Control of Hepatitis B Virus replication by innate response of HepaRG cells

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Control of Hepatitis B Virus replication by innate response of HepaRG cells

Julie Lucifora, David Durantel, Barbara Testoni, Olivier Hantz, Massimo Levrero, Fabien Zoulim

To cite this version:

Julie Lucifora, David Durantel, Barbara Testoni, Olivier Hantz, Massimo Levrero, et al.. Control of Hepatitis B Virus replication by innate response of HepaRG cells. Hepatology, Wiley-Blackwell, 2010, 51 (1), pp.63-72. �10.1002/hep.23230�. �hal-03315490�


1 HEP-09-0579, revised version

1 2

Control of Hepatitis B Virus replication by innate response


of HepaRG cells

4 5 6

Julie Lucifora1,2, David Durantel1,2,3, Barbara Testoni4,5, Olivier Hantz1,2, Massimo 7

Levrero4,5 and Fabien Zoulim1,2,3,*. 8

9 10

1) INSERM, U871, 151 Cours Albert Thomas, 69003 Lyon, France;


2) Université Lyon 1, IFR62 Lyon Est, 69008 Lyon, France ; 12

3) Hospices Civils de Lyon, Hôtel Dieu Hospital, 69002 Lyon, France;


4) Department of internal Medecine and Laboratory of Gene Expression, Fondazione A.


Cesalpino, University of Rome La Sapienza, Rome, Italy 15

5) Laboratoire Associé INSERM, U785, Villejuif, France.

16 17

Short title : 18


HBV and the induction of the IFN pathway 20


Keywords : 22


antiviral state, baculovirus, hepatitis B Virus, Interferon 24


Contact information : 26


*Fabien Zoulim 28

Mailing address: INSERM, U871, 151 cours Albert Thomas, 69003 Lyon, France.


Phone: +33437497410. Fax: +33437497419. E-mail: 30


List of abbreviations : 32


HBV (Hepatitis B Virus), IFN (interferon), ISG (interferon stimulated genes) 34


Financial support : 36


This work was supported by grants from the European Community (ViRgil LSHM-CT- 38

2004-503359), the French National Agency for Research against AIDS and viral 39

hepatitis (ANRS), and INSERM. J.L. was recipient from a PhD bursary from the ANRS.

40 41

Word count and figures number : 42


Abstract : 251 words 44

4990 words in text + reference 45

Figures : 7 + 1 supplementary file 46


No conflict of interest 48


2 Abstract


Background and aims : HBV is currently viewed as a stealth virus that does not 50

elicit innate immunity in vivo. This assumption has not yet been challenged in vitro 51

because of the lack of a relevant cell culture system. The HepaRG cell line, which is 52

physiologically closer to differentiated hepatocytes and permissive to HBV infection, has 53

opened new perspectives in this respect.


Methods : HBV baculoviruses were used to initiate an HBV replication in both 55

HepG2 and HepaRG cells. To monitor HBV replication, the synthesis of encapsidated 56

DNA, and secretion of HBsAg, was respectively analyzed by southern-blot and Elisa.


The induction of a type-I IFN response was monitored by targeted qRT-PCR, low 58

density arrays, and functional assays. The invalidation of type-I IFN response was 59

obtained by either antibody neutralisation or RNA interference.


Results : We demonstrate that HBV elicits a strong and specific innate antiviral 61

response that result in a non-cytopathic clearance of HBV DNA in HepaRG cells.


Challenge experiment showed that transduction with Bac-HBV-WT, but not with control 63

baculoviruses, leads to this antiviral response in HepaRG cells, whereas no antiviral 64

response is observed in HepG2 cells. Cellular gene expression analyses showed that 65

IFN- and other interferon stimulated genes were up-regulated in HepG2 and HepaRG 66

cells, but not in cells transduced by control baculoviruses. Interestingly, a rescue of viral 67

replication was observed when IFN- action was neutralized by antibodies or RNA 68

interference of type-I IFN receptor.


Conclusions : Our data suggest that a strong HBV replication is able to elicit a 70

type-I IFN response in HepaRG-infected cells.

71 72


3 Background and aims


HBV is currently viewed as a non-cytopatic virus and HBV-associated liver 74

damage is thought to be the consequence of a long lasting cytolytic immune response 75

against infected hepatocytes (1). The outcome of HBV infection, as well as the severity 76

of HBV-induced liver disease, varies widely from one patient to another. In around 90- 77

95% of adults, exposure to HBV leads to an acute infection which is rapidly cleared 78

without long-term consequences. The remaining 5-10% fail to control viral infection that 79

consequently evolves to chronicity. The latter predisposes patients to severe liver 80

disease, including cirrhosis and hepatocellular carcinoma (2).


The intracellular innate antiviral response to infection constitutes an early 82

defense mechanism that aims at stopping replication in already infected cells, as well as 83

limiting the spreading of infection to neighboring cells. This response takes place very 84

early after infection of cells and plays an important stimulatory role for the subsequent 85

development of innate and adaptive immune responses. In many cases, cells mounting 86

this response produce type-I (IFN-,  (and type-III; i.e. IFN-) interferons which are 87

cytokines with pleiotropic functions that mediate both direct and indirect antiviral effect 88

(3-5). The direct antiviral effect of type-I IFNs is exerted by a variety of effectors that are 89

expressed by genes whose transcription is directly stimulated by IFNs, i.e. IFN- 90

stimulated genes (ISGs). The production of type-I IFNs can be triggered by virus 91

components and/or replication through cellular sensors that detect the presence of viral 92

RNA, DNA or proteins (4). The indirect antiviral effect of type-I IFNs is due to their 93

stimulatory effect on different cells of the innate and adaptive immune response.


Using experimentally-infected chimpanzees, microarray analyses suggested that 95

HBV, early in infection, does not modulate host cellular gene transcription and would 96

neither induce innate antiviral response in hepatocytes nor intrahepatic innate immune 97

responses (6). Following this study, HBV was qualified as a “stealth virus” (7). However 98


4 a study from the same group showed that HBV could be cleared from the livers of 99

infected animals before any detectable adaptive immune response (8), thus suggesting 100

that innate immunity, i.e. antiviral response at level of infected cells and innate response 101

via specialized cells of immune system (e.g. NK, NKT), could play an important role.


This was eventually demonstrated by a study on two HBV-seronegative blood donors 103

who became HBV DNA positive and were carefully and immediately monitored after the 104

onset of infection. In both patients, NK and NT cells were activated before maximal HBV 105

DNA elevation whereas HBV-specific T cell responses were maximal later when HBV 106

DNA was already declining, thus demonstrating that the innate immune system was 107

able to sense HBV infection (9).


The study of antiviral response in HBV-infected cells was hampered by the 109

difficulty to grow the virus in cell culture systems. Only freshly prepared primary human 110

hepatocytes (PHH) and differentiated HepaRG cells can support a complete HBV life 111

cycle, including early events of infection (10, 11). HepaRG cells are liver bi-potent 112

progenitor cells that are able to differentiate into both biliary and hepatocyte-like cells 113

(12). However, the overall replication level in these cells is rather low with less than 20%


of cells infected, which complicates the study of host/pathogen interaction (13). One 115

could hypothesize that the low level of replication might be the consequence of an IFN 116

response. In this case, the virus would be able to trigger a host antiviral response and 117

would be capable to disarm it in only a low percentage of cells. This low percentage of 118

infected cells is an obstacle for studying the potential ability of HBV to elicit an IFN 119

response. Indeed, in other viral models, when a low multiplicity of infection is used, 120

which is likely the case with HBV, it has been documented that an IFN response may 121

occur in only a low percentage (< 30%) of infected cells (14), thus complicating its 122

analysis. Moreover, some viruses are particularly efficient to counteract this IFN 123

response and may therefore render the analysis of IFN response more difficult (15). In 124


5 this respect, HBV was shown to be very efficient to inhibit IFN signaling pathway (16- 125

19). Another technical obstacle for studying the potential ability of HBV to elicit an IFN 126

response is that an inoculation time of 16 hours is requested to initiate a strong infection 127

of hepatocytes in vitro (20). This is not compatible with the very early post-infection 128

analysis that would be likely necessary to detect a potentially weak and transient IFN 129

response. To gain data in cell culture system on the potential ability of HBV to elicit and 130

then disarm an IFN response, it appears necessary to initiate a timely controlled and 131

high HBV replication in a large number of cells.


One possibility to initiate a timely controlled and high intracellular HBV replication 133

in vitro is to use a recombinant baculovirus carrying full HBV genome to efficiently 134

transduce hepatic cells. This approach led to higher HBV replication levels compared to 135

either transfection of cells or stable cell lines such as HepG2.2.15 (21). In a previous 136

study, we have improved this system by generating a new HBV recombinant 137

baculovirus in which the synthesis of pregenomic RNA was driven by a strong 138

mammalian promoter. The initiation of a complete HBV DNA replication cycle, followed 139

by the production of infectious particles was evidenced in transduced-HepG2 cells (22).


However, HepG2 cells are not ideal for studying IFN response as they are transformed 141

and therefore impaired for many cellular pathways.


The goal of this study was to determine whether HBV could elicit an type-I IFN 143

response in the non-transformed/non-neoplastic HepaRG cells that are functional for 144

type-I IFN signalling pathway (23), and whether this IFN production could be 145

responsible for the control of HBV replication in this cell line.



6 Experimental procedures


Cell culture 148

HepG2 (ATCC) cells and HepaRG cells (11) were maintained in William’s medium 149

(Invitrogen) supplemented with 10 % fetal calf serum (Perbio), penicillin/streptomycin 50 150

U/mL (Invitrogen), glutaMax (Invitrogen) 2mM, insulin bovine 5 µg/mL and 5 x 10-5 M 151

hydrocortisone hemisuccinate (Roche Diagnostics, Boehringer Mannheim) at 37°C in 152

humidified incubators at 5% CO2. To obtain differentiation of HepaRG, cells were 153

maintained for two weeks in standard medium, then for at least two more weeks in 154

medium supplemented with 1,8 % of DMSO (cell culture grade, Sigma).

155 156

Baculovirus and transduction of Mammalian cells 157

Baculoviral constructions (Bac-HBV-WT, Bac-HBV-YPDD, Bac-βGal, Bac-GFP) as well 158

as stock production, titration, and concentration were performed as described previously 159

(22). All the recombinant HBV baculoviruses contain a 1.1x unit-length HBV genome 160

(genotype D, serotype ayw, accession number in GenBank : V01460) and enables the 161

synthesis of pgRNA under the control of chicken β-actin promoter. Baculoviral 162

transduction of mammalian cells has also been performed as previously described (22).

163 164

Analysis of viral DNA 165

Purification of HBV DNA from intracellular core particles and analysis/quantification by 166

southern blotting with radioactive probe were performed as previously described (24, 167


168 169

Analysis of gene expression by qRT-PCR 170

Total RNA was extracted from cells with the “NucleoSpin RNA II” kit according to 171

manufacturer’s instructions (Macherey-Nagel). IFNβ genes expression was analyzed by 172


7 qRT-PCR with the “SYBR GreenER Two-Step qRT-PCR Kit for iCycler” according to the 173

manufacturer’s instructions (Invitrogen). Primers used for the qPCR step are the 174



176 177

Analysis of secreted type I interferon 178

Three millions Huh7.5 cells, that are deficient for RIG-induced type-I IFN production 179

(26), were transfected with 10 g of pISRE-Luc vector (Stratagene) in a 10 cm diameter 180

dish using “Fugene6 tranfection reagent” according to manufacturer’s instructions 181

(Roche). pISRE-Luc plasmid expresses luciferase under type-I IFN inductible promoter.


After 16h of incubation with transfection mixture, cells were trypsinized are re-seeded in 183

96 wells plate at around 3.104 cells /well in a volume of 100 L. Six hours later, 100 L 184

of clarified (13.000g , 5 min) supernatants, that were collected from transduced cells, 185

was added to wells. After 24 h incubation at 37°C, cells of each well were washed with 186

PBS and lysed before luciferase activity was monitored using the Renilla Luciferase 187

Assay System (Promega).

188 189

Analysis of the IFN response by AB Low Density Arrays 190

Total RNA was extracted from cells with the “NucleoSpin RNA II” kit according to 191

manufacturer’s instructions (Macherey-Nagel) and retro-transcribed with the Invitrogen 192

Superscript kit using random examers as template. 200ng of cDNA were loaded in 193

double on the Low Density Arrays (Applied Bioystem) customized with 95 ISGs probes 194

and 18s probe as a control. The LDAs were run on a AB 7900HT and real time PCR 195

data were collected and analyzed through the SDS 2.2 program (Applied Biosystem).

196 197

Neutralization of IFNβ 198


8 Neutralization of IFNβ produced in supernatant from cells was performed using a 199

neutralisazing anti-IFNβ antibody (ab9662, Abcam) with a final concentration of 0,5 200


201 202

Construction of HepaRGshIFNR1 cell line 203

Five different lentivirus enabling the expression of 5 different short-hairpin RNAs 204






GUUGACUCAUUUACACCAUUU) directed against the ifnr1 gene (that codes IFN 208

receptor 1) were obtained as viral stocks from Sigma. HepaRG cells were seeded at 209

1.105 cells in 24 wells plate and transduced with the 5 lentivirus (m.o.i. of 5 for each 210

virus). After 16h of incubation at 37°C with transduction mixture, medium containing 211

viruses was replace by fresh medium containing 20 g/mL of puromycin (Invivogen) to 212

select for stably transduced cells. Resistant cells were expanded in presence of 40 213

g/mL of puromycin until the constitution of a frozen stock of cells. The inhibition of the 214

expression of IFNR1 mRNA was checked by RT-PCR using the following primers 215



217 218


9 Results


Kinetic of HBV replication in HepaRG cells after transduction with Bac-HBV.


As the efficiency of transduction was even in both HepG2 and HepaRG cell lines, a 221

direct comparison of the kinetic of HBV replication after transduction with Bac-HBV-WT 222

was possible. Both proliferating and differentiated HepaRG as well as HepG2 cells were 223

transduced at an m.o.i. of 100 pfu/cell with Bac-HBV-WT. Intracellular encapsidated 224

viral DNA was isolated at various time points post-transduction (p.t.) and analyzed by 225

Southern Blot. HBV DNA was detectable very rapidly after transduction in both HepG2 226

and HepaRG cells (fig. 1). Although the amount of HBV intracellular encapsidated DNA 227

was comparable at day 1 p.t. in both cell lines, kinetics of HBV replication were quite 228

different, as its level decreased slowly in HepG2 cells after 12 days, whereas a rapid 229

and sharp decrease was observed in both proliferating and differentiated HepaRG cells 230

to become nearly undetectable at day 12 p.t. This difference in kinetics could not be 231

attributed to differences in kinetics of clearance of the baculovirus template (data not 232

shown), neither to differential cell death. The fast and non-cytolytic elimination of 233

intracellular encapsidated DNA in HepaRG cells suggested that an antiviral response 234

involving specific cellular factors could be involved.

235 236

Establishment of an antiviral state in Bac-HBV-transduced HepaRG.


To characterize this antiviral state, cells were transduced twice (at day 0 and day 3) with 238

Bac-HBV-WT. As previously shown the amount of HBV intracellular encapsidated DNA 239

was increased by a second transduction in HepG2 cells (21), whereas it remained 240

unchanged in HepaRG cells (fig. 2). This result strongly suggests that HepaRG cells 241

were able to mount a sustained antiviral response following the first transduction with 242




10 To determine whether this antiviral response in HepaRG cells was due to the transgene 244

expression (i.e. synthesis of pgRNA, mRNA, and proteins) and replication of HBV 245

genome, or was induced by the baculovirus vector itself, HepG2 or HepaRG cells were 246

first mock-transduced (inoculation with medium only) or transduced with two different 247

control baculoviruses, containing either the -galactosidase gene (Bac-Gal) or an HBV 248

genome carrying a mutation in the catalytic domain of the polymerase (Bac-HBV- 249

YPDD). The latter baculovirus enables the production of HBV proteins without genome 250

replication (22). Three days after the first transduction, a second transduction was 251

performed with Bac-HBV-WT. In HepG2 cells, HBV intracellular encapsidated DNA was 252

detectable in all experimental conditions (fig. 3A, upper panel), suggesting that no (or 253

weak) antiviral response was induced in these cells after the first transduction. The 254

apparent differences in signal intensity between mock and Bac-Gal (or Bac-HBV- 255

YPDD) for HepG2 in the shown figure was neither significant nor reproducible. What 256

was highly reproducible is the presence of signal in all three conditions. In contrast, 257

HBV intracellular encapsidated DNA was always detectable in HepaRG cells that were 258

first mock-transduced or transduced with Bac-Gal, but never in those first transduced 259

by Bac-HBV-YPDD (fig. 3A, bottom panel; see also fig7B). The results reproducibly 260

obtained by southern blot analysis, were independently confirmed by the analysis of 261

HBsAg secretion in the same conditions (fig. 3B). HBsAg was never detected in the 262

supernatant of HepaRG cells that were first transduced with Bac-HBV-YPDD, 263

suggesting a strong IFN-induced degradation of mRNA encoding HBsAg. Altogether 264

those results demonstrates that the antiviral response observed in HepaRG following 265

transduction is mediated by HBV itself and is independent of the baculovirus vector.


Moreover, as no reverse transcription step can occur with Bac-HBV-YPDD, this result 267

also indicates that the establishment of an HBV-mediated antiviral state was 268


11 independent of HBV DNA replication, but was rather due to HBV protein or RNA 269


270 271

HBV elicits IFN- production that subsequently induces ISGs expression in both 272

HepG2 and HepaRG cells.


As production of type-I interferons, in particular IFN-, is one of the first cellular 274

antiviral defense (4), we first tested whether the ifn- gene was activated in HepG2 and 275

HepaRG cells (proliferating or differentiated). Cells were transduced with Bac-Gal or 276

Bac-HBV-WT and qRT-PCR performed 24 and 48h p.t. The amount of mRNA encoding 277

IFN was strongly increased in cells transduced with Bac-HBV-WT (either HepG2 or 278

HepaRG cells), but not in those transduced with the control baculovirus (Bac-Gal) (fig.


4A). This indicates that HBV, and not the vector, is responsible for the induction of IFN- 280

 expression in both HepG2 and HepaRG cells. Using a functional assay, we also 281

demonstrated that biologically active type-I interferons were produced in the 282

supernatant of Bac-HBV-WT transduced cells (fig. 4B).


Genes activated by type-I IFNs are well known, and their expression represents a 284

signature of the IFN pathway (4). To determine whether the pattern of induction after 285

transduction with Bac-HBV-WT was similar to that already described, HepG2 and 286

HepaRG (proliferating or differentiated) cells were transduced with Bac-Gal or Bac- 287

HBV-WT. Expression of 96 ISGs was evaluated by qRT-PCR analyses. Only selected 288

ISGs are shown in fig. 4C; the results for the whole set of genes is shown in the 289

supplementary file. The control baculovirus did not induce significant changes in the 290

pattern of expression of the genes, thus confirming that the vector is not responsible for 291

the observed IFN response. In contrast, many of the analyzed genes were 292

overexpressed in HepG2, as well as in proliferative and differentiated HepaRG cells 24 293

and 48h after transduction with Bac-HBV-WT.



12 Many of the overexpressed genes, including viperin, mx1, isg15, oas1, isg56, have 295

antiviral properties and may be candidate genes responsible for the inhibition of HBV 296

replication in HepaRG. However, these genes were also activated in HepG2 cells in 297

which the replication of HBV was not inhibited as compared to that observed in 298

HepaRG, suggesting that the IFN signaling in these cells do not translate into antiviral 299

effect and confirming the partially non functional IFN pathway I this cell line (27, 28).


One gene, i.e. CXCL9, was only upregulated in HepaRG cells, thus representing a 301

signature for this line, whereas three genes representated a signature for HepG2 cells.


So far no correlation between the pattern of gene activation (or repression) and the 303

antiviral response has been evidenced.

304 305

Inhibition of type-I IFN pathway results in enhanced HBV replication in HepaRG 306



The next step was to inhibit the IFN pathway in HepaRG cells to determine whether 308

HBV replication could be enhanced. First, proliferating HepaRG cells were transduced 309

with Bac-HBV-WT and immediately treated with a neutralizing anti-IFN antibody.


Intracellular encapsidated viral DNA was isolated at various time points post- 311

transduction (p.t.) and analyzed by Southern Blot. Results showed that neutralization of 312

IFN- clearly increased amount of intracellular encapsidated HBV DNA in proliferating 313

HepaRG (fig. 5), thus confirming the crucial role of IFN- in mediating the observed 314

antiviral response in this cell line. It is worth noting that neutralizing anti-IFN-β antibody 315

did not significantly improve HBV replication in HepG2 cells, as shown in figure 5B that 316

present the fold increase of DNA produced with anti-IFN- treatment compared to non- 317

treated conditions. This result suggest that the IFN- produced by HepG2 cells upon 318

Bac-HBV transduction, which is functional as shown in figure 4B, does not seem to 319

trigger an antiviral response in HepG2.



13 To further demonstrate the role of type-I IFN in inhibiting HBV replication in HepaRG 321

cells, we invalidated the expression of the ifnar1 gene, which codes for subunit 1 of the 322

type-I IFN receptor, using short hairpin RNA strategy. The down regulation of the gene 323

in the resulting cell line, i.e. HepaRGshIFNR1, was verified by RT-PCR (fig. 6A). This 324

down regulation of the expression of type-I IFN receptor is expected to be associated 325

with an inhibition of the amplification of the IFN pathway. To confirm that we significantly 326

invalidated type-I IFN pathway, the expression of ISGs was analyzed by qRT-PCR. We 327

showed that a poly-IC stimulation did not increase the expression of ifn-, oas1 and 328

isg56 genes (fig. 6B). Moreover the levels of mRNA encoding IFN, 2OAS or ISG56 329

were decreased by up to 80 % in HepaRGshIFNR1 after transduction with Bac-HBV- 330

WT compared to parental cell line (fig. 6C).


If type-I IFN pathway is crucial for the control of HBV replication, its inhibition should 332

result in an increased HBV replication. Both HepaRG and HepaRGshINFR1 were 333

transduced with Bac-HBV-WT and encapsidated HBV DNA extracted at different time 334

post transduction. The amount of intracellular encapsidated HBV DNA was increased in 335

HepaRGshINFR1 cells, thus demonstrating an inverse correlation between IFN- and 336

ISGs expression, and HBV replication (fig. 7A). The inhibition of the antiviral state in 337

HepaRGshINFR1 cells was further evidenced by a double transduction experiment, as 338

done previously (fig. 2). Indeed when two transductions with Bac-HBV-WT were 339

performed at three days of interval, this rate of HBV replication was significantly 340

increased in HepaRGshIFNR1 cells as opposed to HepaRG cells (fig. 7A). It is also 341

worth noting that no additional significant increase in HBV DNA accumulation in 342

HepaRGshIFNR1 cells was observed after IFN- neutralization (fig 5B). This inhibition 343

of the antiviral state in HepaRGshINFR1 was confirmed by successive transductions of 344

cells with different baculovirus as previously described. Indeed, we showed that, as 345

opposed to wild type HepaRG cells, HBV intracellular encapsidated DNA could be 346


14 detected in HepaRGshIFNR1 cells in all conditions and especially when cells were first 347

transduced with Bac-HBV-YPDD (fig. 7B). Taken together, these results emphasize a 348

clear correlation between the production of IFN-β, inhibition of intracellular HBV 349

accumulation, and installation of an antiviral state in HBV-transduced HepaRG cells.

350 351


15 Discussion


The belief that HBV may be a “stealth virus”, i.e. not detected by innate immune 353

defences, comes from a longitudinal analysis of the activation of cellular genes 354

performed on liver biopsies of three experimentally-infected chimpanzees. No data in 355

human are available to confirm or challenge this result. In chimpanzees, the expression 356

of cellular genes, including those of antiviral cytokines such as IFN/, remained 357

apparently unchanged within the liver during the lag phase of infection (6). However, 358

these data should be interpreted with caution, as chimpanzees may not be a completely 359

relevant models and moreover one cannot exclude that the inability to detect variation in 360

gene expression may come from the technology used, the stringency of cut off, as well 361

as the timing of the analysis (i.e. the first gene profiling was performed two weeks after 362

the inoculation). The later point is crucial as IFN response to viral infection often takes 363

place very rapidly after infection and can be negatively regulated by the virus. Many 364

studies have shown that HBV, when actively replicating, is able to inhibit IFN signaling 365

pathway (16-19). This capacity to inhibit IFN signaling pathway, in particular in the 366

context of exogenous IFN exposure, may explain at least partially why chronically 367

infected patients do not respond efficiently to IFN- treatment.


There are other indirect evidences that innate immunity, i.e. antiviral response at level of 369

infected cells and innate response via specialized cells of immune system (e.g. NK, 370

NKT), may play an important role to control HBV infection to some extent. A study 371

showed that HBV clearance could be initiated in the liver of infected chimpanzees 372

before the onset of adaptive immune response (8). In addition, several studies on HBV 373

transgenic mice showed that in animal deficient for type-I IFN receptor, PKR or IRF1, 374

HBV replicates at higher levels than in controls mice (29-31). These observations 375

strongly suggest that type-I IFN response contributes to the control of HBV replication in 376

mice. Finally, recent data suggest that the lack of intrahepatic gene induction may be a 377


16 peculiar feature of the chimpanzee model since an early induction of both innate and 378

adaptative response was observed in two patients with acute HBV infection (9).


In vitro data are needed to gain insight in the mechanism by which HBV may induce an 380

innate response which in turn may control viral replication. Transformed hepatic cell 381

lines (i.e. HepG2 and Huh7) have limited interest as they do not have fully functional 382

IFN pathways (27, 28), in contrast to PHH and HepaRG that are non-cancerous, 383

permissive to some extend to HBV infection (10, 11), and functional for IFN signalling 384

(23). The latter may explain the relatively low replication rate in these cells (i.e. less than 385

20% of cells infected) (10, 32). This low level of replication, that might result from a 386

cellular antiviral response, complicates the study of host/pathogen interaction.


To gain insight on the potential ability of HBV to elicit then disarm an IFN response, it 388

appeared necessary to initiate a high HBV replication in a large number of cells. In this 389

study, we present evidence that a strong initial HBV infection can induce a type-I IFN 390

response, which results in the establishment of an antiviral state that is not overcome by 391

the virus in the non-transformed/non-neoplastic HepaRG cell line. To study this antiviral 392

response, we have used recombinant baculoviruses carrying the HBV genome that are 393

able to transduce high percentage of cells and trigger high initial rate of HBV replication.


The comparison of the kinetics of HBV replication after cell transduction showed, that in 395

contrast to that observed in HepG2, the presence of intracellular HBV DNA in both 396

proliferating and differentiated HepaRG was transient.


The mechanism of clearance of encapsidated HBV DNA in HepaRG was non-cytolytic 398

and correlated with the production of IFN- and subsequent activation of ISGs. In 399

HepG2 cells, despite the production of IFN- and activation of ISGs, the amount of 400

intracellular HBV DNA remained stable during time, thus confirming that type-I IFN 401

pathway is not fully functional in this transformed hepatoblastoma-derived cell line, as 402

previously suggested (27, 28, 33). Importantly, we have shown that the establishment of 403


17 the antiviral state in HepaRG was mainly due to the expression of HBV proteins and/or 404

transcripts, as an HBV mutant with a non-functional polymerase was yet able to induce 405

the antiviral state.


The inverse correlation between the activation of type-I IFN pathway and inhibition of 407

HBV replication in HepaRG was definitely demonstrated by experiment in which the 408

action of IFN- was directly neutralized by antibodies and by experiment with 409

engineered HepaRG in which the type-I IFN receptor was down regulated by RNA 410

interference. In both case the rate of HBV replication was greatly increased after 411

transduction. These HepaRGshIFNR1 cells may represent a unique model to study 412

HBV biology and the effect of persistent HBV replication on cell physiology.


Our data demonstrate that, in HepaRG cells, a strong HBV expression and replication 414

induce a potent IFN response that in return restricts infection. It seems that in this model 415

the virus is not able to disarm/counteract the IFN response, despite the demonstrated 416

capacity of some HBV proteins to do so in different experimental conditions (16, 18, 34- 417

36). Further investigations are necessary to determine which viral determinant is 418

responsible for the induction of IFN response in this model and how the virus may 419

overcome it to induce a persistent infection.

420 421

Acknowledgements 422

We would like to thank Dr Isabelle E. Vincent for her advice and critical review of the 423


424 425


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518 519 520


20 Figures legends

521 522

Figure 1 : Kinetic of HBV replication in different cell line after transduction with an 523

HBV recombinant baculovirus (Bac-HBV-WT). Cells were transduced at an m.o.i. of 524

100 pfu/cell with Bac-HBV-WT. Encapsidated DNA was extracted at different times 525

post-transduction and analyzed by Southern Blot. HepaRGp = proliferative HepaRG 526

cells, HepaRGd = differenciated HepaRG cells.

527 528

Figure 2 : Transduction with HBV recombinant baculoviruses leads to an antiviral 529

state in HepaRG cells but not in HepG2 cells. HepG2 and HepaRG cells were 530

transduced once or twice (days 0 and 3) at an m.o.i. of 100 pfu/cell with Bac-HBV-WT.


Encapsidated DNA were extracted at different times post-transduction and analyzed by 532

Southern Blot. Graph represents quantification of the autoradiogram.

533 534

Figure 3 : The Antiviral state is due to HBV. HepG2 and HepaRG cells were first 535

transduced at an m.o.i. of 100 pfu/cell either with Bac-HBV-YPDD, Bac-Bgal or with 536

medium. Three days later, cells were transduced a second time at an m.o.i. of 100 537

pfu/cell with Bac-HBV-WT. Encapsidated DNA were extracted and analyzed by 538

Southern Blot (A), and HBsAg dosed by Elisa (B) at different times post-transduction.

539 540

Figure 4 : Transduction with HBV recombinant baculovirus activates the IFN 541

pathway in both HepG2 and HepaRG cells. Cells were transduced at an m.o.i. of 100 542

pfu/cell with Bac-HBV-WT or with Bac-βGal. Twenty four and 48h after, (A) total RNA 543

were extracted, and IFN expression analyzed by qRT-PCR and (C) ISG expression 544

was analyzed by qRT-PCR using AB LDA technology customized with 95 ISG and 18s 545

probes. Only selected ISGs are shown (the whole set genes is shown in the 546

supplementary file). (B) supernatant from transduced cells was also tested for type I IFN 547


21 activity. All results are expressed in fold change compared to non transduced cells.


HepaRGp = proliferative HepaRG cells, HepaRGd = differenciated HepaRG cells.

549 550

Figure 5 : Neutralization of IFN-β in supernatant from cells enhances HBV 551

replication. HepaRG, HepG2, or HepaRGshIFNR1 (IFN receptor expression 552

invalidated by RNA interference) cells were transduced at an m.o.i. of 100 pfu/cell with 553

Bac-HBV-WT and immediately treated or not with a neutralizing anti-IFN-β antibody.


Treatment was repeated every 3 days when medium was changed. Encapsidated DNA 555

was extracted at different times post-transduction and analyzed by Southern Blot. (A) 556

Autoradiogram of Southern blot obtained with HepaRG cells. (B) Graph showing the 557

ratio between quantity of DNA detected with anti-IFN- treatment and quantity of DNA 558

detected without treatment.

559 560

Figure 6 : Down-regulation of IFN pathway in HepaRGshIFNR1. HepaRG cells and 561

HepaRGshIFNR1 cells were stimulated either by (A, B) polyIC at 10µg/mL or (C) by 562

transduction with Bac-HBV-WT at an m.o.i. of 100 pfu/mL. Cells were lysed (A, B) 8h 563

after stimulation by polyIC or (C) 24h after transduction. Total RNA were extracted and 564

gene expression analyzed by (A) RT-PCR or (B, C) qRT-PCR. Results of qRT-PCR 565

analyses are expressed compared to WT HepaRG cells.

566 567

Figure 7 : Down-regulation of IFN pathway in HepaRGshIFNR1 cells leads to 568

enhanced HBV replication and prevent the establishment of an HBV-mediated 569

antiviral state. HepaRG and HepaRGshIFNR1 cells were first transduced at an m.o.i.


of 100 pfu/cell with either (A) Bac-HBV-WT, (B) Bac-HBV-YPDD, Bac-gal or medium.


Three days later, cells were transduced or not a second time at an m.o.i. of 100 pfu/cell 572


22 with Bac-HBV-WT. Encapsidated DNA were extracted at different times post- 573

transduction and analyzed by Southern Blot.

574 575

Supplementary file : Transduction with HBV recombinant baculovirus activates the 576

IFN pathway in both HepG2 and HepaRG cells. Cells were transduced at an m.o.i. of 577

100 pfu/cell with Bac-HBV-WT or with Bac-βGal. Twenty four and 48h after, total RNA 578

were extracted and ISG expression was analyzed by qRT-PCR using AB LDA 579

technology customized with 95 ISG and 18s probes.

580 581




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