a full replication cycle can be obtained without the need of ectopically expressed hNTCP [START_REF] Hantz | Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells[END_REF]. Moreover, cccDNA can be detected in infected HepaRG cells, and has been shown, in a proof of concept study, to be degradable in an APOBEC3A/B-dependent manner by activation of IFN-or lymphotoxin receptor-(LR-) response pathway(s) [START_REF] Werle-Lapostolle | Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[END_REF]. Therefore, the HepaRG cell line represents a unique model to study the interplay between HBV/HDV and hepatocyte-specific innate immunity, as well as to explore new therapeutic developments. So far, regarding HDV biology, the HepaRG model has mostly been used for studying the entry step [START_REF] Sureau | The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model[END_REF], its inhibition by Myrcludex®, a drug competing with hNTCP viral attachment [START_REF] Ni | Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[END_REF], thus confirming the relevance of this cellular receptor for HDV entry [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF].

In mono-infection with HDV we found that, as expected, only a small percentage of dHepaRG were infected (< 5% in IF). But in contrast to what seen with HBV [START_REF] Hantz | Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells[END_REF], the intracellular level of HDV replication was very high, and could be detected even without amplification, by northern blot. Notably, in a super-infection setting, despite the very low proportion of co-infected cells (1-2%), neo-produced infectious HDV particles were titered at 10 7 vge/mL in supernatant, thus reflecting again the very high efficiency of viral RNA replication [START_REF] Chen | Structure and replication of the genome of the hepatitis delta virus[END_REF]. The rather low proportion of detectable infected cells could be due, at least in part, to cell polarization and accessibility of hNTCP in the basolateral membrane of hepatocytes [START_REF] Schulze | Hepatocyte polarization is essential for the productive entry of the hepatitis B virus[END_REF]. Interestingly, in both mono-infection and super-infection conditions, HDV replication seemed to decline after a peak of replication at day-6 post inoculation. A similar decrease over time has also been described in mice injected with a HDV cDNA construct, in the chimpanzee experimental model and, more recently, in the hNTCP transgenic mouse model [START_REF] Sureau | Cloned hepatitis delta virus cDNA is infectious in the chimpanzee[END_REF][START_REF] He | Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide[END_REF][START_REF] Chang | Replication of the human hepatitis delta virus genome Is initiated in mouse hepatocytes following intravenous injection of naked DNA or RNA sequences[END_REF]. At least two hypotheses might explain such phenotype: the infection is limited in time either by the decrease of available factor needed for replication, such as S-HDAg or some proviral host factors, or by accumulation of inhibitors, such as L-HDAg or cellular negative factor(s). Alternatively, active antiviral innate immune response that was, in our experiments, found temporally related to the peak of RNA accumulation, could also contribute to such inhibition. With respect to the former, it was suggested that, besides mediating virion assembly, L-HDAg could inhibit viral replication, and therefore play crucial role to switch life-cycle from replicative to morphogenetic phase [START_REF] Modahl | The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication[END_REF]. However in our model, L-HDAg may not play this role, as the ratio of S-HDAg and L-HDAg remained constant throughout the kinetics of HDV RNA replication, and in the super-infection setting, in which HDV virion release is observed, the decline of HDV RNA signals after day-6 p.i. is still observed. Regarding the immune hypothesis, we showed that the induction of some ISGs expression occurs at the peak of HDV RNA accumulation, indicating that neo-synthesized HDV-replicative intermediates, rather than inoculum RNA, act as a pathogen-associated molecular pattern (PAMP). Whether the activation of IFN response could lead to the decline of HDV replication after day-6 post infection is still unknown in dHepaRG, but such an hypothesis was not confirmed in the transgenic hNTCP mouse model [START_REF] He | Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide[END_REF]. Interestingly, in the cellular super-infection setting, we were able to confirm that HDV can interfere with HBV replication. The observations that HDV super-infection is associated with a decrease of HBeAg, HBV virion secretion, intracellular HBV DNA and pgRNA, although not HBsAg, total HBV RNA or cccDNA, are in agreement with what has been described in HDV-infected patients [START_REF] Pollicino | Replicative and transcriptional activities of hepatitis B virus in patients coinfected with hepatitis B and hepatitis delta viruses[END_REF]. This is part of the originality of this satellite infection that may often overcome its helper replication, while maintaining its budding trans-complementation. A competition for viral egress through the HBsAg secretory pathway is unlikely, as HBsAg is produced in large excess leading to a high proportion of empty subviral particles, and both viruses may not have the same cellular egress pathway [START_REF] Watanabe | Involvement of host cellular multivesicular body functions in hepatitis B virus budding[END_REF]. Furthermore, this would not account for the specific diminution of the HBV pgRNA that might be due to a modulation of cccDNA transcriptional activity [START_REF] Pollicino | Replicative and transcriptional activities of hepatitis B virus in patients coinfected with hepatitis B and hepatitis delta viruses[END_REF][START_REF] Wu | Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line[END_REF]. In reporter systems and exogenous expression of HD proteins, direct inhibition of both HBV enhancers, especially by L-HDAg has been previously suggested [START_REF] Williams | Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene[END_REF]. In a previous pioneer work using Huh7 cell co-transfected by both HBV-expressing plasmid pA3HBV3.8 and pSVLD3 (or pSVL-HDAg), J.C. Wu and co-workers suggested a possible repressive effect on transcription of the 3.5 kb and the 2.1 kb transcripts of HBV by HDV-replication or HDAg coding gene expression [START_REF] Wu | Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line[END_REF]. But this approach was less physiological than that based on proper infections. We trust the cellular super-infection model described in this study may therefore further contribute to determine at which step (e.g. cccDNA transcription, viral mRNA export and/or stability), and with which kinetic, the HDV-induced HBV-inhibition may occur. Another explanation of this viral interference may be linked to the HDV-induced IFN response. Our results indicate that an increase in HDV MOI was associated with a more significant decrease of HBV replicative intermediates and a dose-dependent increase in ISGs expression. Previous works on HBV have shown that type-I IFN response modulates transcriptional regulation of cccDNA, decreasing pgRNA synthesis through modifications in histone acetylation status and recruitment of chromatin modifying enzymes [START_REF] Belloni | IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome[END_REF]. Whether HDV super-infection could induce such an epigenetic negative regulation of cccDNA transcription remains to be further explored.

During HDV infection (with and without a previous infection by HBV), we identified a pattern of gene activation suggesting the induction of an IFN response, without any effect on NF-B regulated genes. The induction of ISGs expression by HDV is fully in agreement with results obtained in in both the humanized and hNTCP transgenic mice [START_REF] Giersch | Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection[END_REF][START_REF] He | Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide[END_REF]. Among the studied ISGs genes that match HDV replication, RDSA2 was found to be the most activated one. Interestingly, it has been suggested that in the woodchuck hepatitis infection of woodchuck neonates, Viperin was found to be at a higher level in neonates that resolved their infection, than those who progress to chronic carriage [START_REF] Fletcher | Identification of an intrahepatic transcriptional signature associated with self-limiting infection in the woodchuck model of hepatitis B[END_REF]. Another innate-immune mediated mechanism of HBV repression due to HDV superinfection might be linked to a counteract the inhibition of the MxA expression probably linked to the HBV capsid (48-50) Finally, our results demonstrate that, unlike other cellular models, dHepaRG sequentially infected by HBV and HDV represent a relevant model for the evaluation of antiviral drugs. Nucleos(t)ide analogues, such as tenofovir, while widely used in the setting of chronic hepatitis B, have failed to show a beneficial effect on the treatment of HDV infected patients [START_REF] Wedemeyer | Prolonged therapy of hepatitis delta for 96 weeks with pegylated-interferon-α-2a plus tenofovir or placebo does not prevent HDV rna relapse after treatment: the HIDIT-2 study[END_REF][START_REF] Wedemeyer | Peginterferon plus adefovir versus either drug alone for hepatitis delta[END_REF]. Our data support these findings, as no effect of tenofovir was observed on either HDV replication or HBsAg secretion. Whereas an antiviral effect of IFN on HBV replication has been thoroughly studied [START_REF] Lucifora | Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA[END_REF], data obtained in cellular models have been conflicting regarding its mechanism of action on HDV. Indeed, no direct effect of interferon on HDV replication was previously demonstrated [START_REF] Mcnair | Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA[END_REF], and other mechanisms of action have been suggested [START_REF] Taylor | Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus[END_REF][START_REF] Hartwig | Interferon-alpha stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection[END_REF]. Our findings, being consistent with a suppression of HDV replication by interferon, in the absence of cytotoxicity, are in line with the data obtained in vivo, supporting the notion that dHepaRG cells is a more pertinent model than hepatoma derived cells for the evaluation of immune-modulators. We also aimed to evaluate the effect of some investigational drugs, currently undergoing clinical trials. The HBV/HDV entry inhibition we could observe with Myrcludex® treatment confirmed previous data form other groups [START_REF] Lütgehetmann | Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation[END_REF][START_REF] Ni | Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[END_REF]. Although not reaching statistical significance, we reproduced a trend of decreased HDV secretion inhibition by the prenylation inhibitor FTI-277. The fact that this effect was less pronounced than previously described, may be associated with the small number of co-infected cells in the HepaRG cell model. Interestingly, and unlike previous studies, the treatment with FTI-277 in our model was associated with an increased HDV RNA accumulation in the cells. These results, although unexpected, can be explained i) by a possible abrogation of the inhibitory effect of L-HDAg on HDV replication in the absence of prenylation or ii) a defect in assembly of HDV RNP with HBsAg [START_REF] Hwang | Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen[END_REF]. In summary, we demonstrated here the usefulness of the HepaRG cell line model for the study of HDV infection, in mono-and super-infection settings and could show that a robust HDV replication occurs in these cells and is associated with a strong induction of ISG expression. Moreover, upon HDV-super-infection of HBV-infected cells, HDV/HBV viral interference contributing to lowering HBV expression and the production of infectious HDV particles could be confirmed.

Figure legends

Figure 1. MOI-dependent replication of HDV in dHepaRG cells in mono-infection setting. dHepaRG were infected with HDV at different MOIs (ranging from 1 to 500 vge/cell). At day-6 post-infection, (A) levels of intracellular HDV RNA were assessed by RT-qPCR or (B) northern blot analyses using a genomic probe for antigenome detection, (C) HDAg expression was evidenced by western blot. Data in (A) represent the mean +/-SEM of 3 independent experiments. MOI, multiplicity of infection; AG, antigenome; L-HDAg, large hepatitis delta antigen; S-HDAg, small hepatitis delta antigen; n.s., nonsignificant.

Figure 2. Kinetics of HDV mono-infection in dHepaRG cells. dHepaRG cells were infected with HDV at 10 vge/cell and viral parameters were followed over time. As controls, cells were treated or not with Myrcludex ® at 100nM for 2h before and during HDV inoculation. At the indicated time, (A) levels of intracellular HDV RNA were assessed by RT-qPCR or (B) northern blot analyses using a genomic probe for antigenome detection, (C) HDAg expression was evaluated by western blot. Data in (A) represent the mean +/-SEM of 3 independent experiments. Myr, Myrcludex ® ; AG, antigenome; L-HDAg, large hepatitis delta antigen; S-HDAg, small hepatitis delta antigen. HDV or concentrated supernatant from HBV/HDV co-infected dHepaRG cells (HDV-2P) at 10 vge/cell. As controls, cells were treated or not with Myrcludex ® at 100nM for 2h before and during HDV inoculation. 6 days later, levels of intracellular HDV RNA and HDAg were respectively assessed by qRT-PCR or immunofluorescent staining followed by confocal microscopy analyses. Results are presented as ratio to HDV infected cells and represent the mean +/-SEM of one representative experiment performed in triplicate. Figure 5. ISG induction is also present in a super-infection setting. dHepRG cells were either mock (i.e. 0 vge/mL) or infected with HBV at 100 vge/cell and, 6 days later, super-infected with HDV at 0, 10 or 100 vge/cell. IL-6, type-1 IFNs and ISGs expressions were evaluated by RT-qPCR at day-15 post-HBV infection. Results are presented as ratio to the mock condition at each day and represent the mean +/-SEM of 3 independent experiments each performed in triplicate. HDV"was"produced"in"vitro"by"co9transfec;on"of"Huh7" cells" with" T7HB2.7" (coding" for" PreS19PreS29S" from" HBV)" and" pSVLD3" (containing" a" trimer" of" HDV" genotype" 1" genome)." Supernatant" was" collected" every" other" day" from" day99" to" day915" post9 transfec;on." (B)" Northern" blot" analysis" (with" a" full9length" an;9genomic" probe," for" detec;on" of" HDV" genome)"of"HDV"RNA"in"the"supernatant"of"transfected"cells"throughout";me."G,"genome." Serial"109fold"dilu;ons"of"a"full9length"HDV91"RNA"genomic" transcript" are" used" as" a" quan;fica;on" standard," confirm" PCR" linearity" within" a" range" of" 102" to" 108" copies/"reac;on"with"a"PCR"efficiency"of"~2,1."(B)"Electrophoresis"of"PCR"products"evidences"a"unique" band" located" between" 100" and" 200bp," consistent" with" the" predicted" 129bp" amplicon." (C)" Serial" 109 fold" dilu;ons" of" RNA" extracted" from" cell" culture" supernatant," confirm" PCR" linearity" between" 6X101" and"6X105"copies/"reac;on,with"a"PCR"efficiency"of"~2,2."(D)"Serial"39fold"dilu;ons"of"intracellular"total" RNA"confirm"a"linear"HDV"amplifica;on"within"a"range"of"0.4933,3"ng"of"total"RNA"per"PCR"reac;on."(E)" Mel;ng"curve"plot"of"HDV"infected"samples,"confirming"one"single"Tm"peak"consistently"found"at"84ºC." Lad,"DNA"ladder"(100bp,"New"England"Biolabs);"NC,"nega;ve"control;"Tm,"mel;ng"temperature." ,,,,,,,,,,,,,,,,,,,0,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,10,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,100,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,500,,, DAPI, HDAg, Figure ,S3."Increasing,MOI,lead,to,increases,of,HDV,infected,dHepaRG."dHepaRG"cells"were"infected" by"HDV"at"the"indicated"MOI."At"the"indicated";me,"cells"were"fixed,"permeabilized,"labeled"with"an;9 HDAg"an;bodies"and"analyzed"by"epifluorecence"microscopy."

Supplementary,table,and, figures

Figure, S4

." HDV, infecDon, of, different, cells." dHepaRG," PHH" or" HepG29NTCP" cells" were" infected" by" HDV" at" 10" vge/mL." 6" days" later," levels" of" intracellular" HDV" RNA" were"assessed"by"RT9qPCR" ,,,,,,,,,,,,,,,,,,,0,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,10,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,100,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,500,,, HepG2BNTCP,-,day,9,p.i., Figure ,S5."HDV,infecDon,of,different,cells."HepG29NTCP"cells"were"infected"by"HDV"at"the"indicated"MOI." At"the"indicated";me,"cells"were"fixed,"permeabilized"and"labeled"with"an;9HDAg"an;bodies"and"analyzed" by"confocal"microscopy."" A,,B,,C)" dHepaRG" cells" were" infected" with" H D V" a t" 1 0" v g e / c e l l" a n d" ( A )" Apolipoprotein" B" secre;on" as" well" as" (B,, C)" cells" viability" were" assessed" by" ELISA," neutral" red" or" sulforodamin" assays."Results"are"presented"as"ra;o"to" non"infected"cells"(mock)"and"represent" the" mean" +/9SEM" of" 8" replicates" (D)" dHepaRG" were" treated" with" the" i n d i c a t e d" c o n c e n t r a ; o n" o f" staurosporine" and" QVD9OPH" for" 16" hours" "and"ALT"ac;vity"were"measured" with" colorimetric" assays." (E)" dHepaRG" cells"were"infected"by"HDV"and"treated" with" QVD9OPH" for" 12" day." Levels" of" intracellular"HDV"RNA"were"assessed"by" RT9qPCR."Results"are"presented"as"ra;o" to" the" mock" condi;on" and" represent" the" mean" +/9" SEM" of" 3" independent" experiments" each" performed" in" triplicate." " E. , , EvaluaDon, of, the, anDBHBV, effect, of, approved, and, invesDgaDonal, molecules., dHepaRG" cells" were"infected"with"HBV"at"100"vge/cell"for"6"days"and"super9infected"with"HDV"at"the"indicated"at"10"vge/ mL." Three" days" post" HDV" infec;on," cells" were" treated" with" (A)" Tenofovir," (B)" IFNa," (C)" FTI9277," (D)" Tenofovir"and"IFNa,"(E)"FTI9277"and"IFNa"for"10"days."(F)"Cells"were"treated"with"Myrcludex"B"(Myr)"either"2" hours"before"and"during"HDV"inocula;on"(Pre)"or"once"the"infec;on"was"established"as"described"for"the" other" drugs." Levels" of" intracellular" HBV" RNA" (RNAic)" or" secreted" HBV" DNA" (DNA" SN)" were" respec;vely"" assessed"by"qRT9PCR"and"qPCR."HBeAg"and"HBsAg"secre;on"were"assessed"by"ELISA."Results"are"presented" as" ra;o" to" the" non" treated" condi;on" and" represent" the" mean" +/9" SEM" of" at" least" 3" independent" experiments"performed"in"triplicate." ,dHepaRG"cells"were"infected"with"HBV"at"100"vge/cell"for"6"days"and" super9infected"with"HDV"at"the"indicated"at"10"vge/mL."Three"days"post"HDV"infec;on,"cells"were"treated" with"Tenofovir,"IFNa"or"FTI9277"for"10"days."Cells"were"also"treated"with"Myrcludex"B"(Myr)"either"2"hours" before"and"during"HDV"inocula;on"(Pre)"or"once"the"infec;on"was"established"as"described"for"the"other" drugs." Levels" of" secreted" ApoB" were" assessed" by" ELISA." When" a" decrease" was" iden;fied," lactate" desidrogenase" (LDH)" ac;vity" was" measured." Results" are" presented" as" ra;o" to" the" non" treated" condi;on" and" represent" the" mean" +/9" SEM" of" 3" independent" experiments" performed" in" triplicate." TDF," tenofovir;" IFN," interferon;" Myr,myrclydex;" Pre," treatment" before" and" during" inocula;on;" post," treatment" aier" infec;on."

A model of super-infection with HDV on HBV-infected hepatocytes was established;

HDV infection induces a strong IFN response in these immune-competent hepatocytes;

In this model, HDV infection is associated with HBV inhibition, thus access to recapitulating in vivo viral interference;

This super infection model is also suitable for the evaluation of novel drugs/antivirals, including immune-modulators.

Figure 3 .

 3 Figure 3. Kinetics of IL-6, IFNs, and ISGs expression in HDV-infected cells & MOI-

Figure 4 .

 4 Figure 4. HDV super-infection of HBV infected dHepaRG leads to secretion of HDV

Figure 6 .

 6 Figure 6. HDV super-infection of HBV-infected cells leads to inhibition of HBV

Figure 7 .

 7 Figure 7. HDV super-infection affects HBeAg, but not HBsAg, secretions and

Figure 8 .

 8 Figure 8. Evaluation of the anti-HDV effect of approved and investigational

Figure 1

 1 Figure 1.

Figure 4

 4 Figure 4.

Figure 7 .

 7 Figure 7.

Figure 8 .

 8 Figure 8.

  Figure,S1."HDV,viral,inoculums,producDon."(A)"HDV"was"produced"in"vitro"by"co9transfec;on"of"Huh7" cells" with" T7HB2.7" (coding" for" PreS19PreS29S" from" HBV)" and" pSVLD3" (containing" a" trimer" of" HDV" genotype" 1" genome)." Supernatant" was" collected" every" other" day" from" day99" to" day915" post9 transfec;on." (B)" Northern" blot" analysis" (with" a" full9length" an;9genomic" probe," for" detec;on" of" HDV" genome)"of"HDV"RNA"in"the"supernatant"of"transfected"cells"throughout";me."G,"genome."

  Figure,S2.,Set,up,of,the,HDV,RTBqPCR."(A)"Serial"109fold"dilu;ons"of"a"full9length"HDV91"RNA"genomic" transcript" are" used" as" a" quan;fica;on" standard," confirm" PCR" linearity" within" a" range" of" 102" to" 108" copies/"reac;on"with"a"PCR"efficiency"of"~2,1."(B)"Electrophoresis"of"PCR"products"evidences"a"unique" band" located" between" 100" and" 200bp," consistent" with" the" predicted" 129bp" amplicon." (C)" Serial" 109 fold" dilu;ons" of" RNA" extracted" from" cell" culture" supernatant," confirm" PCR" linearity" between" 6X101" and"6X105"copies/"reac;on,with"a"PCR"efficiency"of"~2,2."(D)"Serial"39fold"dilu;ons"of"intracellular"total" RNA"confirm"a"linear"HDV"amplifica;on"within"a"range"of"0.4933,3"ng"of"total"RNA"per"PCR"reac;on."(E)" Mel;ng"curve"plot"of"HDV"infected"samples,"confirming"one"single"Tm"peak"consistently"found"at"84ºC." Lad,"DNA"ladder"(100bp,"New"England"Biolabs);"NC,"nega;ve"control;"Tm,"mel;ng"temperature."

Figure ,

 , Figure,S6."HDV,does,not,induce,specific, death,of,infected,dHepaRG,cells."(A,,B,, C)" dHepaRG" cells" were" infected" with" H D V" a t" 1 0" v g e / c e l l" a n d" ( A )" Apolipoprotein" B" secre;on" as" well" as" (B,, C)" cells" viability" were" assessed" by" ELISA," neutral" red" or" sulforodamin" assays."Results"are"presented"as"ra;o"to" non"infected"cells"(mock)"and"represent" the" mean" +/9SEM" of" 8" replicates" (D)" dHepaRG" were" treated" with" the" i n d i c a t e d" c o n c e n t r a ; o n" o f" staurosporine" and" QVD9OPH" for" 16" hours" "and"ALT"ac;vity"were"measured" with" colorimetric" assays." (E)" dHepaRG" cells"were"infected"by"HDV"and"treated" with" QVD9OPH" for" 12" day." Levels" of" intracellular"HDV"RNA"were"assessed"by" RT9qPCR."Results"are"presented"as"ra;o" to" the" mock" condi;on" and" represent" the" mean" +/9" SEM" of" 3" independent" experiments" each" performed" in" triplicate." "

  Figure, S7., EvaluaDon, of, the, anDBHBV, effect, of, approved, and, invesDgaDonal, molecules., dHepaRG" cells" were"infected"with"HBV"at"100"vge/cell"for"6"days"and"super9infected"with"HDV"at"the"indicated"at"10"vge/ mL." Three" days" post" HDV" infec;on," cells" were" treated" with" (A)" Tenofovir," (B)" IFNa," (C)" FTI9277," (D)" Tenofovir"and"IFNa,"(E)"FTI9277"and"IFNa"for"10"days."(F)"Cells"were"treated"with"Myrcludex"B"(Myr)"either"2" hours"before"and"during"HDV"inocula;on"(Pre)"or"once"the"infec;on"was"established"as"described"for"the" other" drugs." Levels" of" intracellular" HBV" RNA" (RNAic)" or" secreted" HBV" DNA" (DNA" SN)" were" respec;vely"" assessed"by"qRT9PCR"and"qPCR."HBeAg"and"HBsAg"secre;on"were"assessed"by"ELISA."Results"are"presented" as" ra;o" to" the" non" treated" condi;on" and" represent" the" mean" +/9" SEM" of" at" least" 3" independent" experiments"performed"in"triplicate."

  Figure S8.

Table , S1."List,of,primers,and,probes,used,for,qPCR""

 ,

	Designation		Sequence (5'-3')
	HDV	Forward Primer	CGGGCCGGCTACTCTTCT
		Reverse Primer	AAGGAAGGCCCTCGAGAACA
	HBV total	Forward Primer	GCT GAC GCA ACC CCC ACT
		Reverse Primer	AGG AGT TCC GCA GTA TGG
	HBV pgRNA	Forward Primer	GGA GTG TGG ATT CGC ACT CCT
		Reverse Primer	AGA TTG AGA TCT TCT GCG AC
	HBV cccDNA	Forward Primer	CTC CCC GTC TGT GCC TTC T
		Reverse Primer	GCC CCA AAG CCA CCC AAG
		Probe	GTT CAC GGT GGT CTC CAT GCA ACG T
		Probe	AGG TGA AGC GAA GTG CAC ACG GAC C
	GUS	Forward Primer	CGTGGTTGGAGAGCTCATTTGGAA
		Reverse Primer	ATTCCCCAGCACTCTCGTCGG
	RPLP0	Forward Primer	CAC CAT TGA AAT CCT GAG TGA TGT
		Reverse Primer	TGA CCA GCC CAA AGG AGA AG
	B-globin	Forward Primer	ACA CAA CTG TGT TCA CTA GC
		Reverse Primer	CAA CTT CAT CCA CGT TCA CC
			CAA ACA GAC ACC ATG GTG CAC CTG ACT
		Probe	CCT GAG GA
			AAG TCT GCC GTT ACT GCC CTG TGG
		Probe	GGC AA
	IL6	Forward Primer	ACCCCTGACCCAACCACAAAT
		Reverse Primer	AGCTGCGCAGAATGAGATGAGTT
	IFNa	Forward Primer	GTGAGGAAATACTTCCAAAGAATCAC
		Reverse Primer	TCTCATGATTTCTGCTCTGACAA
	IFNb	Forward Primer	GCCGCATTGACCATGTATGAGA
		Reverse Primer	GAGATCTTCAGTTTCGGAGGTAAC
	OAS1	Forward Primer	AGGTGGTAAAGGGTGGCTCC
		Reverse Primer	ACAACCAGGTCAGCGTCAGAT
	ISG15	Forward Primer	ATGGGCTGGGACCTGACG
		Reverse Primer	GCCAATCTTCTGGGTGATCTG
	MxA	Forward Primer	GGTGGTCCCCAGTAATGTGG
		Reverse Primer	CGTCAAGATTCCGATGGTCCT
	RSAD2	Forward Primer	CTTTGTGCTGCCCCTTGAG
		Reverse Primer	TCCATACCAGCTTCCTTAAGCAA
	RIG-I	Forward Primer	GCTGATGAAGGCATTGACATTG
		Reverse Primer	CAGCATTACTAGTCAGAAGGAAGCA
	MDA5	Forward Primer	CCCATGACACAGAATGAACAAAA
		Reverse Primer	CGAGACCATAACGGATAACAATGT

Acknowledgements

The authors would like to thank Dr Alan Kay for the kind gift of anti-HDAg antibodies and Dr Stephen Urban for the gift of Myrcludex® and the HepG2 hNTCP cell line.

Dulce Alfaiate was supported by grants from Fundação Calouste Gulbenkian and Fundação para a Ciência e a Tecnologia. DD, JL, FZ, and PD were supported by grants from ANRS (French national agency for research on AIDS and viral hepatitis; several grants from CSS4), FINOVI (Foundation for innovation in infectiology; project call n°#4), and by INSERM core grants. PD was supported by an INSERM Interface contract. DD and FZ were also supported by FRM (Foundation for medical research; DEQ20110421327), and DEVweCAN LABEX (ANR-10-LABX-0061) of the "Université de Lyon", within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). Author's contributions: -Study concept and design: DA, JL, FZ, PD and DD -Acquisition of data: DA, MM, NAS, JL -Analysis and interpretation of data: DA, JL, PD and DD -Writing of the manuscript: DA, CS, FZ,


Julie Lucifora

Ulrike Protzer

Prof Ulrike Protzer

Attacking Hepatitis B Virus cccDNA -the Holy Grail to Hepatitis B Cure

Keywords: hepatitis B virus, cccDNA, liver no conflict of interest financial support:

table number: 1 list of abbreviations: Hepatitis B virus (HBV), relaxed circular DNA (rcDNA), primary human hepatocyte (PHH), differentiated HepaRG (dHepaRG), Na + -taurocholate cotransporting peptide (NTCP), humanized liver-chimeric (HuHEP), Rolling circle amplification (RCA), protein-free rcDNA (pf-rcDNA), tyrosyl-DNA-phosphodiesterase (TDP), disubstituted sulfonamides (DSS), interferon alfa (IFN-α) tumor necrosis factor (TNF), lymphotoxin beta p (L βR) APOBEC3A (A3A), APOBEC3B (A3B) a a ( FNγ) AL nucleases (TALENs).

Introduction

Hepatitis B virus (HBV) has optimized its life cycle for long-term persistence in the liver. To achieve this, the virus establishes a plasmid-like covalently closed circular DNA form, the socalled cccDNA. HBV cccDNA serves as template for transcription of all HBV RNAs and production of progeny virus. HBV cccDNA resides in the nucleus of infected cells as an episomal (i.e. non-integrated) DNA associated to histones. It drives production of viral antigens such as HBeAg and HBsAg, and progeny virions. In contrast to cccDNA, the DNA contained in infectious virions is an only partially double stranded, relaxed circular DNA (rcDNA) that results from reverse transcription of a pregenomic RNA.

Available antivirals cannot cure hepatitis B because they target reverse transcription as a late step in the HBV life cycle and do not affect the cccDNA transcription template. Nucleos(t)ide analogues such as lamivudine, entecavir or tenofovir can efficiently control progeny release and thus viremia, but neither cccDNA persistence nor HBsAg or HBeAg expression and secretion. Similarly, neither entry inhibitors nor capsid assembly modifiers as novel antivirals under clinical development target HBV cccDNA ( "New antiviral targets for novel treatment concepts for HBV", Durantel and Zoulim). Therefore, long-term treatment is required, which is expensive, challenges patient adherence and may lead to concomitant resistance [START_REF] Trepo | Hepatitis B virus infection[END_REF]. Interferon (IFN)-α, which is also licensed for hepatitis B therapy and has direct antiviral as well as immune modulatory effects, may result in HBsAg loss and even virus clearance in a proportion of patients, but dosing is limited by severe side effects [START_REF] Wursthorn | Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B[END_REF]. Therefore, efficient removal of cccDNA from infected hepatocytes is regarded as the " a " to achieve HBV cure. This review will summarize the different experimental models and detection assays available to study cccDNA, our current knowledge on cccDNA biology as well as potential therapeutic approaches to attack cccDNA.

Experimental models to study the cccDNA fate

For a long time, in vitro and in vivo models to study the fate of HBV cccDNA were rather limited (see Experimental models (cell culture and animals) for studies of human HBV pathobiology and new treatment concepts, M Dandri). Only primary tupaia or human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) were used allowing HBV infection and thus replication of HBV from its natural transcription template, the cccDNA. These models, however, are very demanding. The recent identification of the Na + -taurocholate cotransporting peptide (NTCP) as a crucial HBV receptor [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF] allowed establishing new cell lines that support HBV infection and the study of cccDNA in vitro.

While short-term experiments were enabled by an improved HBV infection rate (and consequently higher levels of cccDNA) in NTCP-overexpressing HuH7, HepaRG and in particular HepG2 cells [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF][START_REF] Ni | Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes[END_REF], experiments to study the fate and regulation of cccDNA over time remain challenging. Only HepaRG cells can be kept infected in culture for more than a month, but they differentiate into two different cell types (biliary like and hepatocyte like cells) and establish only low levels of cccDNA (see Table 1). Other cell lines keep dividing and cell division results in loss of cccDNA (see below). This technically challenges our research. It is furthermore important to note that despite recent improvements, in vitro HBV infections are still rather inefficient, requiring a huge excess of virus (100-10,000 virions/cell depending on the virus inoculum, the cell type and infection protocol used) and polyethylene glycol to facilitate virus fusion. Moreover, there is no evidence of HBV spread in cell culture -unlike for other viruses.

In vivo models are limited by the strict species specificity of HBV that only infects human and humanoid primates. Studies using chimpanzees largely contributed to our understanding of the virus-host interaction [START_REF] Guidotti | Viral clearance without destruction of infected cells during acute HBV infection[END_REF] but have since been banned [START_REF] Harrington | State of the (research) chimp[END_REF]. While chimpanzee hepatocytes support HBV replication [START_REF] Gheit | Experimental transfection of Macaca sylvanus with cloned human hepatitis B virus[END_REF][START_REF] Lucifora | Hepatitis B virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model[END_REF][START_REF] Dupinay | Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island[END_REF], monkeys such as macaques or baboons frequently used for research cannot be infected by HBV due to differences in the NTCP protein sequence [START_REF] Yan | Molecular determinants of hepatitis B and D virus entry restriction in mouse sodium taurocholate cotransporting polypeptide[END_REF]. Macaques expressing the human NTCP will, however, constitute a very promising experimental model. Mouse hepatocytes grafted with NTCP support HBV entry but do not allow cccDNA establishment [START_REF] Li | HBV life cycle is restricted in mouse hepatocytes expressing human NTCP[END_REF], probably because they lack key factors involved in nuclear transport of rcDNA or rcDNA to cccDNA conversion. Humanized liverchimeric (HuHEP) mice allow long-term study of cccDNA [START_REF] Allweiss | Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration[END_REF] but are immune deficient and due to technical and cost issues cannot not be used for large-scale experiments.

Understanding cccDNA biology, regulation and fate would thus greatly benefit from further improvements of the HBV infection models.

The challenging detection of HBV cccDNA

With low copy numbers of cccDNA in vitro (Table 1) and in vivo, detection of cccDNA is a huge challenge for the HBV research field. Mean values from 0.01 to 1 copies per cell in livers of HBV-infected patients [START_REF] Werle-Lapostolle | Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy[END_REF] or HuHEP mice [START_REF] Allweiss | Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration[END_REF] have been reported. This means that only a portion of the cells were actually infected by HBV, but methods to detect cccDNA on a per-cell level are lacking. Moreover, true discrimination of cccDNA from almost identical viral linear DNA or rcDNA increases the challenge, because these exist in vast excess in virions and in viral capsids in the cytoplasm of infected cells. While Southern blot analyses can be used to distinguish between relaxed rcDNA, linear and supercoiled cccDNA, the sensitivity of this technique is rather low. To increase sensitivity, cccDNA selective qPCR methods have been developed that are based on the use of primers (and probes) spanning the nick in rcDNA and hybridizing in its " ap " (Figure 1). With the amazing technical development in PCR-based methods over the past years [START_REF] Mu | A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system[END_REF], the sensitivity of cccDNA detection is no longer an issue. Specificity of the cccDNA PCR methods, however, remains a big concern and correct quantification of cccDNA is hampered by false positive detection of rcDNA HBV genomes that are present in vast excess in infected cells. Rolling circle amplification (RCA) coupled or not with nested PCR has been described to increase specificity of cccDNA detection [START_REF] Margeridon | Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA[END_REF][START_REF] Zhong | Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR[END_REF][START_REF] Zhong | A novel method for detection of HBVcccDNA in hepatocytes using rolling circle amplification combined with in situ PCR[END_REF] but has not been widely accepted. Extraction of nuclear DNA only [START_REF] Pollicino | Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones[END_REF], plasmid safe DNAse that digests linear viral DNA, but not rcDNA [START_REF] Kock | Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner[END_REF] and T5 exonuclease that degrades rcDNA but should leave cccDNA molecules intact (Xia et al, Hepatitis B Virus: Methods and Protocols, in press) can also be used to increase specificity. Of note, these exonucleases have different efficacy depending on the type and a " a a " DNA. So far, laboratories apply different protocols and a consensus or standard has yet to be reached. This, however, should be of high priority given that also the protocol of DNA extraction may influence cccDNA detection by generating nicks in cccDNA molecules that may render them susceptible to exonuclease treatment, may prevent RCA detection and may change the running behavior in gels before Southern blotting.

Establishment of HBV cccDNA in the nucleus

Upon infection, cccDNA is formed as a plasmid-like episome in the host cell nucleus from the rcDNA genome in incoming virions that is linked to the viral polymerase protein.

Alternatively, cccDNA may be established from capsid rcDNA that can be reimported into the nucleus after it has been newly formed in infected cells. cccDNA was recently detected in a mouse hepatocyte cell line immortalized by stable expression of the human transforming growth factor alpha [START_REF] Cui | Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization[END_REF] supporting the fact that human host factors are essential for cccDNA formation and more precisely for destabilization of mature nucleocapsids. Nuclear localization signals within the arginine-rich C-terminal domain of the HBV core protein [START_REF] Li | Nuclear export and import of human hepatitis B virus capsid protein and particles[END_REF] trigger transport of the rcDNA-containing HBV capsid to the nucleus where the capsid attaches to nucleoporin 153 and is disintegrated [START_REF] Schmitz | Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket[END_REF]. Upon translocation into the nucleus, the conversion of the rcDNA genome into cccDNA takes place via a multi-step process that remains largely obscure although it has been linked to DNA repair mechanisms [START_REF] Nassal | HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B[END_REF].

Firstly, the rcDNA needs to be detached from the HBV polymerase and its RNA primer that are remnants from reverse transcription that took place within the viral capsid. Hereby, a protein-free rcDNA form (pf-rcDNA, equivalent to DP-rcDNA for deproteinized rcDNA) is generated. Pf-rcDNA may persist in the nucleus before conversion into cccDNA, and has even been observed in the cytoplasm [START_REF] Guo | Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation[END_REF][START_REF] Guo | Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses[END_REF][START_REF] Guo | Characterization of the host factors required for hepadnavirus covalently closed circular (ccc) DNA formation[END_REF]. Secondly, the incompleteness of the rcDNA (+) a d ( " ap") a d a d da its (-) a d ( " k") b pa d (Figure 2). It has recently been demonstrated that tyrosyl-DNA-phosphodiesterase (TDP) 2 can specifically cleave the Tyr-DNA bond to release the polymerase protein from HBV rcDNA [START_REF] Koniger | Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses[END_REF]. The fact that TDP2 knockout HepG2-NTCP cells engineered by a CRISPR/Cas9-based approach still proved susceptible to HBV infection [START_REF] Cui | Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?[END_REF] indicated that alternative pathway(s) exist involving nucleolytic cleavage by (a) still elusive endonuclease(s). Comparison of HBV and duck hepatitis B virus cccDNA formation revealed an unexpected contribution of the virus itself to cccDNA formation. DHBV but not HBV rcDNA converted efficiently into cccDNA in the same cells [START_REF] Kock | Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner[END_REF]. Linear HBV DNA, a probably accidental by-product of reverse transcription, cannot be transcribed per se but may form cccDNA-like molecules by nonhomologous end joining. Most of these, however, will be functionally defective [START_REF] Nassal | HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B[END_REF].

Structure and regulation of the HBV cccDNA

Once formed, cccDNA persists as an episome in the nucleus of infected cells and viral mRNAs are transcribed by the cellular RNA polymerase II. The cccDNA molecule is organized into a chromatin-like structure that displays the typical beads-on-a-string arrangement in electron microscopy [START_REF] Newbold | The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes[END_REF][START_REF] Bock | Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell[END_REF]. Attached proteins are host histone but also non-histone proteins [START_REF] Bock | Structural organization of the hepatitis B virus minichromosome[END_REF].

During the last years major efforts have been made to demonstrate that the transcriptional a DNA bj " d " b in vitro and in vivo [START_REF] Pollicino | Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones[END_REF][START_REF] Belloni | Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function[END_REF][START_REF] Benhenda | Methyltransferase PRMT1 is a binding partner of HBx and a negative regulator of hepatitis B virus transcription[END_REF]. Using ChIP approaches, the recruitment of H3 and H4 histones, as well as that of several cellular transcription factors (including CREB, ATF, STAT1/2) and histone modifying enzymes (such as CBP, p300, PCAF/GCN5, HDAC1, hSirt1, PRMT1) has been demonstrated and was associated with modulation of cccDNA transcriptional activity [START_REF] Pollicino | Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones[END_REF][START_REF] Belloni | Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function[END_REF][START_REF] Belloni | IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome[END_REF]. Using a chromosome immunoprecipitation approach followed by next generation sequencing, the first genomewide map of histone posttranslational modifications in cccDNA-associated chromatin was etablished [START_REF] Tropberger | Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation[END_REF]. This revealed that HBV chromatin shares some basic features with cellular chromatin, in particular marks linked to active transcription, but lacks significant levels of posttranslational histone modifications associated with transcriptional repression, which, in cellular chromatin, are indispensable for transcriptional control [START_REF] Tropberger | Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation[END_REF]. When applied to de novo infected HepG2-NTCP cells or primary human hepatocytes or to HBV-infected liver tissue the analysis revealed general features of cccDNA chromatin organization as well as features that are unique to the respective source of cccDNA [START_REF] Tropberger | Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation[END_REF]. This emphasizes the importance to repeat experiments and to confirm results using different infection models.

Despite being named a " " B DNA unlike other chromosomes does neither contain an origin of replication nor possess a nuclear retaining signal. It thus cannot replicate by itself in infected hepatocytes and should theoretically be excluded from the nucleus after cell division. cccDNA persistence therefore relies on its stability in the nucleus and on its replenishment via reimport of rcDNA from cytoplasmic capsids after cell division.

The half-life of HBV cccDNA has not been exactly defined and varies with inflammatory activity resulting in hepatocyte turnover. Rebound of HBV replication upon withdrawal from nucleos(t)ide analogue treatment [START_REF] Zoulim | Hepatitis B virus resistance to antiviral drugs: where are we going?[END_REF] and traces of HBV-DNA remaining detectable years after clinical recovery from acute hepatitis [START_REF] Rehermann | The hepatitis B virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response[END_REF], however, indicate that HBV cccDNA is very long-lived in the human liver and can persist for decades. In vitro, cccDNA also appears to be very stable [START_REF] Cai | Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation[END_REF][START_REF] Zhou | Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays[END_REF] independently of its transcriptional activity as demonstrated in HepaRG cells infected with an HBx-deficient HBV unable to transcribe mRNA [START_REF] Lucifora | Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection[END_REF]. Since it is not selfmaintained, cccDNA should theoretically be lost by cell division e.g. in case of liver regeneration. However, cccDNA may survive through mitosis even though it has been observed that induction of cell division results in a dramatic decrease of cccDNA [START_REF] Lutgehetmann | In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice[END_REF][START_REF] Guo | Conditional replication of duck hepatitis B virus in hepatoma cells[END_REF][START_REF] Reaiche-Miller | Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver[END_REF][START_REF] Summers | Hepatocyte turnover during resolution of a transient hepadnaviral infection[END_REF][START_REF] Allweiss | 127 PROLIFERATION OF HEPATITIS B VIRUS INFECTED HUMAN HEPATOCYTES INDUCES SUPPRESSION OF VIRAL REPLICATION AND RAPID cccDNA DECREASE IN HUMANIZED MICE[END_REF]. This a b d a " " a p DNA w d a ap d nucleus.

A combination of in vitro approaches, including electromobility shift and DNA protection assays, has shown that a number of transcription factors bind to HBV regulatory elements [START_REF] Quasdorff | Control of hepatitis B virus at the level of transcription[END_REF]. Among them, NF1α a d NF4α a the crucial nuclear factors for HBV transcription needed at sufficient levels [START_REF] Quasdorff | A concerted action of HNF4alpha and HNF1alpha links hepatitis B virus replication to hepatocyte differentiation[END_REF][START_REF] Palumbo | IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome[END_REF]. Viral proteins are also very important in cccDNA structure and control. In particular, the non-structural protein HBx is associated with cccDNA [START_REF] Belloni | Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function[END_REF] and essential to initiate and maintain transcription from cccDNA [START_REF] Lucifora | Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection[END_REF]. Indeed, using dHepaRG cells in which HBx is expressed in a tetracyclin-regulated fashion and infecting them with an HBx-deficient HBV mutant, we showed that transcription of viral RNA exactly followed the switching on and off of HBx expression [START_REF] Lucifora | Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection[END_REF]. Hereby it appeared that HBV transcription is repressed in the absence of HBx rather than HBx functioning as a typical viral transactivator. This repression is characterized by a more compact structure of cccDNA [START_REF] Riviere | HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase[END_REF] and associated with repressive epigenetic marks [START_REF] Belloni | Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function[END_REF][START_REF] Riviere | HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase[END_REF] on cccDNA that were absent in the presence of HBx [START_REF] Tropberger | Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation[END_REF]. Interestingly, it was shown that HBx supports HBV gene expression by a conserved mechanism that acts specifically on episomal DNA templates and requires HBx binding to the cellular DDB1 E3 ubiquitin ligase [START_REF] Van Breugel | Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates[END_REF]. Altogether these data suggest that HBx may block cellular factor(s) inducing mechanisms that inhibit transcription from cccDNA such as SETDB1 [START_REF] Riviere | HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase[END_REF], Spindlin1 [START_REF] Ducroux | The Tudor domain protein Spindlin1 is involved in intrinsic antiviral defense against incoming hepatitis B Virus and herpes simplex virus type 1[END_REF] as well as essential other factors that were reported at the recent international HBV meeting and will be published soon. In addition to HBx, the HBV core protein (HBc) binds to cccDNA resulting in a rearrangement of the nucleosomal spacing of the HBV nucleoprotein complexes [START_REF] Bock | Structural organization of the hepatitis B virus minichromosome[END_REF]. Whether HBc also regulates transcription from cccDNA is still debated. On one hand, it was shown that HBc alters the cccDNA methylation profile and therefore maintains a permissive epigenetic state in a critical region of the HBV cccDNA [START_REF] Guo | HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state[END_REF]. On the other hand, a decreased recruitment of HBc on HBVx-cccDNA was recently described but did not account for the failure in cccDNA transcription [START_REF] Riviere | HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase[END_REF]. The origin of HBc attached to cccDNA (i.e. whether it stems from the incoming virus or from viral replication), the kinetic of its recruitment onto cccDNA and its stability once bound to cccDNA remain unresolved questions that would certainly help to understand its role on cccDNA regulation. Noteworthy, as HBc is important for cccDNA structure and/or control, capsid assembly modifiers (CpAMs) that are currently in clinical development were hypothesized to also have an effect on cccDNA [START_REF] Belloni | 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[END_REF]. This, however, needs to be proven.

Targeting HBV cccDNA to achieve HBV cure

Different options to target cccDNA to achieve affect HBV persistence are available: inhibition of its formation, inhibition of its function or inducing its degradation (Figure 3).

Two structurally related disubstituted sulfonamides (DSS) were shown to interfere with rcDNA conversion into cccDNA [START_REF] Cai | Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation[END_REF]. Given the proposed contribution of the DNA-damage response in this process, viral proteins or substances interfering with the DNA-damage response should also interfere with rcDNA to cccDNA conversion. Considering the long halflife of cccDNA, however, these compounds should only have an effect if given during the establishment of HBV infection or during phases of high hepatocyte turnover.

Once cccDNA is formed its transcription can be inhibited by cytokines. For instance, we demonstrated that IL-6 inhibits HBV transcription from cccDNA by reducing the levels of the a a p a NF1α a d NF4α a a AP k a pathway [START_REF] Hosel | Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection[END_REF]. This results in a loss of NF1α a d NF4α binding to the cccDNA, which is accompanied by a redistribution of STAT3 binding from cccDNA to cellular IL-6 target genes [START_REF] Palumbo | IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome[END_REF]. It has also been shown that cccDNA transcription can be silenced by the use of IFN-α small molecules targeting chromatin modifying enzymes [START_REF] Belloni | IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome[END_REF][START_REF] Protzer | Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection[END_REF][START_REF] Belloni | Mimicking Interferon-α ( FNα) b a p-atitis B virus (HBV) transcription and replication by tar-geting the epigenetic control of nuclear cccDNA minichromosome with epigenetic small molecules[END_REF][START_REF] Liu | Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes[END_REF]. Genome-wide mapping of histone modifications revealed that transcription and active modifications in HBV chromatin can be reduced when innate immunity pathways are activated, or by a small molecule epigenetic modifying agent [START_REF] Tropberger | Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation[END_REF]. This supports the idea that chromatin-based regulation of cccDNA transcription could be of therapeutic use. A better understanding of the function of HBx protein and its interaction with DDB1 in the E3 ubiquitin ligase complex [START_REF] Li | A promiscuous alpha-helical motif anchors viral hijackers and substrate receptors to the CUL4-DDB1 ubiquitin ligase machinery[END_REF], that potentially results in degradation of host proteins able to control HBV, may allow to identify new targets for antiviral drug discovery. Because these strategies that aim at inhibiting transcription from cccDNA will likely only have a transient effect, they may help to achieve at least a functional cure of HBV.

Purging cccDNA from the nucleus of infected cells is a much more promising approach since this will result in a sustained control of HBV or even in HBV cure. In chimpanzees, it has been shown that clearance of HBV replication, but also HBV cccDNA, occurs before full-blown cytotoxicity of T cells hits the liver in acute hepatitis B [START_REF] Guidotti | Viral clearance without destruction of infected cells during acute HBV infection[END_REF]. This indicates that the cccDNA pool can be affected by non-cytolytic mechanisms involving cytokines such as IFNs and tumor necrosis factor (TNF) [START_REF] Guidotti | Viral clearance without destruction of infected cells during acute HBV infection[END_REF]. Recently, we showed that induction of intrahepatic antiviral immune responses (through high doses of IFN-α L βR-agonists) is able to trigger noncytolytic degradation of cccDNA from infected cells in vitro [START_REF] Lucifora | Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA[END_REF]. Hereby, activation of nuclear deaminases such as APOBEC3A (A3A) and APOBEC3B (A3B) results in cccDNA deamination and a significant reduction of cccDNA [START_REF] Lucifora | Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA[END_REF]. Although A3A or A3B-mediated deamination was essential, the exact pathways and enzymes involved in downstream degradation of HBV-DNA still need to be deciphered. and to a minor extend also A3B in a synergistic fashion [START_REF] Xia | Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis[END_REF]. To study whether these and eventually also other T cell-derived cytokines contribute to cccDNA loss in a non-cytolytic fashion, we set up a transwell system, which allowed us to separate cytolytic and noncytolytic T cell functions. Hereby, we took advantage of chimeric antigen receptor (S-CAR) grafted T cells [START_REF] Bohne | T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes[END_REF][START_REF] Krebs | T Cells Expressing a Chimeric Antigen Receptor That Binds Hepatitis B Virus Envelop Proteins Control Virus Replication in Mice[END_REF] that recognize HBsAg on the surface of infected cells but could confirm the findings when T cells carried the natural T cell receptor. While upon direct contact in co-culture T cells killed HBV infected cells, they were only able to release cytokines when separated by the transwell. cccDNA loss, however, was observed in both settings and clearly A3A/A3B dependent [START_REF] Xia | Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis[END_REF]. Thus, HBV-specific T cells were able to eliminate HBV in a cytolytic and in a non-cytolytic fashion. This shows that cccDNA degradation in a non-cytolytic fashion is possible and may support HBV cure approaches by therapeutic vaccination, T cell redirection or other immune cell activation strategies such as e.g. PD-1 or PD-ligand1 blockade ( "New antiviral targets for novel treatment concepts for HBV", Durantel and Zoulim).

Finally, as an alternative to immune activation that may result in tissue damage on the long term if not successful in eliminating HBV, DNA cleaving enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases, TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, specifically targeted to the HBV cccDNA are currently being engineered [START_REF] Cradick | Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs[END_REF][START_REF] Schiffer | Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach[END_REF][START_REF] Chen | An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases[END_REF][START_REF] Bloom | Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases[END_REF][START_REF] Seeger | Targeting Hepatitis B Virus With CRISPR/Cas9[END_REF][START_REF] Kennedy | Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease[END_REF][START_REF] Dong | Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication[END_REF]. Clinical development of these interesting strategies, however, is limited by potential off-target effects resulting in cellular DNA damage. In addition, efficient delivery strategies targeting all infected hepatocytes are lacking and therapeutic application would currently require a gene therapy approach.

Taken together, further basic research and more detailed molecular studies are needed to evaluate the translational potential of the novel antiviral strategies mentioned to finally achieve a true HBV cure.

Figure and table legends

Maintenance of infec:on

Primary human hepatocytes 20 -100 % 1 to 2 2-3 weeks (58) dHepaRG 5 -20 % 0.2 to 0.5 > 6 months (40) HepG2-NTCP 50 -100 % 1 to 5 10-15 days (4) HuHep mice 100 % 1 to 2 > 4 months (13)

  They are likely contributing to baseexcision or nucleotide-excision repair pathways. It should also be determined if cccDNA can be fully purged or if a portion might be refractory to the treatments. Next we examined whether the mechanism of A3A/B-mediated deamination might contribute to T cellmediated loss of cccDNA. We found that T-cell d d k FNγ a d NF d A3A

Table 1 :

 1 cccDNA levels and estimated time of maintenance of HBV infection in different in vitro and in vivo models.

Figure 1 :

 1 Figure 1: cccDNA qPCR primers spanning the "nick region" of the HBV relaxed circular DNA (rcDNA). Examples of qPCR primers designed to selectively amplify cccDNA over rcDNA.

Figure 2 :

 2 Figure 2: Schematic representation of rcDNA conversion to cccDNA. (i) Removal of viral p a k d 5' d (-) DNA strand and of one of the redundant q ( ) a RNA p k d 5' d (+) a d ( ) a d completion of the viral (+) DNA strand, and (iv) ligations of DNA extremities for both (+) and (-) strands. (v) Once formed, cccDNA is supercoiling and associating with proteins, such as histones, to form the so-called cccDNA mini-chromosome.

Figure 3 :

 3 Figure 3: Targeting cccDNA to achieve HBV cure. *EMA = epigenetic modifying agent; **HBP = HBx binding partner activation

Figure 3

 3 Figure 1

Table 1545

 1545

	$1563''
	Werle%Lapostolle,
	et#al.,,2004,

Acknowledgments

The authors thank Judith Fresquet for help with figure 1.

W k a ' ab a wa pp d b E p a Association of the Study of Liver Diseases (EASL), the German Center for Infection Research (DZIF), the French Agency for Research on AIDS and Hepatitis Viruses (ANRS), the FINOVI Foundation, the French Association for Research on Cancer (ARC), the Helmholtz Association, the German Ministry for Research and Education (BMBF), the German Ministry for Economics (BMWi) and the German Research Foundation (DFG).