Hepatitis delta virus inhibits alpha interferon signaling


  • Potential conflict of interest: Nothing to report.


Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected with hepatitis B virus. Interferon-α (IFN-α) is the only treatment reported to be effective in chronic hepatitis delta, albeit in a minority of patients. The molecular mechanisms underlying resistance to therapy are unclear. IFN-α–induced activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade is essential for the induction of an antiviral state. Interference of HDV with the JAK-STAT pathway could be responsible for the IFN-α resistance in chronic hepatitis delta patients. We analyzed IFN-α–induced signal transduction through the JAK-STAT pathway in human hepatoma cells transfected with the complete HDV genome. The expression of IFN-α–stimulated genes was investigated with reverse transcription real-time polymerase chain reaction (PCR). STATs and JAKs activations were examined by immunofluorescence and immunoblot. The IFN-α–stimulated genes coding for the antiviral proteins myxovirus resistance A, double-stranded RNA (dsRNA)-activated protein kinase and 2′,5′-oligoadenylate synthetase were down-regulated in HDV-transfected hepatoma cells in response to IFN-α treatment. HDV severely impaired the phosphorylation of both STAT1 and STAT2, thus preventing their accumulation in the nucleus. Furthermore, HDV blocked the IFN-α–stimulated tyrosine phosphorylation of IFN receptor-associated JAK kinase Tyk2, without affecting either the tyrosine phosphorylation of Jak1 or the expression of type I IFN receptor subunits. Conclusions: IFN-α–induced intracellular signaling is impaired in HDV-transfected human hepatoma cells. HDV subverts the effect of IFN-α by blocking Tyk2 activation, thereby resulting in selective impairment of activation and translocation to the nucleus of STAT1 and STAT2. Interference of HDV with IFN-α signaling could represent an important mechanism of viral persistence and treatment resistance. (HEPATOLOGY 2008.)

Type I interferons (IFN-α/β) are cytokines that play a key role in mediating a range of diverse functions including antiviral, antiproliferative, antitumor, and immunomodulatory activities.1–3 IFN exerts its effect mainly through the JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway.1, 4, 5 IFN-α/β binds to type I IFN receptor, a cell surface transmembrane protein composed of two subunits, IFN-α receptor 1 (IFNAR1) and IFNAR2.6, 7 IFN binding results in receptor subunits dimerization and activation via tyrosine phosphorylation of two receptor-associated tyrosine kinases of the Janus family, Jak1 and Tyk2, which in turn phosphorylate STAT1 and STAT2. Activated STATs heterodimerize and translocate into the nucleus, where they associate with p48 (also known as IRF9), forming the ISGF3 transcription factor complex that binds the IFN-stimulated response element2, 8 to initiate transcription of IFN-stimulated genes (ISGs).4, 9 The genes that code for the antiviral proteins myxovirus resistance A (MxA), the double-stranded RNA-activated protein kinase (PKR), and the 2′,5′-oligoadenylate synthetase (2′,5′-OAS) are among the best characterized ISGs.10 Stringent mechanisms of down-regulation of IFN-α/β signaling are essential for insuring appropriate, controlled cellular responses.11 Among them, protein tyrosine phosphatases,12–14 protein inhibitors of activated STATs,15, 16 and suppressors of cytokine signaling17–19 inhibit specific and distinct aspects of cytokine signal transduction. In addition, many viruses have developed strategies to replicate in the host by down-regulating or blocking type I IFN signaling at different stages of the pathway,20 thus escaping the host immune response and establishing a persistent infection.

Hepatitis delta virus (HDV) is a hepatotropic defective RNA virus that requires hepatitis B virus for virion assembly and propagation.21, 22 HDV contains an approximately 1.7-kb negative sense single-stranded circular RNA genome.23–25 After entry into hepatocytes, HDV uses host cellular enzymes to replicate its genome through a double rolling-circle mechanism.26, 27 HDV RNA encodes only one protein, the hepatitis delta antigen (HDAg), from the antigenomic strand. HDAg has two isoforms, the small HDAg (HDAg-S; 195 amino acids, 24 kDa) and the large HDAg (HDAg-L; 214 amino acids, 27 kDa). HDAg-L contains an additional 19 amino acids at its C terminus as a consequence of an RNA editing event of the termination codon of HDAg-S during RNA replication.28 Both HDAgs are nuclear phosphoproteins.29, 30 Although these two antigens are more than 90% identical,31, 32 they play different roles during the course of an infection. HDAg-S is required for HDV RNA replication,33 whereas HDAg-L suppresses viral RNA replication and is required for HDV assembly.34–36

Similar to hepatitis B virus infection, HDV is most often transmitted parenterally. Approximately 5% of the global hepatitis B virus carriers are co-infected with HDV, leading to a total of 10 to 15 million HDV carriers worldwide. HDV infection can cause severe acute and chronic liver disease, and in approximately 80% of cases there is progression to cirrhosis and a frequent evolution to end-stage liver disease and hepatocellular carcinoma.37–40

To date, treatment of chronic HDV infection relies on IFN-α. Compared with chronic hepatitis B or C, chronic HDV therapy requires higher doses and longer duration, but only a minority of patients reaches a sustained virological response.41–44 Failure to clear HDV after IFN-α treatment suggests that HDV, like many other viruses, may have developed an anti-IFN strategy. Alternatively, an altered host environment may somehow favor viral survival. The current work addresses the question as to whether HDV impairs the IFN-α–induced signaling cascade. We show that HDV infection interferes with the activation of an early step in the JAK-STAT signal transduction pathway.


cDNA, complementary DNA; HDAg, hepatitis delta antigen; HDAg-L, large hepatitis delta antigen; HDAg-S, small hepatitis delta antigen; HDV, hepatitis delta virus; IFN, interferon; ISG, interferon-stimulated genes; JAK, Janus kinase; MxA, myxovirus resistance A; OAS, oligoadenylate synthetase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PKR, dsRNA-activated protein kinase; STAT, signal transducer and activator of transcription.

Materials and Methods

Plasmids, Antibodies, and Chemicals.

Plasmid pSVL(D3), donated by Maria Lorena Abate, University of Turin, Turin, Italy, contains three copies of the complementary DNA (cDNA) of the HDV genome in the expression vector pSVL (Pharmacia, Uppsala, Sweden).33 Vector pcDNA3.1/Hygro, which carries the hygromycin-resistance gene, was from Invitrogen (Basle, Switzerland).

An antibody that recognizes both HDAg-S and HDAg-L, provided by John M. Taylor, Fox Chase Cancer Center, Philadelphia, PA, was raised in rabbits by inoculation of HDAg expressed in Escherichia coli. Mouse monoclonal antibody to IFNAR1 and rabbit polyclonal antibody to IFNAR2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies to STAT1, STAT2, Jak1, Tyk2, phosphotyrosine 701-STAT1 (pY-STAT1), phosphotyrosine 690-STAT2 (pY-STAT2), phosphotyrosine 1022/1023-Jak1 (pY-Jak1), and phosphotyrosine 1054/1055-Tyk2 (pY-Tyk2) were from Cell Signaling Technology, Inc. (Danvers, MA). Mouse monoclonal antibody to β-actin (clone Ac74) was from Sigma-Aldrich (Steinheim, Germany). For immunofluorescence analyses, staining was performed using mouse monoclonal antibodies to STAT1 from BD Transduction Laboratories (Lexington, KY) or to STAT2 from Santa Cruz Biotechnology (Santa Cruz, CA).

IFN-α2b (Intron A) was a gift of Essex Chemie AG (Luzern, Switzerland). Hygromycin B was obtained from PAA Laboratories (Pasching, Austria).

Cell Culture and Transfection.

Human hepatoma Huh-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (all products were from Invitrogen, Basle, Switzerland).

Subconfluent cultures of Huh-7 cells were transfected with either pSVL(D3) or pcDNA3.1/Hygro or a 10:1 ratio of pSVL(D3) to pcDNA3.1/Hygro using lipofectamine 2000 (Invitrogen, Basle, Switzerland) and incubated at 37°C with Opti-MEM medium for 6 hours in a humidified, 5% CO2 incubator. The medium was then replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum without antibiotics, and transfected cells were incubated for another 9 days, with medium changes every second day. When pcDNA3.1/Hygro was transfected, cells were incubated with medium containing 750 μ/mL hygromycin B starting 2 days posttransfection to select for cells containing expression constructs.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hombrechtikon, Switzerland) and subsequently treated with deoxyribonuclease I. RNA integrity was assessed using RNA 6000 nanochips with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). First-strand cDNA was synthesized from 500 ng purified RNA using the SuperScript II RNase H(−) reverse transcriptase (Invitrogen, Basle, Switzerland) and random hexadeoxynucleotides. For real-time polymerase chain reaction (PCR), the following Human SYBR Green QuantiTect Primer Assays (all purchased from Qiagen) were used: double-stranded RNA-activated protein kinase (PKR, No. QT00022960); myxovirus (influenza virus) resistance A (MXA, No. QT00090895); 2′,5′-oligoadenylate synthetase 1 (OAS1, No. QT00099134). Reactions were set up in 384-well plates using a 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA), and all samples were assayed in triplicate. Optical data obtained were analyzed using the default and variable parameters available in the SDS software package (version 2.2.2; Applied Biosystems, Foster City, CA). Expression level of target genes was normalized using as endogenous control genes the eukaryotic translation elongation factor 1 alpha 1 (forward primer 5′-AGCAAAAATGACCCACCAATG-3′, reverse primer 5′-GGCCTGGATGGTTCAGGATA-3′) and the beta-glucuronidase (forward primer 5′-CCACCAGGGACC- ATCCAAT-3′, reverse primer 5′-AGTCAAAATATGTGTTCTGGACAAAGTAA-3′).

Immunoblot Analysis.

For total protein extracts, cells were washed once with ice-cold phosphate-buffered saline (PBS) and scraped from culture dishes in the presence of lysis buffer [50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol) supplemented with protease inhibitor cocktail (COMPLETE; Roche Diagnostics, Mannheim, Germany), 1 mM phenylmethylsulphonyl fluoride and 1 mM sodium orthovanadate. Equal amounts of protein extracts (20 μg) were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and electroblotted onto nitrocellulose (Whatman, Dassel, Germany). Membranes were blocked for 2 hours at room temperature with PBS containing 0.1% (vol/vol) Tween 20 and 5% (wt/vol) nonfat milk powder before incubation with the above primary antibodies in blocking solution. After three washes with blocking buffer, the membranes were incubated for 90 minutes at room temperature with horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (BioRad, Hercules, CA) diluted 1:4000. Membranes were washed extensively with blocking solution, and antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham Biosciences, Otelfingen, Switzerland), according to the manufacturer's instructions.


Monolayer cultures on glass coverslips were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 for 3 minutes, and blocked with 1.5% bovine serum albumin in PBS for 30 minutes. Cells were double-stained by incubation with a mixture of rabbit polyclonal antibody to HDAg and mouse monoclonal antibody to STAT1 or to STAT2 for 1 hour at room temperature. After three washes with PBS, cells were incubated for 45 minutes at room temperature with a mix of a rhodamine-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and an Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen, Basle, Switzerland). After extensive washing with PBS, coverslips were mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA) containing 4′,6-diamidino-2-phenylindole and viewed with an epifluorescence microscope (Axioskop 2; Carl Zeiss, Oberkochen, Germany). Images were acquired with an Axiocam color charge-coupled device camera (Carl Zeiss, Oberkochen, Germany).


Results were expressed as means ± standard deviation of three independent experiments. For statistical comparison, significance was evaluated using the Student t test. Values of P < 0.05(*) and P < 0.005(**) were considered statistically significant.


Transfection of Huh-7 Cells with HDV.

To investigate whether HDV interferes with IFN signaling, we used an in vitro system that was reported to allow HDV replication.33 Cultures of Huh-7 cells were transfected with plasmid pSVL(D3), coding for full-length, replicating HDV RNA, and, at regular intervals thereafter, proteins were extracted and examined by immunoblot analysis for the presence of the two HDAg isoforms, HDAg-S and HDAg-L (Fig. 1). As expected, both HDAgs were present, and the amount of HDAg-L increased with time and became similar to that of HDAg-S at 9 days posttransfection. Based on this observation and on the fact that in this system HDV replication reaches a maximum at 9 days after transfection,45 all the following analyses were performed at this time point. The efficiency of transfection was comparable among different experiments and typically amounted to approximately 20%, as determined by counting HDAg-positive cells under the fluorescence microscope in six randomly selected fields per each of three independent experiments.

Figure 1.

Kinetics of HDAg-S and HDAg-L synthesis. Huh-7 cells were transfected with HDV cDNA, and the expression of the two isoforms of the HDV antigen was analyzed at 3, 6, and 9 days posttransfection by immunoblot analysis using a polyclonal antibody that recognizes both HDAg-S and HDAg-L.

HDV Down-Regulates IFN-Induced Gene Expression.

We examined whether HDV transfection antagonizes IFN-α activity by interfering with the activation of ISGs, whose protein products mediate a variety of specific IFN-dependent antiviral responses. To this purpose, Huh-7 cells were transfected with a vector carrying the hygromycin-resistance gene alone or along with pSVL(D3) and were selected with hygromycin B to obtain a population of cells, most of which were expressing the viral proteins. Thus, we compared the induction of three IFN-stimulated response element–controlled cellular genes, PKR, MxA, and 2′,5′-OAS, by real-time PCR analysis on untransfected versus HDV-transfected cells treated with IFN-α or left untreated (Fig. 2). In the absence of IFN-α stimulation, for none of the genes analyzed did we observe a significant difference in activation when we compared HDV-transfected and untransfected cells. Conversely, addition of IFN-α on untransfected cells resulted in a significant (>fivefold) induction of PKR, and this effect was even stronger (>50-fold) for both MxA and 2′,5′-OAS. Similar effects were observed after IFN-α treatment of HDV-transfected cells. However, in this case, transcriptional activation of all three genes under examination was significantly weaker as compared with untransfected cells (P < 0.05 for PKR and P < 0.005 for both MxA and 2′,5′-OAS, Student t test). These observations strongly suggest that HDV can interfere with the JAK-STAT signal transduction pathway in response to IFN-α, leading to inefficient gene expression.

Figure 2.

HDV inhibition of IFN-α–stimulated genes. Huh-7 cells were transfected with either pcDNA3.1/Hygro alone (Huh-7) or with a mix of HDV cDNA and pcDNA3.1/Hygro [Huh-7 pSVL(D3)], and then selected with hygromycin B. Nine days after transfection, cells were treated for 8 hours with 1000 U/mL IFN-α (+), or were left untreated (−). The expressions of the IFN-α–stimulated genes MxA, 2′,5′-OAS and PKR were measured by real-time reverse transcription PCR in three independent samples (each sample was measured in triplicate). Shown are the mean values and the standard deviations. The IFN-α–elicited transcriptional induction of all three target genes is inhibited in HDV-transfected compared with untransfected Huh-7 cells (*P < 0.05; **P < 0.005; Student t test).

HDV Prevents Nuclear Targeting of STAT1 and STAT2.

Negative regulation of IFN-α signaling could provide one mechanistic explanation for the observed defect in ISG activation in the presence of HDV. Recent reports have shown that different viruses are able to inhibit the activation of STAT1 and/or STAT2 and their translocation to the nucleus in response to IFN stimulation.46–48 To examine the potential of HDV to influence the cellular distribution of one or both the STAT proteins, we performed an indirect immunofluorescence assay on IFN-α–treated transfected cells, using antibodies that specifically recognize STAT1 and STAT2 (Fig. 3). As expected, in normal cells IFN-α promoted nuclear accumulation of STAT1 and STAT2 (Fig. 3A, B, top rows). In contrast, the analysis of transfected cells clearly revealed that HDV severely impaired the translocation to the nucleus of both STAT proteins (Fig. 3A, B, bottom rows). In fact, the presence of a mixed population of HDV-transfected and untransfected cells allowed clear visualization of STAT1 and STAT2 staining only in the nuclei of untransfected cells, whereas the nuclei of neighboring transfected cells stained just very faintly. The inability of STAT1 and STAT2 to translocate efficiently to the nucleus in response to IFN-α treatment in transfected cells as detected by immunofluorescence was a first indication that activation (in other words, phosphorylation) of these proteins may be inhibited by HDV replication.

Figure 3.

The nuclear accumulation of STAT1 and STAT2 in response to IFN-α is inhibited in HDV-transfected cells. Normal cells (Huh-7) or cells transfected with HDV cDNA [Huh-7 pSVL(D3)] were seeded on glass coverslips and treated with 1000 U/mL IFN-α for 30 minutes. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with antisera to STAT1 (A) or STAT2 (B) and HDAg. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (blue). Arrows point to cells successfully transfected with HDV.

HDV Inhibits STAT1 and STAT2 Activation.

Tyrosine phosphorylation of STAT1 and STAT2 is a key event after binding of IFN-α to its receptor.49 Our immunofluorescence data suggested that the HDV-mediated inhibition of the JAK-STAT pathway could occur at this step. To establish whether HDV replication could block the phosphorylation of one or both STATs, we performed an immunoblot analysis of HDV-transfected Huh-7 cells using antibodies to phosphorylated (activated) and nonphosphorylated forms of both proteins (Fig. 4). Analysis of total STAT1 and STAT2 proteins showed that their accumulation was higher after transfection, irrespective of IFN-α treatment. In contrast, the levels of tyrosine phosphorylated STAT1 and STAT2 were dramatically reduced in IFN-α–treated, HDV-transfected cells as compared with untransfected cells. Nevertheless, some phosphorylated STAT1 and STAT2 could still be detected, even if at very low levels. It is possible that these small amounts of activated STATs could provide sufficient signaling to explain the residual, although greatly reduced, ISG activation (Fig. 2) and STAT1 and STAT2 nuclear accumulation (Fig. 3) observed in IFN-α–treated transfected cells. We examined also the expression of p48 that, together with STAT1 and STAT2, forms the ISGF3 transcription factor complex promoting transcription of ISGs. Figure 4 shows that, similarly to both STATs, the level of p48 was significantly enhanced in transfected cells as compared with normal cells independently of IFN-α stimulation. Taken together, our results showed that HDV inhibited the JAK-STAT signaling cascade in response to IFN-α by impairing the phosphorylation of both STAT1 and STAT2.

Figure 4.

HDV inhibition of IFN-α–induced tyrosine phosphorylation of STAT1 and STAT2. Normal cells (Huh-7) and cells transfected with HDV cDNA [Huh-7 pSVL(D3)] were cultured for 9 days and then treated with 1000 U/mL IFN-α for 30 minutes or left untreated. Equal amounts of cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and examined with antibodies to phosphotyrosine 701-STAT1 (pY-STAT1), STAT1, phosphotyrosine 690-STAT2 (pY-STAT2), STAT2, p48, and HDAg. β-Actin levels served as a loading control.

HDV Blocks IFN-α–Induced Tyrosine Phosphorylation of Tyk2.

The ability of HDV to inhibit STAT1 and STAT2 activations in response to IFN-α suggests that there may be one or more components in the JAK-STAT signaling pathway upstream of the phosphorylation of STATs that are affected by viral replication. A first step in the activation of the IFN signal transduction cascade is the autophosphorylation of the tyrosine kinases Jak1 and Tyk2 in response to a ligand-induced conformational change in the IFN-α/β receptor. Phosphorylated JAKs are then responsible for the activation of STAT1 and STAT2. To examine whether the JAKs were activated by IFN-α stimulation in transfected cells, we performed immunoblot analysis and estimated the levels of tyrosine-phosphorylated JAKs in response to IFN-α (Fig. 5A,B). Total cell extracts from normal and HDV-transfected cells were probed with antibodies recognizing either the phosphorylated or the nonphosphorylated forms of Jak1 and Tyk2. The steady-state levels of both Jak1 and Tyk2 were stable, irrespective of IFN-α treatment and HDV transfection (Fig. 5A, B). After IFN-α stimulation, no inhibition of tyrosine phosphorylation of Jak1 was observed in transfected cells as compared with untransfected cells (Fig. 5A, lanes 2 and 4). In contrast, although tyrosine-phosphorylated Tyk2 was detected in lysates of untransfected cells stimulated with IFN-α (Fig. 5B, lane 2), samples from IFN-α–treated HDV-transfected cells did not contain pY-Tyk2 (Fig. 5B, lane 4). These results suggest that HDV inhibits phosphorylation, and thus activation, of Tyk2 in response to IFN-α. Because Tyk2 is responsible for the phosphorylation of both the IFNAR1 subunit, to create a docking site for STAT2, and of STAT2 itself (which is required for any phosphorylation of STAT1 in response to type I IFNs), this block is likely to result in the observed marked suppression of STAT phosphorylation in response to IFN-α.

Figure 5.

HDV inhibition of JAK-STAT signaling in response to IFN-α is associated with inhibition of Tyk2 phosphorylation. Normal cells (Huh-7) and cells transfected with HDV cDNA [Huh-7 pSVL(D3)] were stimulated with 1000 U/mL IFN-α for 30 minutes or left untreated. Cell extracts were subjected to Western blotting analysis using antibodies to phosphotyrosine 1022/1023-Jak1 (pY-Jak1) and Jak1 (A), phosphotyrosine 1054/1055-Tyk2 (pY-Tyk2) and Tyk2 (B), IFNAR1 and IFNAR2 (C). β-Actin levels served as a loading control.

To examine whether the inhibition by HDV of the receptor-associated kinase was attributable to down-regulation of the IFN-α/β receptor, we checked also the expression levels of IFNAR1 and IFNAR2. This analysis showed that the total levels of both IFNAR1 and IFNAR2 were similar in HDV-transfected cells as compared with untransfected cells (Fig. 5C). Therefore, our results imply that the inhibition of Tyk2 phosphorylation attributable to HDV replication is not mediated by the suppression of IFNAR1 and/or IFNAR2 expression and that HDV likely interferes with signaling between the IFN receptor and the JAKs.


The interplay between viruses and the IFN-mediated immune response is a subject of intense research, because it may account for the mechanisms used by these pathogens to establish persistent infection. The effects of HDV on IFN-α/β signaling have been scantily investigated. Here, we report that HDV is likely to have acquired the ability to target the JAK-STAT transduction cascade as a countermeasure for evading the IFN response. IFN-α, especially in its pegylated form, represents the standard treatment for patients with chronic HDV infection. This therapy is effective only in a reduced number of patients, and the reason of failure in viral eradication is not understood. The findings reported in the current study could provide an explanation as to why and how HDV resists IFN-α treatment.

Signaling elicited by IFN-α through the JAK-STAT pathway mediates rapid and robust transcriptional induction of genes encoding antiviral proteins. This process is a highly evolved system that protects animals from viral infection. Interactions between viral proteins and components of the innate immune response are major determinants in viral pathogenesis. This fact is best exemplified with genetically modified mice that are unable to respond to IFN-α because of a lack of functional IFN-α/β receptors or STAT proteins and, as a consequence, rapidly succumb to viral infections.50–52 Conversely, many viruses capable of establishing persistent infections depend on mechanisms to attenuate the IFN response for their survival.53 Both RNA and DNA viruses use various strategies to inhibit IFN-α–stimulated host defense mechanisms.20, 54, 55 Some viruses reduce the basal levels of proteins of the JAK-STAT pathway, such as human cytomegalovirus that targets Jak1 and p48 for degradation via the proteasome.56, 57 Several viruses inactivate components of the JAK-STAT signaling cascade. Measles virus, for example, inhibits the IFN-induced phosphorylation of Jak1,58 whereas herpes simplex virus type 1 blocks the phosphorylations of JAKs and STATs.59, 60 Our results indicate that HDV is capable of lowering the level of IFN-stimulated gene activation (Fig. 2). Furthermore, HDV impairs the nuclear accumulation of STAT1 and STAT2 as a consequence of the inhibition of the IFN-α–induced tyrosine phosphorylation, but not of the total protein expression levels, of both STATs and Tyk2 (Figs. 3, 4, and 5B).

The exact molecular mechanism by which HDV inhibits JAK-STAT signaling is not yet known. In unstimulated normal cells, STAT1 and STAT2 preassociate with the cytoplasmic tail of IFNAR2. IFN-α–mediated aggregation of the IFN receptor subunits, IFNAR1 and IFNAR2, results in phosphorylation of Jak1 and Tyk2. Activated Tyk2 phosphorylates IFNAR1, which then serves as a binding site for STAT2. STAT2 is then phosphorylated by Tyk2, providing a docking site for STAT1, which is subsequently phosphorylated by Jak1. The observations that HDV alters neither the IFN-stimulated tyrosine phosphorylation of Jak1 (Fig. 5A) nor the expression levels of IFNAR1 and IFNAR2 (Fig. 5C) suggest that binding of IFN-α to the receptor and subsequent oligomerization of the receptor subunits occur normally. HDV could counteract the IFN-α signaling by promoting a direct association with the IFN receptor subunits or with Tyk2 such that the tyrosine phosphorylation of the latter is impaired and further signal transduction cannot occur. For example, human papillomavirus type 18 E6 protein was found to physically interact with Tyk2 through a domain that is important for its binding to the cytoplasmic portion of IFNAR1 and therefore impairs Tyk2 activation on IFN-α stimulation.61 HDV encodes a single protein, HDAg, and one or both of its isoforms, HDAg-S and HDAg-L, might possess the IFN-α antagonist activity. During infection, both HDAgs have nuclear localization29, 30 (Fig. 3). Because activations of STATs and JAKs occur in the cytoplasm, it seems unlikely that the mechanism by which HDV inhibits JAK-STAT signaling is the result of a direct physical interaction between HDAg and IFNARs or Tyk2. Nevertheless, HDV could promote the expression or activity of one or more factors that might act in the cytoplasm. Another possibility that could explain the observed HDV-mediated inhibition is that viral replication may lead to the modulation of negative-feedback mechanisms that could suppress Tyk2 phosphorylation. Some of these mechanisms rely on the activity of suppressors of cytokine signaling62 and of protein tyrosine phosphatases, such as the SH2 domain-containing tyrosine phosphatases.13, 63 For example, herpes simplex virus type 1 was shown to induce suppressor of cytokine signaling 3 expression, leading to inhibition of the IFN response.64 It is also possible that the mechanism of HDV inhibition is indirect. For example, it may be the consequence of changes and rearrangements in the cytoplasm caused by viral replication, as reported for some Flaviviridae.65, 66 However, because hepatitis C virus (HCV) replication also induces similar changes and yet is very sensitive to IFNs,48, 67 we do not favor this possibility.

In summary, our study reveals that HDV may impair the IFN-α–stimulated JAK-STAT signaling pathway. The mechanism adopted by HDV to interfere with IFN-α/β signal transduction relies on tyrosine phosphorylation inhibition of STAT1, STAT2 and receptor-associated kinase Tyk2, without down-regulation of the expression levels of the IFN-receptor subunits or of other components in the signaling cascade. These results add HDV to a populated list of viruses that evolve strategies to counteract the actions of type I IFNs. This study may prove helpful to better understand the observed resistance to IFN action of chronic HDV patients and could provide useful indications for the identification of new antiviral intervention strategies.


The authors thank Maria Lorena Abate for providing the vector pSVL(D3), John M. Taylor for kindly donating the anti-delta antigen antibody, the Genomics Platform of the University of Geneva for performing the real-time PCR experiments, and to Alessandra Calabrese and Fabio Carrozzino for invaluable help.