Retinoic acid–inducible gene-I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) belong to the RIG-I–like receptors family of pattern recognition receptors. Both RIG-I and MDA5 have been shown to recognize various viral RNAs, but whether they mediate hepatitis B virus (HBV) infection remains unclear. In this study, we demonstrated that the expression of MDA5, but not RIG-I, was increased in Huh7 cells transfected with the HBV replicative plasmid and in the livers of mice hydrodynamically injected with the HBV replicative plasmid. To further determine the effect of RIG-I–like receptors on HBV replication, we cotransfected the HBV replicative plasmid with RIG-I or MDA5 expression plasmid into Huh7 cells and found that MDA5, but not RIG-I at a similar protein level, significantly inhibited HBV replication. Knockdown of endogenous MDA5, but not RIG-I, in Huh7 cells transfected with the HBV replicative plasmid significantly increased HBV replication. Of particular interest, we found that MDA5, but not RIG-I, was able to associate with HBV-specific nucleic acids, suggesting that MDA5 may sense HBV. Finally, we performed in vivo experiments by hydrodynamic injection of the HBV replicative plasmid into wild-type, MDA5−/−, MDA5+/−, or RIG-I+/− mice, and found that MDA5−/− and MDA5+/− mice, but not RIG-I+/− mice, exhibited an increase of HBV replication as compared with wild-type mice. Collectively, our in vitro and in vivo studies both support a critical role for MDA5 in the innate immune response against HBV infection.

Human hepatitis B virus (HBV) is a small (3.2-kb), enveloped, noncytopathic DNA virus characterized by its pronounced species and liver tropism (1). HBV infection is worldwide with a high prevalence in Asia and Africa, and it causes a wide spectrum of liver diseases, including acute/chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). Although HBV vaccine has been available for three decades, the innate immune response to HBV infection remains to be elucidated.

The genomic arrangement of HBV is unique among viruses. The HBV genome comprises a relaxed circular partially dsDNA that contains four overlapping reading frames encoding the envelope, precore/core, polymerase, and X proteins (2). After entry and uncoating of HBV in hepatocytes, the HBV genome is transported into the nucleus, and the relaxed circular DNA (rcDNA) is converted into covalently closed circular DNA (cccDNA). The cccDNA serves as a transcriptional template for the synthesis of four viral RNAs, which are exported to the cytoplasm and used as mRNA for translating HBV proteins. The largest viral RNA (3.5 kb), known as the viral pregenomic RNA (pgRNA), is assembled with HBV polymerase and core proteins to form nucleocapsids, and functions as the template for reverse transcription within nucleocapsids in the cytoplasm, ultimately generating newly synthesized rcDNA. The nucleocapsids can be either enveloped during their passage through the endoplasmic reticulum and Golgi complex followed by secretion from the cells or retransported into the nucleus for the amplification of the cccDNA pool to generate more viral RNAs (1, 2).

Hosts infected by viruses usually elicit a rapid and potent innate immune response to produce antiviral molecules to limit viral replication and to prevent viral spreading before the adaptive immune response is generated (35). Pattern recognition receptors (PRRs), which recognize various pathogen-associated molecule patterns, have been shown to play a critical role in the innate immune response against pathogens (6). Among PRRs, the endosomal TLRs including TLR3, TLR7/8, and TLR9, and cytosolic RIG-I–like receptors (RLRs) including retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5) are important for sensing viral RNA during viral infection (3, 5, 6). After the recognition of virus-associated molecules by PRRs, PRRs activate their specific adaptor proteins: TIR domain-containing adapter-inducing IFN-β for TLR3 (7), MyD88 for TLR7/8 and TLR9 (8), and IFN-β promoter stimulator 1(IPS-1), also known as mitochondrial antiviral signaling protein, CARD adaptor-inducing IFN-β, and virus-induced signaling adaptor for RLRs (912). The activation of adaptor proteins of PRRs ultimately activates downstream transcription factors, IFN regulatory factors (IRFs), and NF-κB, to induce genes that are critical for antiviral functions, as well as the dictation of adaptive immune responses (3, 5, 6). The inhibition of HBV replication and the induction of antiviral effects by TLR signaling are primarily mediated by nonparenchymal cells, such as dendritic cells, Kupffer cells, and liver sinusoidal endothelial cells (13, 14). Given that TLRs are expressed on plasma or endosomal membranes to recognize ligands from extracellular compartments, and that HBV is a noncytopathic DNA virus whose nucleic acids may not be present in extracellular compartments, one would expect that TLR signaling may not be so critical for the innate response to HBV in hepatocytes. Because RLRs are cytosolic viral sensors and HBV nucleic acids may be present in the cytosol in addition to the nucleus as described in the aforementioned paragraph, HBV is more likely to be recognized by RLRs. Consistent with this notion, recent studies have demonstrated that overexpression of IPS-1 in a hepatoma cell line transfected with the HBV replicative plasmid significantly suppresses HBV replication (15), and that HBV X protein interacts with IPS-1 and disrupts the downstream signaling of RLRs to prevent the production of type I IFNs induced by Sendai virus, vesicular stomatitis virus (VSV), or poly(dA:dT) (1619). Furthermore, two studies have shown that HBV pol impairs the activation of TBK1/IKKε, the downstream signaling molecule of IPS-1 in the RLR signaling pathway (20, 21). Altogether, these studies suggest that IPS-1 is likely involved in the innate response against HBV infection. Given that RIG-I and MDA5 are upstream molecules of IPS-1 (22) and that IPS-1 is involved in the innate response against HBV (1521), RIG-I and MDA5 may play a role in the regulation of HBV infection.

In this study, we intended to investigate the involvement of RIG-I and MDA5 in HBV infection using both in vitro and in vivo experiments. Experimentally studying HBV infection has been challenging because HBV fails to infect commonly used cell lines of hepatocyte origin. Moreover, it becomes difficult to perform in vivo study of the immune response to HBV infection because HBV fails to infect mice, the main animal model widely used for studying immune response to pathogens because of the availability of reagents and the similarity of immune response to humans. Transient transfection of the HBV replicative plasmid into human hepatoma cell lines has been widely used for in vitro studies of HBV replication and of HBV interaction with host (2325), to overcome the obstacles, whereas hydrodynamic injection to deliver the HBV replicative plasmid into mouse livers has been developed for the study of the host response to HBV infection (2630). For in vitro studies, we transfected Huh7 cells with the HBV replicative plasmid and the expression plasmid for MDA5 or RIG-I and found that MDA5, but not RIG-I at a similar protein level, significantly activated downstream signaling to inhibit HBV replication. In addition, we performed in vivo studies by hydrodynamic injection of the HBV replicative plasmid into mice to mimic acute HBV infection (26). In agreement with the in vitro data, the heterozygous and homozygous MDA5 knockout mice, but not the heterozygous RIG-I knockout mice, receiving the HBV replicative plasmid showed increased HBV replication as compared with littermate control wild-type mice. Our study clearly demonstrates that MDA5, a well-known cytosolic sensor for RNA viruses, plays a crucial role in the innate immune response against HBV infection.

BALB/c mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed under specific pathogen-free conditions at the Institute of Biomedical Sciences, Academia Sinica (Taipei, Taiwan). The original breeders of MDA5+/− and RIG-I+/− mice were kindly provided by Dr. Shizuo Akira (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) (31, 32). The MDA5+/− and RIG-I+/− mice were crossed with C57BL/6 mice, and the offspring mice were intercrossed and maintained under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Utilization Committee at Academia Sinica and were performed in accordance with institutional guidelines.

Huh7 cells were cultured in DMEM supplemented with 10% FBS (Life Technologies, Grand Island, NY), 2 mM l-glutamine, 1% nonessential amino acids, and 1% sodium pyruvate (Life Technologies) at 37°C under 5% CO2 in a humidified atmosphere.

The pSV2ANeo-HBVx2 plasmid, an HBV ayw dimer DNA containing plasmid that has two head-to-tail copies of the HBV genome of ayw subtype (33), and the polymerase-null HBV mutant 2310, a point mutation converting the first ATG codon of HBV pol at nucleotide 2310 into ACG in pSV2ANeo-HBVx2 plasmid (34, 35), were kindly provided by Dr. Chiaho Shih (Institute of Biomedical Sciences, Academia Sinica). To generate pKRX-HBVx2, we subjected pSV2ANeo-HBVx2 to EcoRI partial digestion to obtain the HBV dimer DNA fragment of HBV ayw subtype, which was then subcloned into the pKRX vector at the EcoRI site. To construct pCAGGS-RIG-I-Flag2, we used pEF-BOS-RIG-I plasmid (provided by Dr. Takashi Fujita, Department of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan) containing the coding region of RIG-I as the template together with a primer set (forward, 5′-CCCTCGAGATGACCACCGAGCAGCGACGC-3′; reverse, 5′-CGGGGTACCTTTGGACATTTCTGCTGGATCA-3′) to amplify the fragment of the coding region of RIG-I by PCR, and the resulting PCR product was purified and subcloned into pCAGGS-MCS-Flag2 (provided by Dr. Steve S.-L. Chen, Institute of Biomedical Sciences, Academia Sinica) at the XhoI and KpnI sites. To generate pCAGGS-MDA5-Flag2, we performed RT-PCR amplification of total RNA from HepG2 cells to obtain the full-length MDA5 cDNA, which was used as the template together with a primer set (forward, 5′-CCCTCGAGATGTCGAATGGGTATTCCACAG-3′; reverse, 5′- GGGGTACCATCCTCATCACTAAATAAAC-3′) to amplify the fragment of the coding region of MDA5 for subsequent cloning into pCAGGS-MCS-Flag2.

One nontargeting control small interfering RNA (siRNA), four siRNAs targeting human MDA5, and four siRNA targeting human RIG-I were purchased from Dharmacon (Lafayette, CO). Four sequence-specific siRNAs targeting human MDA5 were tested for their silence efficacy on the specific inhibition of exogenous and endogenous MDA5 gene, and the siRNA (5′-UGACACAAUUCGAAUGAUA-3′) with the highest silence efficacy was used for experiments. Similarly, four sequence-specific siRNAs targeting human RIG-I were also subjected to the determination of the silence efficacy and the siRNA (5′-CCACAACACUAGUAAACAA-3′) with the highest silence efficacy was used for experiments. The negative control siRNA and the siRNA targeting human MxA (5′-UCCGUUAGCCGUGGUGAUUUA-3′) were purchased from Qiagen (Germantown, MD) and used to knock down endogenous MxA. Transfection of plasmids and siRNAs into cells was performed using Lipofectamine 2000 (Invitrogen) under optimized conditions.

Total RNA was extracted from mouse livers or Huh7 cells by MaestroZol RNA Extraction Reagent (Maestrogen, Las Vegas, NV) according to the manufacturer’s instructions. Total RNA (5 μg) was reverse transcribed into cDNA by SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cDNA was subjected to real-time PCR analysis. An appropriate amount of the cDNA was mixed with Maxima SYBR Green/ROX qPCR Master Mix (Fermentas, ON, Canada) supplemented with gene-specific primers. The thermal cycling protocol was 1 cycle at 50°C for 2 min and then 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The resultant PCR products were analyzed by ABI 7500 software (Applied Biosystems, Foster City, CA). The levels of gene expression were analyzed with the ΔΔCt method, and all quantifications were normalized to the level of β-actin (forward, 5′-TGGACTTCGAGCAAGAGATG-3′; reverse, 5′-TTGCTGATCCACATCTGCTG-3′), MDA5 (forward, 5′-AGTTTGGCAGAAGGAAGTGTC-3′; reverse, 5′-GGAGTTTTCAAGGATTTGAGC-3′), RIG-I (forward, 5′-GAATCTGCAAAGACCTCGAA-3′; reverse, 5′-TCTGAGTAAGATCTTGCTCAATC-3′), HBV core gene (forward, 5′-CGTTTTTGCCTTCTGACTTCTTTC-3′; reverse, 5′-ATAGGATAGGGGCATTTGGTGGTC-3′), and MxA (forward, 5′-GCTACACACCGTGACGGATATGG-3′; reverse, 5′-CGAGCTGGACTGGAAAGCCC-3′). The real-time PCRs for OAS1, CXCL10, and IFN-β were performed using the TaqMan probes (Applied Biosystems). The levels of gene expression were analyzed with the ΔΔCt method, and all quantifications were normalized to the level of GAPDH.

Cells were lysed on ice in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM aprotinin, 1 mM leupeptin, and 1 mM PMSF), and cell lysates were centrifuged at 14,000 × g at 4°C for 15 min. The resulting cell lysates were subjected to 8% SDS-PAGE and electrotransferred onto nitrocellulose membranes (PerkinElmer, Norwalk, CT) followed by Western blotting. The membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 at room temperature for 1 h, and all incubations and washes were done in the presence of blocking solution. Blots were incubated with specific primary Abs, washed, and incubated with HRP-conjugated secondary Abs (Pierce, Rockford, IL), washed again, and visualized by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). The primary Abs used for Western blotting were rabbit polyclonal anti-FLAG (Sigma-Aldrich, St. Louis, MO) to detect FLAG-MDA5 and FLAG–RIG-I, rabbit anti–β-actin polyclonal Ab (Sigma-Aldrich) to detect β-actin, rabbit anti-IRF3 phospho (pS386) mAb (Epitomics, Burlingame, CA) to detect phosphorylated IRF3, rabbit polyclonal anti-IRF3 (Santa Cruz Biotechnology, Santa Cruz, CA) to detect total IRF3, rabbit polyclonal anti–NF-κB/p65 (Santa Cruz Biotechnology) to detect NF-κB/p65, rabbit polyclonal anti–poly(ADP-ribose) polymerase (PARP) (Cell Signaling, Danvers, MA) to detect the nuclear marker PARP, and rabbit monoclonal anti–α-tubulin (Epitomics) to detect the cytosolic marker α-tubulin.

Total RNA (20 μg) was separated in 1% formaldehyde-agarose gels, transferred to IMMOBILON NY+ charged nylon membranes (Millipore, Billerica, MA), and prehybridized in ULTRAhyb Hybridization buffer (Ambion, Austin, TX) at 42°C for 1 h. The membranes were then hybridized with 1 × 106 cpm/ml of [32P]-labeled specific probe at 42°C overnight. The radioisotope-labeled probe was generated by labeling 25 ng purified full-length 3.2-kb HBV DNA fragment with α-[32P]-dCTP (PerkinElmer) using the Amersham Rediprime II DNA labeling System (GE Healthcare Life Sciences, Piscataway, NJ), and the specificity of probes was ∼1 × 109 cpm/μg. The membrane was washed with 2× SSC/0.1% SDS at room temperature for 1 h followed by three washes with 0.1× SSC/0.1% SDS at 65°C for 30 min. The signals were detected by autoradiography film (MIDSCI, St. Louis, MO).

Viral DNA was isolated from intracellular viral capsids and detected with specific isotope-labeled probe as described previously (36). In brief, cells were lysed in lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 50 mM NaCl, 0.25% Nonidet P-40 [NP-40], and 8% sucrose) at 37°C for 20 min. The cell lysates were centrifuged at 13,000 × g for 15 min at room temperature. The supernatants were collected and brought to a final concentration of 8 mM CaCl2 and 6 mM MgCl2 followed by digestion with 30 U/ml micrococcal nuclease (New England Biolabs, Ipswich, MA) and 3 U/ml RQ1 DNase (Promega, Madison, WI) at 37°C for 20 min. Then the solution (26% polyethylene glycol 8000, 1.4 M NaCl, and 60 mM EDTA) was added into the digested supernatants (1/3, v/v). After incubation at 4°C for 2 h, the viral core particles were pelleted by centrifugation at 10,000 × g at 4°C for 10 min. The pellet was resuspended in buffer containing 10 mM Tris, pH 7.5, 8 mM CaCl2, and 6 mM MgCl2, and subjected to digestion with 30 U/ml micrococcal nuclease and 3 U/ml RQ1 DNase at 37°C for 20 min. The nuclease-digested core particles were lysed in SDS lysis buffer (25 mM Tris, pH 7.5, 10 mM EDTA, and 1% SDS) that contained 400 μg/ml Proteinase K (MDBio, Taipei, Taiwan) at 50°C overnight, and the core particle–associated DNA was released and extracted by phenol/chloroform followed by ethanol precipitation. The core particle–associated DNA was separated in a 1% agarose gel, transferred onto IMMOBILON NY+ charged nylon membranes (Millipore), and prehybridized in ULTRAhyb Hybridization buffer (Ambion) at 42°C for 1 h. The membranes were then hybridized with 1 × 106 cpm/ml [32P]-labeled specific probes at 42°C overnight. The membranes were washed with 2× SSC/0.1% SDS at room temperature for 1 h, followed by three washes with 0.1× SSC/0.1% SDS at 65°C for 30 min. The signals were detected by autoradiography film.

Cells were harvested and washed twice with PBS. Cell pellets were resuspended in hypotonic buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1 mM DTT, and 0.5 mM PMSF) and incubated on ice for 15 min. After incubation, 10% NP-40 was added to the cell suspensions (1/16, v/v) and mixed by vortexing for 20 s. The lysates were then centrifuged at 9000 × g at 4°C for 2 min to pellet the nucleus, and the supernatants were saved as cytosolic fractions. The nuclear pellets were washed with hypotonic buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1 mM DTT, and 0.5 mM PMSF) and then resuspended in hypertonic buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 0.5 M NaCl, 1 mM DTT, and 0.5 mM PMSF). The nuclear lysates were incubated at 4°C for 30 min followed by centrifugation at 15,000 × g for 5 min at 4°C to pellet the debris. The supernatants were saved as nuclear fractions.

Cells were harvested, resuspended in PBS, and fixed with 1% formaldehyde in PBS at room temperature for 15 min followed by the addition of 2.5 M glycine (1/10, v/v) to neutralize the cross-linking. The fixed cells were then centrifuged at 800 × g at 4°C for 5 min and washed three times with PBS. The cell pellets were resuspended in ice-cold FA lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM leupeptin, 1 mM aprotinin, 1 mM PMSF, and 20 μg/ml RNase inhibitor), and the cells were lysed with repeated freezing and thawing. The cell lysates were then centrifuged at 15,000 × g at 4°C for 15 min, and the supernatants were collected and subjected to immunoprecipitation with anti-FLAG M2 affinity gel (Sigma-Aldrich). The immunoprecipitates were sequentially washed three times with FA lysis buffer, once with FA500 (50 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM leupeptin, 1 mM aprotinin, 1 mM PMSF, and 20 μg/ml RNase inhibitor), once with LiCl wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.1% sodium deoxycholate, 1 mM EDTA, 1 mM leupeptin, 1 mM aprotinin, 1 mM PMSF, and 20 μg/ml RNase inhibitor), and finally once with TE/0.1 M NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 1 mM leupeptin, 1 mM aprotinin, 1 mM PMSF, and 20 μg/ml RNase inhibitor). The nucleic acids associated with the immunoprecipitates were eluted from anti-FLAG M2 affinity gels by elution buffer (100 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM leupeptin, 1 mM aprotinin, 1 mM PMSF, and 20 μg/ml RNase inhibitor), and the formaldehyde cross-links were reversed in elution buffer by incubation at 70°C for 1 h. To detect HBV DNA present in immunoprecipitates, we subjected the eluent to DNA purification with the DNA Clean/Extraction Kit (GeneMark Technology, Tainan, Taiwan) followed by the detection of HBV sequence by real-time PCR using a primer set specific for the HBV core gene (forward, 5′-CGTTTTTGCCTTCTGACTTCTTTC-3′; reverse, 5′-ATAGGATAGGGGCATTTGGTGGTC-3′). To detect HBV RNA present in immunoprecipitates, we pretreated the eluent with DNase I for 30 min at 37°C, and the RNA was extracted by MaestroZol RNA Extraction Reagent (Maestrogen). The RNA was then reverse-transcribed into cDNA by SuperScript III Reverse Transcriptase (Invitrogen), and the cDNA was subjected to the detection of HBV sequence by real-time PCR using a primer set specific for the HBV core gene as described earlier.

HEK293T cells were transfected with pCAGGS-MDA5-FLAGx2 or pCAGGS-RIG-I-FLAGx2 by Lipofectamine 2000. At 48 h posttransfection, transfectants were lysed on ice in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM aprotinin, 1 mM leupeptin, and 1 mM PMSF), and cell lysates were centrifuged at 14,000 × g at 4°C for 15 min. The supernatants were collected and subjected into immunoprecipitation with the M2 anti-FLAG affinity gel. After three washes with lysis buffer, the FLAG-tagged proteins were eluted from the immunoprecipitates with lysis buffer containing 100 μg/ml FLAG peptide at 4°C. After centrifugation at 900 × g at 4°C for 5 min, the supernatants containing purified FLAG-tagged proteins were collected and coated onto Nunc MAXISORP Immuno plate (Nunc, Roskilde, Denmark) at 4°C overnight. The wells were washed three times with PBST (PBS containing 0.1% Tween 20) and then blocked with 1% BSA in PBS at room temperature for 1 h. Biotinylated HBV DNA was generated by PCR with pSV2ANeo-HBVx2 as template and with 5′-biotinylated primer pairs (forward: 5′-AATTCCACAACCTTCCACCAAACTC-3′; reverse: 5′-CCACTGCATGGCCTGAGGATGAGTG-3′), and the resultant PCR products were purified using the DNA Clean/Extraction Kit. The purified biotinylated HBV DNA was added into the wells coated with or without FLAG-tagged proteins, and incubated at room temperature for 1 h. After three washes with PBST, the biotinylated HBV DNA associated with FLAG-MDA5 or FLAG–RIG-I was detected using HRP-conjugated streptavidin with tetramethylbenzidine (TMB) as a substrate.

The HBV surface Ag (HBsAg) concentrations in sera or culture supernatants were determined using the SURASE B-96 ELISA kit (General Biologicals, Hsinchu, Taiwan).

Cells were washed three times with PBS, fixed with 4% formaldehyde in PBS at room temperature for 30 min, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min. The cells were then washed three times with PBS, incubated with 5% BSA in PBS at room temperature for 1 h, and immunostained with specific primary Abs. After three washes with PBS, cells were further incubated with fluorochrome-conjugated secondary Abs for 30 min followed by counterstaining with 0.5 μg/ml DAPI for 10 min. Images were obtained using a confocal microscope (LSM 700; Carl Zeiss AG, Oberkochen, Germany).

Mice (6–8 wk old) were anesthetized by i.m. injection of 30 μl per mouse of atropine/ketamine/xylazine mixture (1 mg/ml atropine/100 mg/ml ketamine/2% xylazine/saline, 2/1/1/1, v/v/v/v). Thirty micrograms of pKRX or pKRX-HBVx2 in a volume of isotonic saline equivalent to 8% of body weight were injected into the tail veins of mice within 8 s.

The unpaired Student t test was used to evaluate the significance of the difference between two experimental results. A p value <0.05 was considered statistically significant.

To investigate the involvement of RLRs in HBV infection, we delivered the HBV replicative plasmid into Huh7 cells and into mouse liver by transfection and hydrodynamical injection, respectively, and measured the expression of MDA5 and RIG-I in the Huh7 cells and mouse liver. To ensure successful transfection of the HBV replicative plasmid, we measured HBsAg from the culture supernatants of Huh7 cells and from the sera of mice (Fig. 1A, 1B, left panels). Of interest, the expression of MDA5, but not RIG-I, was significantly increased in Huh7 cells transfected with the HBV replicative plasmid as compared with the vector (p < 0.05; Fig. 1A). Likewise, the in vivo experiments also showed significantly increased expression of MDA5, but not RIG-I, in the livers of mice receiving the HBV replicative plasmid as compared with the vector on day 3 postinjection (p < 0.05; Fig. 1B). Taken together, both in vitro and in vivo experiments suggest that MDA5 plays a role in HBV infection.

FIGURE 1.

Mice or cells transfected with the HBV replicative plasmid increase MDA5 expression. (A) Huh7 cells were transfected with the HBV replicative plasmid. At 45 h posttransfection, the culture supernatants were collected and subjected to HBsAg measurement by ELISA (left panel), and the cells were harvested for total RNA extraction followed by the first-strand cDNA synthesis by reverse transcription. The cDNAs were used to determine the expression of MDA5 and RIG-I by real-time PCR (middle and right panels). (B) The HBV replicative plasmid was delivered into BALB/c mice by hydrodynamic injection. On day 3 posttransfection, HBsAg in the sera was determined by ELISA (left panel), and the expression of MDA5 and RIG-I in the liver was determined by reverse transcription followed by real-time PCR (middle and right panels). Data are presented as the mean ± SEM. *p < 0.05.

FIGURE 1.

Mice or cells transfected with the HBV replicative plasmid increase MDA5 expression. (A) Huh7 cells were transfected with the HBV replicative plasmid. At 45 h posttransfection, the culture supernatants were collected and subjected to HBsAg measurement by ELISA (left panel), and the cells were harvested for total RNA extraction followed by the first-strand cDNA synthesis by reverse transcription. The cDNAs were used to determine the expression of MDA5 and RIG-I by real-time PCR (middle and right panels). (B) The HBV replicative plasmid was delivered into BALB/c mice by hydrodynamic injection. On day 3 posttransfection, HBsAg in the sera was determined by ELISA (left panel), and the expression of MDA5 and RIG-I in the liver was determined by reverse transcription followed by real-time PCR (middle and right panels). Data are presented as the mean ± SEM. *p < 0.05.

Close modal

Knowing that MDA5 expression is upregulated after HBV transfection (Fig. 1) and that MDA5 signaling pathway is responsible for the antiviral effect (32, 37, 38), we next investigated the effect of MDA5 on HBV replication by gain-of-function experiments. Huh7 cells were cotransfected with the HBV replicative plasmid and the plasmid encoding MDA5 or RIG-I or the control plasmid. With comparable protein levels of MDA5 and RIG-I, only cells transfected with the MDA5 expression plasmid showed significant inhibition of the levels of HBV RNA (3.5-, 2.4-, and 2.1-kb RNA) and intracapsid HBV DNA including rcDNA and ssDNA in Huh7 cells (Fig. 2A). Using immunofluorescence staining to detect HBV core Ag (HBcAg) expression in cells that showed positive for MDA5 or RIG-I, we found that the percentage of cells expressing HBcAg in MDA5 transfectants was significantly lower than that in RIG-I transfectants (14.58 ± 2.95 versus 95.27 ± 1.63%, p < 0.01; Fig. 2B). Consistently, the level of secreted HBsAg in the culture supernatants of MDA5 transfectants was significantly reduced as compared with control or RIG-I transfectants (p < 0.01; Fig. 2C). Altogether, MDA5 overexpression significantly reduces the levels of DNA, RNA, and protein of HBV, suggesting that the MDA5 signaling pathway mediates the suppression of HBV replication. The exogenously expressed MDA5 was knocked down by specific siRNA and the level of secreted HBsAg was measured to further confirm that the suppression of HBV replication is mediated by MDA5 expressed in cells transfected with the HBV replicative plasmid. Knockdown of ectopic MDA5 expression by siRNA in cells cotransfected with the HBV replicative plasmid and MDA5 expression plasmid restored the levels of secreted HBsAg (Fig. 2D, 2E).

FIGURE 2.

Overexpression of MDA5 in Huh7 cells leads to the suppression of HBV replication. Huh7 cells were transfected with the HBV replicative plasmid together with either the expression vector encoding FLAG-tagged MDA5 or FLAG-tagged RIG-I or the corresponding control vector. (A) At 48 h posttransfection, the cells were harvested and the expression of MDA5 and RIG-I were detected by Western blotting using an anti-FLAG Ab (top panel), the levels of HBV RNAs were detected by Northern blotting (middle panel), and the levels of HBV DNAs were detected by Southern blotting (bottom panel). (B) The expression of HBcAg in the transfectants was detected by immunofluorescence staining, and the images were taken by a confocal microscope. The percentage of HBcAg+ cells in cells expressing exogenous MDA5 or RIG-I was quantified by counting cells from three different fields per sample (bar graph at right). (C) The levels of HBsAg in culture supernatants were determined by ELISA. (D and E) Huh7 cells cotransfected with the HBV replicative plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the corresponding control vector were transfected with either control siRNA or MDA5 siRNA. (D) At 48 h posttransfection, the level of FLAG-MDA5 was detected by Western blotting using the anti-FLAG Ab, and β-actin was used as a loading control. (E) The levels of HBsAg in culture supernatants were determined by ELISA. Data represent the mean ± SEM from three independent experiments. *p < 0.01. RC, Relaxed circular HBV DNA; SS, single-stranded HBV DNA.

FIGURE 2.

Overexpression of MDA5 in Huh7 cells leads to the suppression of HBV replication. Huh7 cells were transfected with the HBV replicative plasmid together with either the expression vector encoding FLAG-tagged MDA5 or FLAG-tagged RIG-I or the corresponding control vector. (A) At 48 h posttransfection, the cells were harvested and the expression of MDA5 and RIG-I were detected by Western blotting using an anti-FLAG Ab (top panel), the levels of HBV RNAs were detected by Northern blotting (middle panel), and the levels of HBV DNAs were detected by Southern blotting (bottom panel). (B) The expression of HBcAg in the transfectants was detected by immunofluorescence staining, and the images were taken by a confocal microscope. The percentage of HBcAg+ cells in cells expressing exogenous MDA5 or RIG-I was quantified by counting cells from three different fields per sample (bar graph at right). (C) The levels of HBsAg in culture supernatants were determined by ELISA. (D and E) Huh7 cells cotransfected with the HBV replicative plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the corresponding control vector were transfected with either control siRNA or MDA5 siRNA. (D) At 48 h posttransfection, the level of FLAG-MDA5 was detected by Western blotting using the anti-FLAG Ab, and β-actin was used as a loading control. (E) The levels of HBsAg in culture supernatants were determined by ELISA. Data represent the mean ± SEM from three independent experiments. *p < 0.01. RC, Relaxed circular HBV DNA; SS, single-stranded HBV DNA.

Close modal

We next conducted the loss-of-function experiments to confirm the inhibitory effect of MDA5 on HBV replication. Huh7 cells were cotransfected with the HBV replicative plasmid and MDA5 siRNA or RIG-I siRNA followed by the determination of HBV RNA and intracapsid DNA expression, and HBsAg secretion. The siRNA that targeted MDA5 or RIG-I showed ∼40% knockdown efficiency as evaluated by mRNA expression (Fig. 3A). Knockdown of endogenous MDA5 significantly increased HBsAg secretion (p < 0.05; Fig. 3B), and substantially increased HBV RNA expression and intracapsid DNA expression (Fig. 3C). In contrast, knockdown of endogenous RIG-I failed to alter the secretion of HBsAg (Fig. 3B), and the expression of HBV RNA and intracapsid DNA (Fig. 3C). These results clearly demonstrate that MDA5, but not RIG-I, mediated the suppression of HBV replication. To further demonstrate that the suppression of HBV replication by the MDA5 signaling pathway was not limited to Huh7 cells, we performed the loss-of-function experiments in another hepatoma cell line, HepG2 cells. Knockdown of endogenous MDA5 in HepG2 cells increased HBsAg secretion and the HBV RNA expression in a dose-dependent manner (Fig. 3D–F).

FIGURE 3.

Knockdown of endogenous MDA5 in Huh7 cells increases HBV replication. (AC) Huh7 cells were transfected with the HBV replicative plasmid together with control siRNA, MDA5 siRNA, or RIG-I siRNA. At 48 h posttransfection, the culture supernatants were collected and the cells were harvested. (A) The levels of endogenous MDA5 and RIG-I expression were determined by real-time PCR. (B) The levels of HBsAg in culture supernatants were determined by ELISA. (C) The levels of HBV RNAs and HBV DNAs were detected by Northern and Southern blotting, respectively. (DF) HepG2 cells were cotransfected with the HBV replicative plasmid and MDA5 siRNA. At 48 h posttransfection, the culture supernatants were collected and the cells were harvested. (D) The levels of endogenous MDA5 were determined by real-time PCR. (E) The levels of HBsAg in culture supernatants were determined by ELISA. (F) The levels of HBV RNAs were detected by Northern blotting. Data represent the mean ± SEM from four independent experiments. *p < 0.05.

FIGURE 3.

Knockdown of endogenous MDA5 in Huh7 cells increases HBV replication. (AC) Huh7 cells were transfected with the HBV replicative plasmid together with control siRNA, MDA5 siRNA, or RIG-I siRNA. At 48 h posttransfection, the culture supernatants were collected and the cells were harvested. (A) The levels of endogenous MDA5 and RIG-I expression were determined by real-time PCR. (B) The levels of HBsAg in culture supernatants were determined by ELISA. (C) The levels of HBV RNAs and HBV DNAs were detected by Northern and Southern blotting, respectively. (DF) HepG2 cells were cotransfected with the HBV replicative plasmid and MDA5 siRNA. At 48 h posttransfection, the culture supernatants were collected and the cells were harvested. (D) The levels of endogenous MDA5 were determined by real-time PCR. (E) The levels of HBsAg in culture supernatants were determined by ELISA. (F) The levels of HBV RNAs were detected by Northern blotting. Data represent the mean ± SEM from four independent experiments. *p < 0.05.

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Given that with similar levels of protein expression of MDA5 and RIG-I, only MDA5 significantly mediated the suppression of HBV replication (Figs. 2, 3) and that the key transcription factors activated by RLR signaling are IRF3 and NF-κB (3, 5), we then examined whether Huh7 cells cotransfected with the HBV replicative plasmid and the MDA5 or RIG-I expression plasmid could activate IRF3 and NF-κB. Huh7 cells transfected with the HBV replicative plasmid alone did not significantly increase IRF3 phosphorylation (Fig. 4A, lane 4). Overexpression of MDA5 alone substantially increased IRF3 phosphorylation (Fig. 4A, lane 2), and IRF3 phosphorylation was further enhanced profoundly in the presence of the HBV replicative plasmid (Fig. 4A, lane 5). Overexpression of RIG-I alone barely increased IRF3 phosphorylation (Fig. 4A, lane 3); however, IRF3 phosphorylation was not significantly increased even in the presence of the HBV replicative plasmid (Fig. 4A, lane 6). IRF3 activation was further confirmed by examining IRF3 nuclear translocation by immunostaining. As shown in Fig. 4B, the percentage of cells positive for nuclear IRF3 was significantly higher in Huh7 cells cotransfected with the HBV replicative plasmid and the MDA5 expression plasmid than in cells cotransfected with the HBV replicative plasmid and the RIG-I expression plasmid (89.81 ± 13.13 versus 36.86 ± 14.42%, p < 0.05). To examine the NF-κB activation, we determined the nuclear translocation of NF-κB (Fig. 4C). Transfection of Huh7 cells with the HBV replicative plasmid, the MDA5 expression plasmid, or the RIG-I expression plasmid alone, or the HBV replicative plasmid plus the RIG-I expression plasmid did not induce nuclear translocation of NF-κB. In contrast, Huh7 cells cotransfected with the HBV replicative plasmid and the MDA5 expression plasmid significantly increased the translocation of NF-κB into the nucleus. These results indicate that overexpression of MDA5, but not RIG-I, in Huh7 cells transfected with the HBV replicative plasmid triggered RLR downstream signaling pathway.

FIGURE 4.

Ectopic expression of MDA5 leads to IRF3 and NF-κB activation in HBV-transfected cells. Huh7 cells were cotransfected with the HBV replicative plasmid and empty vector, the MDA5, or the RIG-I expression plasmid. (A) The phosphorylation of IRF3 and the expression of MDA5 and RIG-I were detected by Western blotting. β-actin was used as a loading control. (B) Nuclear translocation of IRF3 in transfectants was detected by immunofluorescence staining, and the images were observed by confocal microscopy. The percentage of cells with nuclear IRF3 staining in MDA5- or RIG-I–expressing cells was quantified by counting cells from three different fields per sample, as shown in the bar graph at right. Data are presented as the mean ± SEM from three individual fields. *p < 0.05. (C) The lysates of transfectants were separated into cytoplasmic and nuclear fractions by subcellular fractionation. Whole-cell lysates, cytoplasmic fractions, and nuclear fractions were run on SDS-PAGE followed by immunoblotting with Abs to NF-κB, PARP, and α-tubulin. PARP and α-tubulin are markers for the nucleus and the cytoplasm, respectively.

FIGURE 4.

Ectopic expression of MDA5 leads to IRF3 and NF-κB activation in HBV-transfected cells. Huh7 cells were cotransfected with the HBV replicative plasmid and empty vector, the MDA5, or the RIG-I expression plasmid. (A) The phosphorylation of IRF3 and the expression of MDA5 and RIG-I were detected by Western blotting. β-actin was used as a loading control. (B) Nuclear translocation of IRF3 in transfectants was detected by immunofluorescence staining, and the images were observed by confocal microscopy. The percentage of cells with nuclear IRF3 staining in MDA5- or RIG-I–expressing cells was quantified by counting cells from three different fields per sample, as shown in the bar graph at right. Data are presented as the mean ± SEM from three individual fields. *p < 0.05. (C) The lysates of transfectants were separated into cytoplasmic and nuclear fractions by subcellular fractionation. Whole-cell lysates, cytoplasmic fractions, and nuclear fractions were run on SDS-PAGE followed by immunoblotting with Abs to NF-κB, PARP, and α-tubulin. PARP and α-tubulin are markers for the nucleus and the cytoplasm, respectively.

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We demonstrated that cells cotransfected with MDA5 expression plasmid and the HBV replicative plasmid activated the downstream transcription factors (Fig. 4). We next examined whether activation of the downstream transcription factors of the MDA5 signaling pathway eventually led to the induction of IFN-stimulated gene (ISG) expression, transcriptional targets of the RLR signaling pathway. Huh7 cells were cotransfected with the HBV replicative plasmid and the MDA5 or RIG-I expression plasmid, and total RNAs were extracted from the transfectants 12 and 24 h posttransfection and subjected to the reverse-transcriptase PCR assay. The expression of ISGs, including MxA, OAS1, CXCL10, and IFN-β, known to be induced by the RLR signaling pathway was determined (Fig. 5A). The expressions of MxA, OAS1, and CXCL10 were upregulated in cells transfected with the MDA5 expression plasmid only and further increased in the presence of the HBV replicative plasmid; the increases were more evident at 24 h posttransfection (Fig. 5A). Interestingly, the level of IFN-β expression was slightly higher in Huh7 cells transfected with the HBV replicative plasmid and the empty vector as compared with cells cotransfected with the HBV replicative plasmid and the MDA5 or RIG-I expression plasmid at 12 h posttransfection (Fig. 5A, left bottom panel). However, the IFN-β expression was slightly increased in Huh7 cells cotransfected with the HBV replicative plasmid and MDA5 or RIG-I expression plasmid as compared with cells transfected with the HBV replicative plasmid and the empty vector at 24 h posttransfection (Fig. 5A, right bottom panel).

FIGURE 5.

Overexpression of MDA5 leads to induction of ISGs in HBV-transfected cells. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with the empty vector, or the MDA5 or RIG-I expression plasmid. At indicated time points, total RNAs were extracted from the transfectants and reversed transcribed into cDNA followed by quantitative PCR analysis to determine the levels of gene expression. The expression levels were analyzed with the ΔΔCt method, and all quantifications were normalized to the level of GAPDH mRNA. (B) Huh7 cells were transfected with the HBV replicative plasmid together with the empty vector or the MDA5 expression plasmid at one fourth of the plasmid used in (A) and control siRNA or siRNA-specific targeting to MxA. At 24 h posttransfection, cells and culture supernatants were collected. Cells were subjected to total RNA extraction followed by cDNA synthesis and PCR analysis for determining the level of MxA expression. The levels of HBsAg in the culture supernatants were determined by ELISA.

FIGURE 5.

Overexpression of MDA5 leads to induction of ISGs in HBV-transfected cells. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with the empty vector, or the MDA5 or RIG-I expression plasmid. At indicated time points, total RNAs were extracted from the transfectants and reversed transcribed into cDNA followed by quantitative PCR analysis to determine the levels of gene expression. The expression levels were analyzed with the ΔΔCt method, and all quantifications were normalized to the level of GAPDH mRNA. (B) Huh7 cells were transfected with the HBV replicative plasmid together with the empty vector or the MDA5 expression plasmid at one fourth of the plasmid used in (A) and control siRNA or siRNA-specific targeting to MxA. At 24 h posttransfection, cells and culture supernatants were collected. Cells were subjected to total RNA extraction followed by cDNA synthesis and PCR analysis for determining the level of MxA expression. The levels of HBsAg in the culture supernatants were determined by ELISA.

Close modal

Given that MxA is known to suppress HBV replication (3942) and that MxA expression is upregulated in cells cotransfected with the HBV replicative plasmid and the MDA5 expression plasmid (Fig. 5A, top panel), we examined whether MxA contributed to the suppression of HBV replication mediated by the MDA5 signaling pathway. Knockdown of MxA in Huh7 cells cotransfected with the HBV replicative plasmid and the MDA5 expression plasmid restored the suppression of HBV replication mediated by the MDA5 signaling pathway (Fig. 5B). This result indicates that MxA expression induced by the MDA5 signaling pathway participates in the suppression of HBV replication.

We demonstrated that the MDA5-mediated innate signaling pathway suppressed HBV replication (Figs. 2, 3) and induced the activation of both IRF3 and NF-κB (Fig. 4), which subsequently induced the expression of antiviral ISGs (Fig. 5). We reasoned that HBV might be sensed by MDA5. We next investigated whether MDA5 was associated with HBV RNA by conducting RNA immunoprecipitation. Huh7 cells were cotransfected with the HBV replicative plasmid and the plasmid encoding FLAG-MDA5 or FLAG-RIG-I, and the transfectants were lysed and subjected to immunoprecipitation with an anti-FLAG Ab. RNA was extracted from RLR immunoprecipitates and subjected to cDNA synthesis followed by real-time PCR analysis using primers specific to the HBV core gene. The amplification of core gene was found only in cDNAs that were reverse transcribed from the RNAs extracted from MDA5 immunoprecipitates, but not from RIG-I immunoprecipitates (Fig. 6A). To show the RNA purified from RLR immunoprecipitates was not contaminated with HBV genomic DNA, we also conducted RT-PCR in the absence of reverse transcriptase. No PCR product was detected in the absence of reverse transcriptase, indicating no HBV genomic DNA contamination in the purified RNA (Fig. 6B). These results demonstrate that MDA5, but not RIG-I, is associated with HBV RNAs, suggesting that MDA5 may sense HBV RNA. To ensure the lack of association of HBV RNA with RIG-I in RNA immunoprecipitation assay was not due to an experimental artifact, we performed RNA immunoprecipitation assay simultaneously with cells transfected with the RIG-I or MDA5 expression plasmid followed by infection with VSV, known to be sensed by RIG-I (31, 43). As expected, the result showed that RIG-I, but not MDA5, was associated with VSV RNA (Supplemental Fig. 1). To further confirm that HBV RNAs indeed activated MDA5 signaling, we cotransfected Huh7 cells with the plasmid encoding MDA5 or RIG-I together with the HBV replicative plasmid or the polymerase-mutated HBV replicative plasmid (pol-null) that does not express HBV polymerase and only produce HBV RNAs. Overexpression of MDA5 modestly increased the level of IRF3 phosphorylation (Fig. 7A, lane 2), and the level of IRF3 phosphorylation was further increased in cells cotransfected with the HBV replicative plasmid (Fig. 7A, lane 5). Of interest, replacing the HBV replicative plasmid with the polymerase-mutated HBV replicative plasmid, which produced no HBV DNA, induced comparable levels of IRF3 phosphorylation (Fig. 7A, lane 8). Cotransfection of Huh7 cells with the RIG-I expression plasmid together with the HBV replicative plasmid or the polymerase-mutated HBV replicative plasmid slightly increased the levels of IRF3 phosphorylation (Fig. 7A, lanes 6, 9). The supernatants of these transfectants were collected for determining the levels of HBsAg. As expected, HBsAg secretion was significantly inhibited in cells cotransfected with the MDA5 expression plasmid together with the HBV replicative plasmid or the polymerase-mutated plasmid (Fig. 7B). These results demonstrate that HBV RNAs may be sufficient to activate MDA5-mediated signaling, which subsequently suppresses HBV replication.

FIGURE 6.

MDA5 associates with HBV RNAs. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or FLAG-tagged RIG-I or the corresponding control vector. At 48 h posttransfection, cells were cross-linked by formaldehyde followed by cell lysis, and cell lysates were immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were washed with stringent condition, and the cross-linking was reversed by heating. Total RNAs extracted from the immunoprecipitates were reverse transcribed into cDNAs. The cDNAs were used as templates to amplify HBV DNA by real-time PCR using primers specific for the HBV core gene. The copy number reflected the amount of the HBV nucleic acid in the immunoprecipitates from the cells cultured in a 6-cm dish. Data are presented as the mean ± SEM and are representative of three independent experiments. (B) Similar to (A) except that additional experiments in the absence of reverse transcriptase (RT) were performed to rule out possible HBV genomic DNA contamination in the RNA samples. The resultant PCR products were visualized with ethidium bromide staining.

FIGURE 6.

MDA5 associates with HBV RNAs. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or FLAG-tagged RIG-I or the corresponding control vector. At 48 h posttransfection, cells were cross-linked by formaldehyde followed by cell lysis, and cell lysates were immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were washed with stringent condition, and the cross-linking was reversed by heating. Total RNAs extracted from the immunoprecipitates were reverse transcribed into cDNAs. The cDNAs were used as templates to amplify HBV DNA by real-time PCR using primers specific for the HBV core gene. The copy number reflected the amount of the HBV nucleic acid in the immunoprecipitates from the cells cultured in a 6-cm dish. Data are presented as the mean ± SEM and are representative of three independent experiments. (B) Similar to (A) except that additional experiments in the absence of reverse transcriptase (RT) were performed to rule out possible HBV genomic DNA contamination in the RNA samples. The resultant PCR products were visualized with ethidium bromide staining.

Close modal
FIGURE 7.

HBV RNAs activate MDA5 signaling. Huh7 cells were transfected with the HBV replicative plasmid (HBV wild-type) or the polymerase-mutated HBV replicative plasmid (Pol-null), or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the FLAG-tagged RIG-I or the corresponding control vector. (A) The phosphorylation of IRF3 and the expression of MDA5 or RIG-I were detected by Western blotting. β-Actin was used as a loading control. (B) The cultured supernatants from (A) were collected and subjected to the determination of HBsAg by ELISA.

FIGURE 7.

HBV RNAs activate MDA5 signaling. Huh7 cells were transfected with the HBV replicative plasmid (HBV wild-type) or the polymerase-mutated HBV replicative plasmid (Pol-null), or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the FLAG-tagged RIG-I or the corresponding control vector. (A) The phosphorylation of IRF3 and the expression of MDA5 or RIG-I were detected by Western blotting. β-Actin was used as a loading control. (B) The cultured supernatants from (A) were collected and subjected to the determination of HBsAg by ELISA.

Close modal

Although MDA5 is recognized as a cytosolic RNA sensor, some studies showed that MDA5 interacts with DNA (44, 45). We sought to determine whether HBV DNA was associated with RLR immunoprecipitates by performing DNA immunoprecipitation. Huh7 cells were cotransfected with the HBV replicative plasmid and the plasmid encoding FLAG-MDA5 or FLAG-RIG-I, and the transfectants were lysed and subjected to immunoprecipitation with an anti-FLAG Ab. DNAs associated with the RLR immunoprecipitates were extracted and subjected to real-time PCR analysis using primers specific to the HBV core gene. Interestingly, HBV DNAs were detected in MDA5, but not RIG-I, immunoprecipitates (Fig. 8A). We also developed a plate-based in vitro binding assay to further confirm the association of HBV DNA with MDA5. FLAG-MDA5 or FLAG–RIG-I purified from HEK293T cells overexpressing the protein was coated onto ELISA plates. The biotinylated HBV DNA generated from PCR was then added into the coated plates. After incubation followed by several washes, the biotinylated HBV DNA associated with FLAG-tagged RLRs was detected using HRP-conjugated streptavidin with TMB as a substrate. Consistent with the result obtained from DNA immunoprecipitation, the plate-based in vitro binding assay showed that only purified MDA5, but not RIG-I, was associated with HBV DNA (Fig. 8B, 8C). Based on the results obtained from both assays, we conclude that MDA5 is associated with HBV DNA. Collectively, the results shown in Figs. 68 demonstrate that MDA5, but not RIG-I, is associated with not only HBV RNA but also HBV DNA, suggesting that MDA5 may sense HBV nucleic acids.

FIGURE 8.

MDA5 associates with HBV DNAs. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the FLAG-tagged RIG-I or the corresponding control vector. Cells were cross-linked by formaldehyde followed by cell lysis, and cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitates were washed, and the cross-linking was reversed. Total DNAs were extracted from the immunoprecipitates and used as templates to amplify HBV DNAs by real-time PCR using primers specific for the HBV core gene. The copy number reflected the amount of the HBV nucleic acid in the immunoprecipitates from the cells cultured in a 6-cm dish. Data are presented as the mean ± SEM and are representative of three independent experiments. (B) HEK293T cells were transfected with FLAG-MDA5 or FLAG–RIG-I expression plasmid. The FLAG-MDA5 or FLAG–RIG-I proteins were purified from transfectants using M2 anti-FLAG affinity gel followed by the elution of bound proteins with FLAG peptides. The purity of the purified proteins was determined by Coomassie brilliant blue staining (left panel) and Western blotting (right panel). (C) Purified FLAG-MDA5 or FLAG–RIG-I was coated onto ELISA plates, and increased concentrations of biotinylated HBV DNA were added into the coated plates. After incubation followed by several washes, the HBV DNA that remained on the plates was detected using HRP-conjugated streptavidin with TMB as a substrate.

FIGURE 8.

MDA5 associates with HBV DNAs. (A) Huh7 cells were transfected with the HBV replicative plasmid or the corresponding control plasmid together with either the expression vector encoding FLAG-tagged MDA5 or the FLAG-tagged RIG-I or the corresponding control vector. Cells were cross-linked by formaldehyde followed by cell lysis, and cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitates were washed, and the cross-linking was reversed. Total DNAs were extracted from the immunoprecipitates and used as templates to amplify HBV DNAs by real-time PCR using primers specific for the HBV core gene. The copy number reflected the amount of the HBV nucleic acid in the immunoprecipitates from the cells cultured in a 6-cm dish. Data are presented as the mean ± SEM and are representative of three independent experiments. (B) HEK293T cells were transfected with FLAG-MDA5 or FLAG–RIG-I expression plasmid. The FLAG-MDA5 or FLAG–RIG-I proteins were purified from transfectants using M2 anti-FLAG affinity gel followed by the elution of bound proteins with FLAG peptides. The purity of the purified proteins was determined by Coomassie brilliant blue staining (left panel) and Western blotting (right panel). (C) Purified FLAG-MDA5 or FLAG–RIG-I was coated onto ELISA plates, and increased concentrations of biotinylated HBV DNA were added into the coated plates. After incubation followed by several washes, the HBV DNA that remained on the plates was detected using HRP-conjugated streptavidin with TMB as a substrate.

Close modal

To investigate the physiological importance of RLRs in HBV infection, we conducted in vivo experiments by inducing acute HBV infection using hydrodynamic injection to deliver the HBV replicative plasmid into the livers of MDA5+/−, MDA5−/−, and RIG-I+/− mice and their wild-type littermate controls. The mouse sera on days 1, 3, and 5 postinjection were collected to monitor the serum levels of HBsAg. The serum levels of HBsAg in acute HBV infection induced by hydrodynamical injection of the HBV replicative plasmid into livers consistently peaked on day 3 postinjection and diminished within a week. Interestingly, the serum levels of HBsAg in MDA5+/− and MDA5−/− mice were significantly higher than those in littermate control mice on day 3 postinjection (Fig. 9A). Consistent with the result of serum HBsAg, the levels of HBV RNAs and intracapsid HBV DNAs in livers from MDA5+/−and MDA5−/− mice were markedly increased as compared with littermate control mice (Fig. 9B). Because RIG-I−/− mice rarely survive and show massive liver degeneration (31), we had difficulties obtaining enough RIG-I−/− mice for the experiments. We then compared wild-type littermate control mice with RIG-I+/− mice and found their serum levels of HBsAg to be comparable (Fig. 9C). Consistent with the result of serum HBsAg, the levels of HBV RNAs and intracapsid HBV DNAs were similar in livers between RIG-I+/− mice and littermate control mice (Fig. 9D). Altogether, the in vivo findings indicate that MDA5, but not RIG-I, senses HBV and subsequently activates the innate immune signaling pathway to suppress HBV replication.

FIGURE 9.

MDA5 deficiency increases HBV replication in vivo. (A) MDA5+/−, MDA5−/− mice, and wild-type littermate controls were hydrodynamically injected with 30 μg of the HBV replicative plasmid. The mouse sera were collected on days 1, 3, and 5 postinjection, and the levels of HBsAg were determined by ELISA. The numbers in parentheses indicate the number of mice in each group. Data are presented as the mean ± SEM. *p < 0.05. (B) On day 3 postinjection, the livers from MDA5+/−, MDA5−/− mice, and wild-type littermate controls were harvested and subjected to the detection of HBV RNA and DNA levels by Northern and Southern blotting, respectively. (C and D) Similar to (A) and (B) except that RIG-I+/− mice and their littermate controls were used.

FIGURE 9.

MDA5 deficiency increases HBV replication in vivo. (A) MDA5+/−, MDA5−/− mice, and wild-type littermate controls were hydrodynamically injected with 30 μg of the HBV replicative plasmid. The mouse sera were collected on days 1, 3, and 5 postinjection, and the levels of HBsAg were determined by ELISA. The numbers in parentheses indicate the number of mice in each group. Data are presented as the mean ± SEM. *p < 0.05. (B) On day 3 postinjection, the livers from MDA5+/−, MDA5−/− mice, and wild-type littermate controls were harvested and subjected to the detection of HBV RNA and DNA levels by Northern and Southern blotting, respectively. (C and D) Similar to (A) and (B) except that RIG-I+/− mice and their littermate controls were used.

Close modal

In this study, both loss-of-function and gain-of-function experiments demonstrated that MDA5, but not RIG-I, activates the innate immune signaling pathway to suppress HBV replication. Importantly, hydrodynamic injection of the HBV replicative plasmid into homozygous and heterozygous MDA5 knockout mice, but not in heterozygous RIG-I knockout mice, significantly increased HBV replication as compared with wild-type mice. Thus, our in vitro and in vivo studies clearly indicate a critical role of the MDA5-mediated innate immune signaling in the suppression of HBV infection.

Recognition of viruses by PRRs is essential to initiate the innate antiviral immune response and, subsequently, to dictate the adaptive immune response. RIG-I and MDA5 are cytoplasmic viral sensors that play important roles in host defense against viral infection, particularly RNA virus infection (31, 37, 38, 46). Although MDA5 and RIG-I have similar structures and share high sequence homology, they recognize distinct RNA species (32). It is generally thought that RIG-I recognizes RNAs with uncapped 5′-triphosphate structures (47, 48), short dsRNA molecules (49, 50), and RNAs with panhandle structures (50), whereas MDA5 recognizes long dsRNAs (49) and high m.w. RNAs (51). RIG-I recognizes a wide variety of RNA viruses, whereas MDA5 recognizes mainly picornaviruses whose RNAs do not bear 5′-triphosphates (32, 37). Intriguingly, several recent studies have revealed that RIG-I and MDA5 are able to induce an antiviral response to viruses containing dsDNA genomes, such as EBV (52, 53), vaccinia virus (51, 54), and HSV (55, 56). RIG-I is proposed to induce an antiviral response to DNA viruses via RNA polymerase III–mediated conversion of microbial DNA into 5′-triphosphate dsRNA (57, 58). Recognition of the DNAs of HSV and EBV by RIG-I is mediated via this pathway (52, 55, 58). Of note, one report showed that innate recognition of HSV in macrophages is mediated via an MDA5-dependent and RNA polymerase III–independent pathway (56). In addition to HSV, MDA5 also mediates an antiviral response to another DNA virus, vaccinia virus. RNAs generated during vaccinia virus infection have higher-order RNA structures, which activate an MDA5-dependent antiviral response (51). Of interest, this study demonstrated that immunoprecipitates of MDA5, but not RIG-I, were associated with HBV RNA, suggesting that MDA5, but not RIG-I, acts as the cytoplasmic sensor for HBV RNA and triggers downstream signaling to suppress HBV replication. Given that the 3.5-kb HBV pgRNA does not bear a 5′-triphosphate and that pgRNA is predicted to have a complicated secondary structure (59), it is plausible that HBV pgRNA may be a potential ligand for MDA5. However, we cannot exclude the possibility that the 2.4-, 2.1-, and 0.7-kb HBV RNAs are also MDA5 ligands because these viral RNA sequences are also part of HBV pgRNA.

It is of interest that we found MDA5, but not RIG-I, is associated with HBV DNA. Although some studies mentioned the interaction of MDA5 with DNA (44, 45), whether the association of MDA5 with DNA is required for MDA5-mediated signaling remains unknown. HBV cccDNA, a transcriptional template for the synthesis of viral RNA during HBV replication, may not be the candidate DNA ligand for MDA5 because cccDNA is localized to the nucleus and is absent from the cytosol. However, a recent study showed that an intracellular deproteinized rcDNA (DP-rcDNA) of HBV is present in both the cytoplasm and the nucleus (60). The presence of some naked DP-rcDNA in the cytosol raises the possibility that DP-rcDNA may be sensed by MDA5.

Guo et al. (15) demonstrated that overexpression of IPS-1 suppresses HBV replication, and our study showed that MDA5, an upstream molecule of IPS-1, also suppresses HBV replication. Together, these results provide the complete RLR pathway mediating the innate response to HBV infection. Importantly, our data showing that MDA5 selectively associates with both HBV RNAs and DNAs, and subsequently initiates the downstream signaling pathway provide one more piece of evidence on the nonredundant role of MDA5 and RIG-I in virus recognition.

In this study, we demonstrated that MDA5 not only recognizes HBV nucleic acids, but also initiates a signaling pathway that leads to the activation of transcription factors IRF3 and NF-κB to induce the expression of ISGs-MxA, OAS1, and CXCL10. Among these ISGs, MxA and OAS1 are antiviral genes, whereas CXCL10 is a chemokine gene responsible for the recruitment of effector CD8 T cells and NK cells to the site of viral infection. MxA likely plays an important role in HBV infection because it has been shown to inhibit HBV replication (39, 41, 42). Our results show that knockdown of endogenous MxA reversed the inhibition of HBV replication mediated by MDA5 signaling (Fig. 5B). It is plausible that induction of MxA expression during HBV infection is likely via the MDA5-mediated innate signaling pathway.

Interestingly, we found that unlike other ISGs (MxA, OAS1, and CXCL10), the induction of IFN-β seems not evident in cells cotransfected with the HBV replicative plasmid and the MDA5 or RIG-I expression plasmid at 24 h posttransfection, although the IFN-β induction in cells cotransfected with the HBV replicative plasmid and RIG-I expression plasmid is somewhat increased as compared with cells cotransfected with the HBV replicative plasmid and MDA5 expression plasmid. This observation is reminiscent of the clinical observation that type I IFN induction is not always observed in patients infected with HBV (6163). The fact that type I IFN is not significantly induced in HBV infection may suggest that the ISGs induction by HBV-mediated activation of the MDA5 signaling pathway may be independent of IFN-β. Supporting this notion, a recent study demonstrated that IPS-1 is located on peroxisomes and mitochondria (64), and that peroxisomal IPS-1 induces IFN-independent expression of ISGs, whereas mitochondrial IPS-1 activates an IFN-dependent expression of ISGs (64). Whether the peroxisomal IPS-1–mediated MDA5 signaling is more favorable than the mitochondrial IPS-1–mediated MDA5 signaling in HBV-infected hepatocytes requires further investigation.

Intriguingly, although cells cotransfected with the HBV replicative plasmid and RIG-I expression plasmid slightly activate IRF3 and NF-κB, this RIG-I–mediated signaling pathway failed to suppress HBV replication (Fig. 7). Given that HBV DNA directs the synthesis of a 700-base RNA (HBV 700) by RNA polymerase III (65, 66), that RNA polymerase III–transcribed RNAs contain a 5′-triphosphate, and that RIG-I recognizes 5′-triphosphate RNAs and subsequently activates downstream signaling (47, 48), it is possible that RIG-I might sense HBV 700 and activate IRF3 and NF-κB. Of note, HBV 700 is transcribed by RNA polymerase III at a very low level (65); therefore, HBV 700 may slightly induce the activation of RIG-I signaling, which is not efficient enough to suppress HBV replication. Alternatively, IRF3 phosphorylation alone may not be sufficient to inhibit HBV replication; other molecules triggered by the MDA5 signaling pathway are required to work in concert with activated IRF3 to effectively inhibit HBV replication.

We have proved the physiological importance of MDA5 in innate immunity to HBV infection by in vivo experiments. Of interest, both MDA5+/− and MDA5−/− mice hydrodynamically injected with the HBV replicative plasmid to mimic acute HBV infection had comparable increases in HBV replication (Fig. 9A, 9B), suggesting that a single functional copy of MDA5 is not sufficient for suppressing HBV replication. The haploinsufficiency of MDA5 further strengthens the notion that MDA5 plays a critical role in innate immunity to HBV replication. Given that innate immunity dictates adaptive immunity and that our study shows the importance of MDA5 in innate immunity to HBV infection, MDA5 likely plays a role in regulating adaptive immunity to HBV infection as well. Supporting this notion, MDA5−/− mice have a reduced number of Ag-specific CD8+ cells during acute infection of lymphocytic choriomeningitis virus, resulting in persistent infection (67). The expression and/or signaling of MDA5 may be critical for determining the outcome of HBV infection. When MDA5 expression and/or signaling are robust upon HBV infection, hosts should be able to efficiently eliminate the virus. In contrast, hosts may not efficiently clear the virus if MDA5 expression and/or signaling are attenuated, eventually leading to chronic HBV infection. In this regard, genetic polymorphism of MDA5 may be one of the factors accounting for the variation in an individual’s susceptibility to HBV infection and outcome of HBV infection. Some MDA5 variants that affect MDA5 expression are significantly associated with type 1 diabetes (68, 69). Because this study demonstrates the importance of MDA5 in suppressing HBV replication, one would expect that MDA5 variants with high MDA5 expression may control HBV infection, whereas MDA5 variants with low MDA5 expression may lead to chronic HBV infection. Thus, the association of MDA5 genetic polymorphisms with the outcome of HBV infection should be investigated.

We are grateful to Dr. Chiaho Shih for providing pSV2ANeo-HBVx2 plasmid and pSV2ANeo-HBV mutant 2310, Dr. Steve S.-L. Chen for providing pGAGGS-MCS-Flag2 plasmid, Dr. Takashi Fujita for providing pEF-BOS-RIG-I plasmid, Dr. Shizuo Akira for providing breeding pairs of MDA5+/− mice and RIG-I+/− mice, Drs. Mei-Yi Lu and Kuo-Ming Lee for technical consulting, and Dr. Pauline Yen for critical reading of the manuscript.

This work was supported by grants from Academia Sinica in Taiwan.

The online version of this article contains supplemental material.

Abbreviations used in this article:

cccDNA

covalently closed circular DNA

DP

deproteinized

HBcAg

HBV core Ag

HBsAg

HBV surface Ag

HBV

hepatitis B virus

IPS-1

IFN-β promoter stimulator 1

IRF

IFN regulatory factor

ISG

IFN-stimulated gene

MDA5

melanoma differentiation–associated gene 5

NP-40

Nonidet P-40

PARP

poly(ADP-ribose) polymerase

pgRNA

pregenomic RNA

PRR

pattern recognition receptor

rcDNA

relaxed circular DNA

RIG-I

retinoic acid–inducible gene-I

RLR

RIG-I–like receptor

siRNA

small interfering RNA

TMB

tetramethylbenzidine

VSV

vesicular stomatitis virus.

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The authors have no financial conflicts of interest.