Shared and Unique Functions of the DExD/H-Box Helicases RIG-I, MDA5, and LGP2 in Antiviral Innate Immunity

Cells, DNA transfection, preparation of cell extracts, and luciferase assay

L929 cells were maintained in MEM with 5% FBS and penicillin/streptomycin. 293T cells, Huh7 cells, and mouse embryonic fibroblasts (MEFs) were maintained in DMEM with 10% FBS and penicillin/streptomycin. The transient transfection of the L929 and 293T cells was performed as described previously (14). The 293T cells in Fig. 1⇓C, Huh7 and MEFs, were transfected by FuGENE6 (Roche). The stable transformants of the L929 cells were established by the transfection of a linearized empty vector (pEF-BOS) or an expression plasmid for RIG-I (pEF-flagRIG-Ifull), RIG-IC (pEF-flagRIG-IC), MDA5 (pEF-flagMDA5full), or LGP2 (pEF-flagLGP2) with pCDM8neo, followed by selection with G418 (1 mg/ml). Virus infection, poly(rI):poly(rC) (poly(I:C)) transfection, and preparation of cell extracts were performed as reported previously (14). Luciferase assay was performed with a dual-luciferase reporter assay system (Promega). As the internal control for the dual-luciferase assay, pRL-TK (Promega) was used.

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FIGURE 1.

CARDs of RIG-I and MDA5 participate in a common signaling cascade. A, Schematic representation of RIG-I family helicases. Amino acid identities in the CARD and helicase domain are shown. B, L929 cells were transiently transfected with reporter constructs containing repeated IRF binding sites (p-55C1BLuc; C1B-Luc) together with the empty vector pEF-BOS (control), the RIG-IN expression construct (RIG-IN), or the MDA5N construct (MDA5N). At 48 h after transfection, the cells were subjected to the dual-luciferase assay. The relative firefly luciferase activity, normalized by the Renilla luciferase activity, is shown. Error bars show the SDs of triplicate transfections. C, 293T cells were transfected with a control vector, RIG-IN, or MDA5N. At 24 h after transfection, the IFN-β in the culture medium was quantified by ELISA. D, L929 cells were transfected with p-55UAS_GLuc and pEFGal4/IRF-3 together with pEF-BOS (control), RIG-IN, or MDA5N vector. Luciferase activity was normalized by the internal control pRL-TK. Error bars represent the SDs of triplicate transfections. E, The DNA-binding activity of the IRF-3-containing holocomplex was detected by EMSA using the ³²P-labeled IFN-stimulated response element oligonucleotide of the isg15 gene as probe. The extract was prepared from the 293T cells that were transiently transfected with the indicated plasmids. F, The MEF cells prepared from tbk1^+/+ or tbk1^−/− mice were transiently transfected with p50-tagged human IRF-3 along with the control vector, RIG-IN, or MDA5N plasmid. Extracts were subjected to native-PAGE, and the transfected IRF-3 was visualized by immunoblotting using an anti-p50 antiserum. G, 293T cells were transfected with p50-tagged IRF-3 together with the indicated plasmids. Extracts were subjected to native-PAGE followed by immunoblotting using an anti-p50 Ab (upper panel) and an anti-phophoSer³⁸⁶ antiserum (lower panel). H, Huh7 cells were cotransfected with plasmids encoding the IFN-β luciferase reporter construct, Renilla luciferase, and MDA5N or RIG-IN vector with increasing amounts (0, 50, 100, and 200 ng) of the plasmid encoding NS3/4A (hepatitis C virus 1a), and subjected to luciferase assay. I, Reporter assay was performed as shown in D, except that pEFGal4/IRF-7 was used as effector. J, Reporter assay as in B, except that the NF-κB reporter plasmid (p-55A2Luc) was used.

Plasmid constructs

p-55C1BLuc, p-55UAS_GLuc, p-55A2Luc, p-125Luc, pEFGal4/IRF-3, pEFGal4/IRF-7, pEF-flagRIG-I, pEF-flagRIG-IC, pEF-flagRIG-IKA, pEF-flageIF4A, pcDNA3.1-nonstructural protein 3/4A (NS3/4A), pKS336-Sv-V, and pKS336-Sv-Vu were described previously (14, 18, 19). The pEF-flagRIG-IN, which contains the N-terminal 229 aa of RIG-I, was obtained by the insertion of oligonucleotides at the EcoRI sites of RIG-I cDNA. The cDNA of MDA5 was isolated by the RT-PCR technique, and inserted into the XbaI/ClaI sites of pEF-BOS⁺ with oligonucleotides for the N-terminal flag tag (pEF-flagMDA5). The nucleotide sequence of the cDNA was confirmed with a BigDye DNA-sequencing kit (Applied Biosystems). The cDNA of LGP2 (GenBank accession no. AK021416) was purchased from the Biological Resource Center of National Institute of Technology and Evaluation, and inserted into the XbaI sites of pEF-BOS with oligonucleotides for the N-terminal flag tag (pEF-flagLGP2). The deleted and site-directed mutants of MDA5 were obtained by the insertion of the appropriate oligonucleotides and the Kunkel method (20), respectively (pEF-flagMDA5N, pEF-flagMDA5C, and pEF-flagMDA5KA).

EMSA

EMSA was performed as described previously (14).

Abs, ELISA, immunoprecipitation, and immunoblotting

Anti-murine IRF-3, anti-p50-tag, and anti-V protein Abs were described in our previous reports (14, 19). The anti-flag (M2; Sigma-Aldrich) Ab and anti-actin Ab (Chemicon) are commercial products. The anti-LGP2 antiserum was obtained by immunizing the rabbits with synthetic peptides (IQAKKWSRVPFSVC) of LGP2 conjugated to bovine thyroglobulin. The Ab was affinity-purified by using immobilized Ag peptide. We confirmed that this anti-LGP2 antiserum specifically recognized recombinant LGP2 expressed in 293T cells. ELISA, SDS-PAGE, native-PAGE, and immunoblotting were performed as described previously (14). Immunoprecipitation was performed as shown in previous report using anti-flag M2 affinity gel (Sigma-Aldrich) (5).

Poly(I:C)-pulldown assay

Poly(I:C)-pulldown assay was performed as described previously (14).

RNA interference

The vector for small interfering RNA (siRNA), piGENE hU6, and constructs for control or RIG-I siRNA were described previously (14). The sequences for the siRNA targeting murine MDA5 and LGP2 mRNA are 5′-GTCATTAGTAAATTTCGCACT-3′ and 5′-GGATGGAGTTGGAAGATGA-3′, respectively. For the reporter assay, the L929 cells were transiently transfected with p-55C1BLuc and pRL-TK as reporters, together with the siRNA vectors. The cells were mock-infected or Newcastle disease virus (NDV)-infected, and subjected to dual-luciferase assay. The quantitative assay for endogenous mRNA was performed as described previously (14).

Virus yield titration

Stable L929-derived transformants were mock-infected or infected with vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV). The virus yield in culture supernatants was determined by plaque assay as described previously (14).

Mouse IFN titration

MEFs were stimulated by NDV infection or poly(I:C)/DEAE dextran transfection. After 24 h, culture media were collected, ultracentrifuged to remove viruses and poly(I:C) (436,000 × g, 10 min), and subjected to IFN assay. The mouse IFN was titrated using the L929 cells in a 96-well plate and EMCV as a challenge virus. The IFN titer was normalized using an international standard.

CARDs of RIG-I and MDA5 participate in a common signaling cascade

RIG-I and MDA5 share a limited homology in their overall primary structure (Fig. 1⇑A). Notably, the two proteins contain tandem CARD-like regions at their N-termini as well as C-terminal DExD/H-box helicase domains. The previous observation that the CARD of RIG-I and those of Nod1 and Nod2 target a distinct set of downstream molecules (14) prompted us to compare the functions of the CARDs of RIG-I and MDA5. The overexpression of the CARD of either RIG-I (RIG-IN) or MDA5 (MDA5N) resulted in the constitutive activation of the reporter p-55C1BLuc, which is essentially regulated by multimerized IRF binding sites (Fig. 1⇑B). Furthermore, these CARDs activated the endogenous IFN-β gene, resulting in the secretion of the IFN-β protein without exogenous stimuli, such as viral infection (Fig. 1⇑C). The fusion transcription factor containing the DNA-binding domain of Gal4 and the C-terminal regulatory domain of IRF-3 (Gal4-IRF-3) was activated by the expression of RIG-IN or MDA5N (Fig. 1⇑D), showing that IRF-3 is a downstream target. Consistent with this, RIG-IN and MDA5N induced the IRF-3 holocomplex (Fig. 1⇑E) and IRF-3 dimer (Fig. 1⇑F). These results show that the CARD of MDA5 is capable of activating IRF-3 similarly to that of RIG-I. Interestingly, tbk1^−/− cells are defective in IRF-3 dimer formation induced by RIG-IN or MDA5N, suggesting that these CARDs signal through the protein kinase TBK1 (Fig. 1⇑F). This is consistent with our observation that RIG-IN and MDA5N result in the phosphorylation of Ser 386 of IRF-3 (6) (Fig. 1⇑G). It has been reported that the NS3/4A of the hepatitis C virus blocks RIG-I-mediated signaling through its protease activity (18). Although the target of the NS3/4A protease has not yet been identified, this hypothetical molecule functions downstream of MDA5N and RIG-IN, because the activation of IRF-3 was blocked by NS3/4A (Fig. 1⇑, G and H). Moreover, Gal4-IRF-7 and NF-κB were activated by RIG-IN and MDA5N (Fig. 1⇑, I and J). In summary, these results strongly suggest that the CARDs of RIG-I and MDA5 activate the overlapping cascade.

Positive and negative signalings by the full-length RIG-I, MDA5, and LGP2

The overexpression of the full-length RIG-I and MDA5 failed to activate the reporter genes, p-55C1BLuc and pIFNβLuc; however, considerable activation was observed after stimulation by virus infection or dsRNA transfection (Fig. 2⇓A). Similarly, the dimerization of IRF-3 induced by dsRNA was increased by the overexpression of RIG-I or MDA5 without significant basal activation (Fig. 2⇓B). These results show that RIG-I and MDA5 act as positive regulators of virus-induced signaling and are under strict negative regulation in the absence of stimuli.

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FIGURE 2.

Stimulation-dependent activation of full-length RIG-I and MDA5. A, L929 cells were transfected with p-55C1BLuc (C1B-Luc) or the natural IFN-β gene promoter (p-125Luc; IFNβ-Luc) together with pEF-BOS (control), full-length RIG-I, or MDA5. After stimulation with poly(I:C) transfection (6 h) or NDV infection (12 h), the cells were subjected to dual-luciferase assay. The relative firefly luciferase activity normalized by the Renilla luciferase activity is shown. Error bars show the SDs of triplicate transfections. B, L929-derived stable transformants expressing the neo gene alone (control) or flag-RIG-I or flag-MDA5 were stimulated with poly(I:C) transfection for 4 h. Endogenous IRF-3 was visualized by native-PAGE/immunoblotting using an anti-mouse IRF-3 antiserum (upper panel). The expressed RIG-I and MDA5 were detected by SDS-PAGE/immunoblotting using an anti-flag Ab (lower panel).

The database search revealed another DExD/H-box helicase, LGP2, related to RIG-I and MDA5, but lacking CARD (Figs. 1⇑A and 3A). The LGP2 protein is markedly inducible by IFN-β, similarly to RIG-I and MDA5 (14, 21), whereas the house keeping actin expression was unaffected (Fig. 3⇓B). To explore the functions of these proteins, we generated mutants of RIG-I and MDA5 lacking CARD (RIG-IC and MDA5C) as well as having an inactivated Walker’s ATP binding motif by the Lys-to-Arg substitution of the respective helicases (RIG-IKA and MDA5KA). For comparison, a distantly related DExD/H helicase, eukaryotic initiation factor 4A (eIF4A), was used. Under these conditions, full-length RIG-I and MDA5 enhanced the virus-induced gene induction (Fig. 3⇓C). eIF4A did not affect the virus-induced activation of the reporter; however, as reported earlier, RIG-IC and RIG-IKA strongly inhibited the virus-induced gene activation (14). In contrast, MDA5C and MDA5KA neither augmented nor suppressed the gene activation by the virus. In either case, the helicase domain alone is incapable of transducing positive signaling. Moreover, the results show that ATPase activity is critical to the functioning of RIG-I and MDA5 as positive regulators. LGP2 strongly inhibited the gene activation, similarly to RIG-IC. In cells stably expressing RIG-IC or LGP2, virus-induced dimerization of endogenous IRF-3 was severely impaired (Fig. 3⇓D). These results suggest that LGP2 functions as a physiological negative feedback regulator. Consistent with this, knockdown of LGP2 markedly enhanced gene activation induced by NDV infection (Fig. 3⇓E). The binding of these helicases to dsRNA was investigated by pulldown assay (Fig. 3⇓F). All of the tested helicases bound to poly(I:C)-agarose, except eIF4A with a relative affinity of LGP2≫RIG-I>MDA5, confirming that the RIG-I family recognizes dsRNA.

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FIGURE 3.

Functional analysis of RIG-I family proteins. A, Schematic representation of full-length and mutant helicases. B, L929 cells were cultured with a control medium (−) or a medium containing recombinant mouse IFN-β (+, 1000 IU/ml) for 12 h. Endogenous LGP2 and actin were detected by immunoblotting using an anti-LGP2 antiserum and anti-actin Ab, respectively. C, L929 cells were transfected with p-55C1BLuc (C1B-Luc) together with the indicated constructs. After stimulation with NDV infection (12 h), the cells were subjected to dual-luciferase assay. The relative firefly luciferase activity, normalized by the Renilla luciferase activity, is shown. Error bars show the SDs of triplicate transfections. D, L929-derived stable transformants expressing RIG-IC or LGP2 were stimulated with NDV for 9 h. Endogenous IRF-3 was separated by native PAGE and detected by immunoblotting using an anti-mouse IRF-3 antiserum. The positions of monomeric IRF-3 and dimeric IRF-3 are indicated. E, Knockdown of LGP2 expression by siRNA. L929 cells (5 × 10⁵) were cotransfected with the reporter genes (250 ng of p-55C1BLuc and 10 ng of pRL-TK) and empty vector (vector, 2.5 μg) or vector for siRNA targeting LGP2 (LGP2, 2.5 μg). Cells were infected with NDV and subjected to dual-luciferase assay at the indicated time. Error bars show SD of triplicate transfections. F, Flag-tagged RIG-I, MDA5, LGP2, and eIF4A were produced in 293T cells, precipitated with poly(I:C)-agarose, and detected by immunoblotting using an anti-flag Ab (right panel). One-thirtieth of each extract used for pulldown assay was similarly separated and detected (left panel).

Suppression of viral yield by RIG-I and MDA5

In our previous work, we showed that the stable expression of the RIG-I protein in cultured cells severely inhibits viral replication (14). We compared the antiviral activity of the RIG-I family by the same assay using EMCV and VSV as infecting viruses (Fig. 4⇓). These viruses cause a severe “shutoff” of the host RNA and protein syntheses; thus, they are fully competent to induce lytic infection in L929 cells. RIG-I and MDA5 significantly reduced the viral yield 100- to 1000-fold, whereas LGP2 did not affect the viral yield. Likewise, the artificial inhibitor RIG-IC did not affect the viral yield (our unpublished observations).

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FIGURE 4.

Antiviral activities of RIG-I and MDA5. L929-derived stable transformants expressing RIG-I, MDA5, or LGP2 were infected with EMCV (upper panel) or VSV (lower panel) at the indicated multiplicity of infection (m.o.i). After 24 h of infection, virus yield in the supernatant was determined by plaque assay.

Effect of inhibition of RIG-I and MDA5 on signal transduction

The above-mentioned results using mutant proteins indicate that, in addition to their structural homology, RIG-I and MDA5 share functional similarities. To explore the loss-of-function phenotype of these proteins, we performed knockdown of RIG-I and MDA5 using siRNA. We used luciferase reporter (Fig. 5⇓A) and quantification of endogenous target gene expression (Fig. 5⇓B) as readout. Vector and control siRNAs did not affect the virus-induced activation of the reporter p-55C1BLuc and endogenous IFN gene expression. The siRNA targeting endogenous mouse RIG-I or MDA5 blocked the gene induction in a dose-dependent manner (Fig. 5⇓A). Furthermore, the expression of endogenous IFN mRNA was specifically inhibited by siRNA for either RIG-I or MDA5 (Fig. 5⇓B). These results indicate the requirement of endogenous RIG-I and MDA5 in the signaling.

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FIGURE 5.

Knockdown of RIG-I and MDA5 expression by siRNA. A, L929 cells (2 × 10⁵) were cotransfected with the reporter genes (200 ng of p-55C1BLuc and 4 ng of pRL-TK) and increasing amounts of vectors for siRNA (400, 600, and 800 ng). The cells were mock-infected or NDV-infected for 12 h, and subjected to dual-luciferase assay. Error bars show the SDs of triplicate transfections. B, L929 cells were transfected with the indicated siRNA vectors together with the plasmid for puromycin-resistant gene. After cultivation in the presence of puromycin (10 μg/ml) for 3 days, cells were mock-infected or NDV-infected for 12 h. Total RNA was prepared, and subjected to real-time PCR using the primer-probe sets for mouse IFN-α4 and IFN-β genes.

Recently, a report has shown that V proteins encoded by paramyxoviruses inhibit the MDA5-induced activation of type I IFN genes (16). The inhibition is apparently mediated by the direct association between V proteins and MDA5. We investigated the effect of the Sendai virus (SeV) V protein on signaling by RIG-I and MDA5 (Fig. 6⇓A). We used an expression vector for the entire coding region of the V protein (sharing the N-terminal 316 aas with the P protein: 384 aa) and a partial clone encompassing the C-terminal region unique to the V protein (Vu: 68 aa). The expression of the SeV V protein caused a marginal (∼30%) suppression of the NDV-induced reporter gene expression, suggesting that the NDV-induced signal is largely insensitive to the V-protein-mediated inhibition. The expression of RIG-I resulted in a marked augmentation of the NDV-induced reporter gene activation; however, it was not notably affected by the V protein expression. In contrast, the enhancement of the virus-induced gene activation by MDA5 was completely abrogated by the V protein as reported (16). A Vu protein exhibited an activity indistinguishable from that of the V protein, indicating that the C-terminal region is sufficient for the inhibition. The selective inhibition of MDA5 by V protein is likely due to selective physical interaction as revealed by coprecipitation experiment (Fig. 6⇓B), in which RIG-I did not interact with V protein.

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FIGURE 6.

The V protein of SeV selectively inhibited MDA5-mediated signaling. A, L929 cells were transfected with reporter constructs (p-55C1BLuc and pRL-TK) along with the indicated combination of expression vectors. Cells were mock-infected or NDV-infected for 12 h. The relative firefly luciferase activity normalized by the Renilla luciferase activity is shown. B, 293T cells were transfected with empty vector (pEF-BOS), flag-tagged full-length RIG-I, flag-tagged full-length MDA5, or SeV V protein vectors. Expression of the proteins was confirmed by immunoblotting using anti-flag or anti-V protein Abs (input). The indicated extracts were mixed on ice for 10 min and subjected to immunoprecipitation (IP) using anti-flag M2 Gel (Sigma-Aldrich). The precipitated RIG-I, MDA5, and V protein were detected by immunoblotting.

Recently, Balachandran et al. (22) have reported that Fas-associated protein with death domain (FADD) plays an essential role in the poly(I:C)-induced induction of type I IFN genes as well as in apoptosis. We used fibroblasts derived from wild-type and knockout mice for irf-3, tbk1, and fadd genes. These cells were either infected with NDV or transfected with poly(I:C) by the DEAE dextran method, and the secreted IFN was quantified (Table I⇓). The wild-type cells efficiently produced IFN after NDV infection; however, this production was markedly reduced in cells deficient in IRF-3 and TBK1, consistent with previous reports (4, 10, 11, 23). FADD-deficient cells produced IFN at a level comparable to that of the wild-type cells, showing that FADD is not necessary in the signaling by NDV. However, when these cells were transfected with poly(I:C), FADD was observed to be apparently necessary for the IFN production along with other signaling components such as IRF-3 and TBK1. This result shows that virus and a synthetic dsRNA trigger the partially shared but distinct signaling cascade.