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. 2020 Nov 18;48(20):11664-11674.
doi: 10.1093/nar/gkaa935.

Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

Ivana Duic  1   2 , Hisashi Tadakuma  3   4 , Yoshie Harada  3 , Ryo Yamaue  1   2 , Katashi Deguchi  1 , Yuki Suzuki  5 , Shige H Yoshimura  1 , Hiroki Kato  2   6 , Kunio Takeyasu  1 , Takashi Fujita  1   2   6
Affiliations

Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

Ivana Duic et al. Nucleic Acids Res. .

Abstract

Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.

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Figures

Figure 1.
Figure 1.
LGP2 increases IFN-β promoter activity through MDA5 in response to poly(I:C), EMCV or BPEVdsRNA. (A) Experimental design to analyze IFN-β promoter activity in HEK293T cells in response to different stimuli. (B–D) HEK293T cells in 12-well plate were transfected with a reporter plasmid containing the luciferase gene under the control of the IFN-β promoter (p-125Luc, 1 μg), Renilla luciferase construct (pRL-tk, 40 ng) as an internal control, 25 ng of plasmid expressing MDA5 and 4 ng of plasmid expressing LGP2. Twenty-four hours after transfection, cells were mock treated or (B) transfected with 3 μg of poly(I:C), (C) infected with EMCV (MOI = 1), and (D) transfected with 300 ng of BPEVdsRNA. After 8 h, cells were subjected to the dual-luciferase assay. Data represent relative firefly luciferase activity normalized to Renilla luciferase activity in one of at least three independent experiments, and error bars indicate ± S.D. *** P <0.001, * P <0.05, unpaired Student's t test.
Figure 2.
Figure 2.
LGP2 increases fiber formation by MDA5. (A) AFM images of the time course of binding of different proteins with BPEVdsRNA. AFM image of BPEVdsRNA (top). BPEVdsRNA was mixed with the proteins indicated on the left and observed at indicated time points by AFM. (B) The average fiber length measured from at least three independent experiments. Error bars: ± S.D. MDA5 fiber formation. (C) Height quantification of BPEVdsRNA alone (top) and fibers formed by MDA5 (middle) or MDA5/LGP2 (bottom). (D) Average of BPEVdsRNA height (gray), MDA5 fiber height in the absence (blue) and presence (red) of LGP2, measured from 100 sections. Error bars: ± S.D. **** P < 0.0001, unpaired Student's t-test.
Figure 3.
Figure 3.
Incorporation of LGP2 into MDA5 fiber. (A) AFM images of Qdot (upper left), and MDA5/BPEVdsRNA fiber with or without LGP2 probed with αLGP2-Qdot or αGFP-Qdot as indicated. (B) Quantification of average number of Qdots/1 μm of MDA5 fiber. Values are the average of four Qdot counts in 1 μm of MDA5 fiber. Error bars: ±S.D. * P < 0.05, unpaired Student's t-test. (C) Representative AFM image of distance measurements between αLGP2-Qdot attached to the fiber; H = height, A = amplitude. (D) Distances of 10 Qdot pairs (left). Schematic model of fiber composed of MDA5 and LGP2 (right).
Figure 4.
Figure 4.
MDA5 fiber turnover by LGP2 and ATP hydrolysis. (A) SEC fractionation of free protein and dsRNA/protein complexes. Fractionation of MDA5 (a) and the reaction mixture of MDA5 and BPEVdsRNA (b) by Sepharose 4B. The high-molecular weight fractions (fractions 8–10 of b) were pooled as MDA5/BPEVdsRNA fibers. The fibers were re-chromatographed without incubation (c) or after incubation with ATP (d), LGP2 (e), ATP+LGP2 (f), ATP+LGP2 K30G (g), AMP-PNP+LGP2 (h). Samples were analyzed by immunoblotting (IB) using anti-Flag antibody. (B) ATPase assay. Recombinant proteins were analyzed for ATPase activity in the presence or absence of dsRNA (Materials and Methods). Values are the average of 3 independent experiments. Error bars: ±S.D. ****P <0.0001, *P <0.05, NS = P > 0.05, unpaired Student's t-test.
Figure 5.
Figure 5.
LGP2 regulates molecular conformation of MDA5. (A) Schematic representation of MDA5 domain structure (top). Limited trypsin digestion of MDA5 yields 4 major cleavage fragments (silver staining). FLAG-tagged MDA5 was produced in HEK293T cells and digested with trypsin, followed by immunoblotting with anti-FLAG or anti-CTD antibodies. Identification of bands 1–4 is shown. (B) Time course of trypsin digestion (TPCK trypsin 35 ng) of recombinant proteins. The indicated recombinant proteins in the presence or absence of poly(I:C), AMP-PNP or ATP were treated with trypsin. The digestion was terminated by adding SDS sample buffer and subjected to SDS-PAGE, followed by silver staining. (C) Densitometry of bands 2 and 4 generated by the digestion of MDA5, poly(I:C) and AMP-PNP. (D) Densitometry of bands 2 and 4 generated by the digestion of MDA5, LGP2, poly(I:C) and AMP-PNP normalized to non-digested FL-MDA5 as 100%. (E) Native PAGE analysis of poly(I:C)/MDA5 complex. MDA5 was mixed with poly(I:C) and the complex was isolated by SEC as in Figure 4Ab. The complex was incubated with ATP in the presence or absence of LGP2. The mixture was fractionated by centrifugation into poly(I:C)-bound complex (ppt) and released proteins (sup). The fractions were analyzed by native (top) and SDS (bottom) PAGE followed by immunoblotting with anti-MDA5 antibody (top) and-Flag antibody (bottom). (F) Trypsin digestion of MDA5. Naïve recombinant MDA5 and MDA5 recovered by dissociation from poly(I:C) (lane 4 sample of Fig. 5E) were analyzed by limited trypsin digestion as in (B). Positions of full length MDA5 (1) and its digestion products (2–4) are shown in the right. (G–I) AFM images of MDA5 protein under different conditions. Wild type MDA5 protein was incubated with reaction buffer (G) or with dsRNA (H) or with dsRNA and LGP2 (I), in the presence of 1 mM ATP for 30 min and was observed by AFM. Left: 300 × 300 nm2 images, Right: 75 × 75-nm2 images. From these images, MDA5 monomers (objects with total volume of 300 ± 30 nm3) were selected and quantified for diameter (bottom).
Figure 6.
Figure 6.
Model for activation of MDA5 by LGP2 and ATP. (A) Naïve MDA5 has an open structure and is hypersensitive to trypsin, however the tandem CARD is masked. (B) Upon binding with viral dsRNA localized within the viral replication complex, MDA5 forms fibers, in which it has a closed structure with partially exposed CARDs. (C) ATP hydrolysis weakly promotes MDA5 fiber dissociation. (D) In the presence of LGP2 and ATP, MDA5 further changes conformation to fully expose its CARD1. (E) Upon ATP hydrolysis, the fiber efficiently dissociates. (F) Released MDA5 retains its open structure as monomer/oligomer mixture and exits from the viral replication complex and migrates to its downstream adaptor, MAVS. (G) MDA5 forms a complex with MAVS on mitochondria. (H) Aggregation of MDA5 and MAVS results in the activation of transcription factors, including IRF-3 and NF-κB.
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