Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections
© Wei et al.; licensee BioMed Central Ltd. 2014
Received: 29 November 2013
Accepted: 27 May 2014
Published: 18 June 2014
Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.
The innate immune system (also known as the non-specific immune system) is an evolutionarily conserved system that protects the host from invading microbial pathogens and other potential threats through germline-encoded pattern recognition receptors . Pattern recognition receptors are located on multiple types of innate immune cells and are capable of responding to specific pathogen associated molecular patterns exclusively present on microbes, such as viruses, bacteria, parasites and fungi. Post virus infection, some pattern recognition receptors such as toll-like receptors (TLRs; e.g., TLR-3, -7, -8 and -9), retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) and nucleotide oligomerization domain-like receptors are activated, which specifically recognize various types of viral nucleotides [1, 2].
The RLR family contains three members: RIG-I, melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), which are located in the cytoplasm . RLR family members harbor one central ATPase and helicase domain and one regulatory domain (RD) in the carboxy terminus. RIG-I and MDA5 also have two caspase activation and recruitment domains (CARD) at the N terminus, which is absent in LGP2 [4, 5]. RIG-I recognizes short double-stranded RNA (dsRNA; < 1 kb) and uncapped 5′-triphosphate ssRNA (5′ppp-ssRNA) [6, 7]. However, MDA5 senses longer dsRNA (>1 kb) and synthetic dsRNA, such as polyinosinic-polycytidylic acid (poly [I:C]) .
The MDA5 pathway has been well characterized in mammals. After MDA5 is activated, the signal is transmitted to interferon-β (IFN-β) promoter-stimulator-1 (IPS-1, also known as MAVS/VISA/Cardif) via a CARD-CARD interaction on the mitochondrial membrane [9–12]. This association coordinates a serine kinase-mediated cascade that activates latent transcription factors, including interferon-regulatory factor 3 (IRF-3) and nuclear factor-κB (NF-κB), culminating in the expression of IFN-β and a number of other crucial antiviral effector genes .
Avian influenza viruses (AIV) cause a serious and economically significant disease in domestic poultry, such as chickens, quails and pheasants. Ducks and wild birds have naturally high resistance to AIV infections [14, 15], although recently there have been severe outbreaks in ducks caused by H5N1 highly pathogenic (HP) AIV [16–20]. Natural resistance to AIV in ducks has been linked molecularly to RIG-I . However, whether MDA5 also has this function in ducks has not been explored until now.
Recently, a partial MDA5 sequence was deposited in GenBank for the duck (accession number: GU936632) . However, full-length duck MDA5 cDNA has not been cloned and sequenced. In the present study, we cloned the full-length duck MDA5 cDNA and investigated the role of the predicted functional domains of MDA5. In addition, we investigated the MDA5-mediated signaling pathway in primary duck embryonic fibroblast (DEF) cells and examined its antiviral function. We also demonstrate that nonstructural protein 1 (NS1) of H5N1 HPAIV inhibits the MDA5-mediated signaling pathway.
Materials and methods
Cells, viruses, and animals
Primary DEF cells were prepared using 14-day-old Muscovy duck eggs as described previously . All cells, including human embryonic kidney 293 T (China Center for Type Culture Collection, China) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotics, and were incubated at 37 °C in 5% CO2.
A/Duck/Guangdong/212/2004(H5N1) virus (designated as DK212) was isolated from ducks in the Guangdong Province of China in 2004, and identified as H5N1 avian influenza A virus using hemagglutination inhibition and neuraminidase inhibition tests . It was purified and propagated in the allantoic cavity of 10-day-old specific pathogen-free embryonated hen eggs. Allantoic fluid pooled from multiple eggs was clarified by centrifugation and frozen in aliquots at -70 °C. All experiments with H5N1 HPAIV were performed under animal biosafety level 3 (ABSL-3) conditions.
One-day-old healthy Muscovy ducks were purchased from a duck farm in Guangzhou and housed in isolators. Muscovy ducks were confirmed as serologically negative for avian influenza by agar gel precipitation tests and hemagglutinin inhibition assays.
Identification of muscovy duck MDA5, PKR and OAS genes
Gene cloning PCR primers used in this study
Sequence of Oligonucleotide (5′ → 3′)
pCAGGS-MDA5ΔCARD + ΔRD-f
MDA5ΔCARD + ΔRD cloning
pCAGGS-MDA5ΔCARD + ΔRD-r
Cloning and bioinformatics analysis of the full-length cDNA of MDA5
The full-length MDA5 cDNA was isolated using 5′- and 3′-SMART RACE PCR (Clontech, Mountain View, CA, USA), according to the manufacturer’s protocol, with gene-specific primers and the SMART universal primer (Table 1). The structure of deduced amino acid sequences of Muscovy duck MDA5 was analyzed using the SMART program . Amino acid sequences were aligned using ClustalW2  and edited with BOXSHADE .
Construction of plasmids
Quantitative real-time PCR primers used in this study
Sequence of Oligonucleotide (5′ → 3′)
The luciferase reporter plasmid for the chicken IFN-β (chIFN-β) promoter (pGL3-chIFNβ-Luc) has been previously described . The chicken NF-κB (chNF-κB) and chicken IRF-7 (chIRF-7) binding positive regulatory domains were predicted by the TFSEARCH: Searching Transcription Factor Binding Sites . The chicken pGL3-chNF-κB-Luc and pGL3-chIRF-7-Luc contain four copies of the NF-κB- (sequence: GGGAATTCTC) or IRF-7- (sequence: TTCACTTTCAATA) positive regulatory domains motif of the chicken IFN-β promoter in front of a luciferase reporter gene, respectively.
Transient transfections and luciferase assays
DEF cells were transfected with firefly luciferase reporter plasmid, such as pGL3-chIFNβ-Luc, pGL3-chNF-κB-Luc or pGL3-chIRF-7-Luc. In addition, an internal control plasmid to normalize transfection efficiency, pTK-RL (Promega, Madison, WI, USA), encoding the Renilla luciferase protein, was transfected into the cells. The reporter gene plasmids pGL3-chIFNβ-Luc, pGL3-chNF-κB-Luc or pGL3-chIRF-7-Luc and the pTK-RL plasmids were cotransfected along with each of the pCAGGS expression plasmids into 80% confluent cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 24 h post-transfection, the cells were lysed and luciferase activities were determined with a dual-luciferase reporter assay system (Promega) and normalized on the basis of the Renilla luciferase activities.
Generation of reverse genetic reassorted viruses
To establish eight-plasmid reverse genetic systems for the DK212 and DK212-ΔNS1 viruses, a bidirectional transcription vector (pDL) was used. Reassorted viruses were generated by reverse genetics as described previously . The DK212 virus devoid of the NS1 gene (DK212-ΔNS1) was rescued by transfecting of 293 T cells with the plasmid pDL-NS-ΔNS1 and the seven parent DK212 plasmids. To produce a viral stock, rescued DK212 (rDK212) and rDK212-ΔNS1 were further inoculated to embryonated eggs. The rescued viruses were detected by hemagglutination assay, and RNA was extracted and analyzed by reverse transcription PCR. Each viral segment was sequenced to confirm the identity of the reassorted viruses. Viral titers were measured by 50% tissue culture infective dose (TCID50) on DEF cells. All experiments with H5N1 HPAIV were performed under ABSL-3 conditions.
Quantitative real-time PCR analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the QuantiFast SYBR Green PCR kit (Qiagen, Hilden, Germany). Primers used for qRT-PCR were designed using the primer3 software , based on published target sequences and have been previously reported . Primer pairs (Table 2) were selected based on their specificity, as determined by dissociation curves. qRT-PCR was carried out using a 7500 Fast Real-Time PCR system (Applied Biosystems, Rotkreuz, Switzerland). The relative expression ratios of target genes in the tested group versus those in the control group were calculated by the 2-ΔΔCt method using the duck housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; AY436595) as the endogenous reference gene to normalize the level of target gene expression .
For tissue distribution analyses, three uninfected ducks (aged 4 weeks) were killed and tissues were collected, including the brain, crop, trachea, heart, liver, spleen, lung, kidney, muscular stomach, glandular stomach, muscle, skin, duodenum, ileum, colon, cecum, rectum and bursa.
To examine the response of duck MDA5 infected with DK212, groups of nine ducks (aged 4 weeks) were inoculated intranasally with 106 EID50 of DK212 or mock infected with phosphate-buffered saline at a volume of 0.2 mL. At 1, 2 and 3 days post-infection (dpi), three individuals from each group were sacrificed and the brain, spleen, and lung tissues were harvested immediately for RNA extraction. The remaining ducks were observed for clinical symptoms for 14 days. All experiments were carried out in ABSL-3 facilities and were conducted under the guidance of CDC’s Institutional Animal Care and Use Committee and the Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility. The ABSL-3 Committee of South China Agricultural University approved the animal experiments in this study.
Data were expressed as means ± standard deviations. Statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). A value of P < 0.05 was considered significant.
Muscovy duck MDA5 cDNA and gene structure
Tissue distribution of Muscovy duck MDA5 expression
Quantitative analysis of tissue distribution of MDA5 transcripts in healthy Muscovy ducks 1
MDA5 gene relative expression level (mean ± SD)
MDA5 gene relative expression level (mean ± SD)
0.62 ± 0.09a
8.16 ± 0.71a
19.61 ± 1.57a
0.02 ± 0.004a
373.68 ± 13.83a
0.07 ± 0.006a
0.13 ± 0.01a
22.26 ± 1.89a
1.62 ± 0.19a
23.76 ± 0.66a
2.68 ± 0.17a
13.88 ± 0.84a
1.45 ± 0.13a
6.98 ± 0.58a
2.02 ± 0.17a
15.02 ± 0.86a
0.41 ± 0.05a
1.00 ± 0.11
Expression of MDA5 in muscovy ducks infected with DK212
Function of the domain of muscovy duck MDA5 in type I IFN induction
Muscovy duck MDA5 signaling through the IRF-7 pathway
Antiviral molecules and proinflammatory cytokine responses induced by CARD overexpression
Overexpression of CARD upregulates the expression of RIG-I and LGP2
Regulation of the expressions of other RLR genes by overexpression of the CARD of the Muscovy duck was studied in vitro using DEF cells. Upon overexpression of the CARD of the Muscovy duck, the levels of RIG-I and LGP2 mRNA transcripts increased compared with their levels in cells transformed with the empty plasmid (Figure 5). These data indicate that activation of the Muscovy duck MDA5 pathway upregulated the mRNA expression of RIG-I and LGP2.
Suppression of viral yield by CARD overexpression
The NS1 of DK212 inhibits the muscovy duck MDA5 signaling pathway
Taken together, these data show that the NS1 functions to prevent MDA5-mediated innate immunity in Muscovy duck cells and underlines the need for a functional NS1 for full virulence.
Here, we successfully cloned the 3012 bp MDA5 cDNA from the Muscovy duck. The amino acid sequence alignment shows that duck MDA5 is highly homologous to the antiviral molecule chicken MDA5 (86.1%), suggesting that duck MDA5 may have a similar function. Based on the predicted amino acid sequence, bioinformatic analysis was performed. Like other MDA5 [4, 31, 32], duck MDA5 contained three main structural domains: two CARD motifs at the N terminus, one DExD/H-box RNA helicase domain and an RD at the C-terminus.
The functional profile of the predicted domains of Muscovy duck MDA5 was examined in this study. Overexpression of the CARD of duck MDA5 significantly induced the chIFN-β promoter, which is consistent with the function of MDA5 in mammalian systems [33, 34]. However, overexpression of the Muscovy duck MDA5 lacking the CARD failed to induce the chIFN-β promoter (Figure 3). These results demonstrate the CARD of Muscovy duck MDA5 has a similar function as its counterpart in mammals, which is involved in immune signaling [4, 5, 30]. In addition, overexpression of the Muscovy duck MDA5 lacking the predicted C-terminal RD showed a stronger ability to induce the chIFN-β promoter than expression of the full-length duck MDA5 (Figure 3). This result demonstrates that the C-terminal RD of Muscovy duck MDA5 has the self-repression ability. However, the C-terminal RD of human MDA5 neither stimulates nor inhibits IFN-β promoter expression .The overexpression of the CARD of Muscovy duck MDA5 significantly activated the IRF-7-dependent signaling pathway (Figure 4). In addition, the expressions of several antiviral molecule genes, such as Mx, PKR and OAS, were significantly induced by overexpression of the CARD of Muscovy duck MDA5. The expression of the Mx gene was induced more strongly than others (Figure 5). In addition, the expressions of IL-2, IL-6, IFN-α and IFN-γ were significantly induced by overexpression of the CARD of Muscovy duck MDA5 (Figure 5). Therefore, Muscovy duck MDA5 activates signaling pathways that mediate both antiviral and proinflammatory responses.
Overexpression of the CARD of Muscovy duck MDA5 also increased the expressions of RIG-I and LGP2 transcripts (Figure 5). It was reported that IFN-α and IFN-β treatment of a human lung adenocarcinoma epithelial cell line and human umbilical vein endothelial cells resulted in activation of MDA5, RIG-I, and TLR3 mRNA expression . Previous research also showed that the MDA5 and RIG-I genes are upregulated by IFN-α [33, 36, 37]. Thus, the upregulation of RIG-I and LGP2 genes induced by overexpression of CARD may be caused by the upregulation of IFN-α and IFN-β.
Muscovy duck MDA5 mRNA was constitutively expressed in all tested tissues of healthy ducks (Table 3), and was induced post infection with H5N1 HPAIV (Figure 2). These findings indicate that duck MDA5 might be an important receptor for recognizing H5N1 HPAIV in the antiviral innate immune response in Muscovy ducks. The results of in vitro antiviral assays of duck MDA5 show that virus titers of DK212 in the supernatant of CARD-overexpressing DEF cells were significantly lower than in the control (Figure 6). These results suggest that activation of the MDA5 pathway inhibited the replication of H5N1 HPAIV in vitro. It was reported that chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, used MDA5 to sense AIV . Chickens lack RIG-I, but can regulate the production of IFN-β through the MDA5 pathway . Our results suggest that Muscovy duck MDA5 has a similar function to chicken MDA5; i.e., it is involved in the host antiviral innate immunity.
In mammals, influenza NS1 is described as a multifunctional protein with regulatory activities, able to inhibit the innate antiviral immune response of infected cells . In ducks, the interaction between NS1 and the innate immune response remains to be evaluated . Here, we showed that in the duck system, NS1 interferes with the duck MDA5-mediated pathway. The plasmid-expressed NS1 of DK212 suppressed duck MDA5-mediated chIFN-β promoter activation. This was similar to the results reported in chicken cells, where plasmid-expressed NS1 suppressed chicken MDA5-mediated chIFN-β promoter activation . In addition, plasmid-expressed NS1 of DK212 also suppressed the duck MDA5-mediated proinflammatory and antiviral signaling pathways. These results were similar to those reported in mammals .
In previous works, several groups have shown that viruses expressing a truncated NS1 elicited a major type I IFN response in infected cells compared with their wild-type counterparts [42–47]. Using reverse genetics, we successfully created a mutant H5N1 AIV with a deleted NS1 protein (rDK212-ΔNS1). The mutant virus, but not the wild-type virus, was a strong IFN inducer in DEF cells (Figure 7C). A similar result was reported in DEF cells in which a mutant H7N1 AIV expressed a truncated NS1 protein that induced high titers of type I IFN . In addition, the mutant virus (rDK212-ΔNS1) was an effective inducer of both antiviral molecules and proinflammatory cytokines. These results once again demonstrated that NS1 of DK212 inhibited the duck MDA5-mediated signaling pathway.
In summary, we cloned the Muscovy duck MDA5 cDNA and demonstrate that the CARD of Muscovy duck MDA5 has a similar function as its mammalian counterpart, which is involved in immune signaling. In addition, overexpression of the CARD of Muscovy duck MDA5 activated the IRF-7-dependent signaling pathway and mediated both antiviral and proinflammatory responses in DEF cells. Importantly, overexpression of the CARD of the Muscovy duck MDA5 led to induction of the chIFN-β promoter and inhibition of the replication of H5N1 HPAIV in vitro. We also demonstrate that NS1 of H5N1 HPAIV inhibits the duck MDA5-mediated signaling pathway. This molecular cloning and functional characterization of the Muscovy duck MDA5 increases our understanding of the host immune response of ducks infected with H5N1 HPAIV.
This work was supported by grants from the Natural Science Foundation of Guangdong Province (No.10251064201000004), the National Natural Science Foundation of China (No.31172343), the Science and Technology Projects of Guangdong Province (No.2012B020306003), the Earmarked Fund for Modern Agro-Industry Technology Research System (nycytx-42-G3-03), and High-level Talents in University Project of Guangdong Province (2010).
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