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. 2012 Dec 4;109(49):E3340-9.
doi: 10.1073/pnas.1208618109. Epub 2012 Nov 5.

Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments

Alys Peisley  1 , Myung Hyun Jo, Cecilie Lin, Bin Wu, McGhee Orme-Johnson, Thomas Walz, Sungchul Hohng, Sun Hur
Affiliations

Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments

Alys Peisley et al. Proc Natl Acad Sci U S A. .

Abstract

The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ~0.5-7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here, we demonstrate that filament formation alone is insufficient to explain its length specificity, because the intrinsic affinity of MDA5 for dsRNA depends only moderately on dsRNA length. Instead, MDA5 uses a combination of end disassembly and slow nucleation kinetics to "discard" short dsRNA rapidly and to suppress rebinding. In contrast, filaments on long dsRNA cycle between partial end disassembly and elongation, bypassing nucleation steps. MDA5 further uses this repetitive cycle of assembly and disassembly processes to repair filament discontinuities, which often are present because of multiple, internal nucleation events, and to generate longer, continuous filaments that more accurately reflect the length of the underlying dsRNA scaffold. Because the length of the continuous filament determines the stability of the MDA5-dsRNA interaction, the mechanism proposed here provides an explanation for how MDA5 uses filament assembly and disassembly dynamics to discriminate between self vs. nonself dsRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ATP hydrolysis increases the binding preference of MDA5 for longer dsRNA. (A) EMSA of MDA5 and RIG-I with increasing amounts of 112-bp dsRNA. Protein (0.3 μM) was incubated with 112-bp dsRNA [30 nM (lane 1)–150 nM (lane 5)]. 3′-Fluorescein-labeled dsRNA was maintained at 10 nM, and the ratio of protein to binding sites (BS) was calculated assuming that each monomer occupied 14 bp. (B) Representative electron micrographs of MDA5 in complex with 2,012-bp dsRNA at a protein-to-binding site ratio of 1.8 (Left) or 0.31 (Right) in the presence of 0.5 mM ADP⋅AlF4. (C) EMSA and representative class averages of filaments formed by the CARD deletion mutant MDA5h on 112-bp dsRNA in the presence of 0.5 mM ADP⋅AlF4. EMSA was performed as in A. (D) Competition binding assay (mean ± SD, n = 2–3). Fluorescein-labeled reporter dsRNA (112 bp, 0.18 μg/mL) and unlabeled competitor dsRNA (21–2,012 bp, 0.06–43.74 μg/mL) were premixed and incubated with MDA5 (80 nM). The level of the reporter complex was monitored by EMSA in the presence of increasing amounts of competitor dsRNA (Fig. S2) and was plotted with fitted competition binding curves, which yielded the IC50. (E) Competition EMSA of MDA5 with dsRNAs of various lengths with ADPCP or ATP (2 mM). Experiments were performed as in A with a fixed concentration of competitor dsRNAs (0.54 μg/mL). The relative level of labeled complex with respect to 62-bp competitor dsRNA was plotted (mean ± SD, n = 3). (F) Competition EMSA of RIG-I as in B (mean ± SD, n = 3). The two bands corresponding to RIG-I:RNA complexes with ADPCP suggest binding to each end of dsRNA. The presence of multiple ill-defined bands with ATP is consistent with the translocation of RIG-I on dsRNA (37).
Fig. 2.
Fig. 2.
The ATP hydrolysis rate reflects the length-dependent RNA binding by MDA5. (A) ATP hydrolysis rates of MDA5 (0.3 μM) and RIG-I (30 nM) with model dsRNAs prepared by in vitro transcription (▲) and rotavirus genomic dsRNAs (●) (4.8 μg/mL) (mean ± SD, n = 3). (B) Relative ATP hydrolysis rate of MDA5 with rotavirus dsRNAs at different concentrations of RNA and MDA5 (mean ± SD, n = 4). Rates were normalized against the rate measured with 3.3 kb dsRNA. (C) Relative ATP hydrolysis rates of MDA5 (0.3 μM) with 112- or 1,012-bp dsRNA (4.8 μg/mL) containing variant 5′ functional groups, different sequences (Table S1), and different internal structures (a mismatch, bulge, or gaps) (mean ± SD, n = 3–4). Rates were normalized against the rate measured with intact 112-bp or 1,012-bp dsRNAs.
Fig. 3.
Fig. 3.
ATP hydrolysis promotes dissociation of MDA5, but not RIG-I, from dsRNA at a rate inversely proportional to dsRNA length. (A) Schematic of pull-down dissociation kinetic assay. The level of MDA5 bound to biotinylated dsRNA was monitored at discrete time points during dissociation by using streptavidin magnetic bead pull-down. MDA5 was fluorescently labeled for quantitation on SDS/PAGE. See Fig. S3 for details. (B) A representative SDS/PAGE image of MDA5 (Left) or RIG-I (Right) from dissociation pull-down assays at t = 0 or 2 min (2 m) using 512-bp dsRNA with either ATP or ADPCP (2 mM). Nonspecific binding (BG) was measured by using nonbiotinylated dsRNA. (C) Analysis of dissociation kinetics of MDA5 with ATP or ADPCP. Biotin pull-down was performed as in B using dsRNAs of 112–2,012 bp (mean ± SD, n = 3).
Fig. 4.
Fig. 4.
Slow dsRNA binding amplifies the length dependence of MDA5. (A) Analysis of the RNA-binding kinetics of MDA5 using biotin pull-down. As with the dissociation assays in Fig. 3, the level of MDA5 bound to dsRNA was monitored using streptavidin magnetic beads and was quantitated by using the fluorescein tag on MDA5 (Methods and Materials). On the right is the time course of the bound fractions and fitted single exponential curves (mean ± SD, n = 3). (B) The binding rate constant (kon) was obtained from the first-order approximation of the apparent binding rate (kobs, obtained from Fig. S4B) against MDA5 concentration. (C) Time evolution of the ATP hydrolysis reaction initiated by the addition of ATP to the preformed complex of MDA5 (0.3 μM) and 112-bp dsRNA (0.6 μg/mL) (dsRNA→ATP) or by the addition of dsRNA to the preformed complex of MDA5 and ATP (ATP→dsRNA) (mean ± SD, n = 3). (D) Initial lag period of the ATP hydrolysis reactions of MDA5 (0.3 μM) using different concentrations of 112-bp dsRNA at 100 or 150 mM NaCl (mean ± SD, n = 3). The lag period was estimated from the linear extrapolation of the reaction time course as in C. (E) Relative ATP hydrolysis rates of MDA5 (0.3 μM) bound to model dsRNAs of 62- to 2,012 bp (4.8 μg/mL) at 100 or 150 mM NaCl (mean ± SD, n = 3). Rates are normalized against the rate measured with 2,012-bp dsRNA.
Fig. 5.
Fig. 5.
MDA5 binds to dsRNA via multiple, rate-limiting nucleation steps to assemble discontinuous filaments on a single dsRNA. (A) Schematic of the single-molecule fluorescence assay. The filament assembly reaction was monitored using TIRF single-molecule microscopy upon the addition of fluorescently labeled MDA5 or MDA5h to surface-immobilized dsRNA (t = 0) (Methods and Materials). (B) Representative traces of filament assembly reactions using Alexa 647-labeled MDA5hSNAP (50 nM) and 2,012-bp dsRNA. The immediate increase in fluorescence upon MDA5hSNAP injection (t = 0) indicates the background noise, which also was observed without dsRNA. (C) A model of stepwise assembly reaction. The alternation between elongation and pause in assembly traces indicates the involvement of multiple nucleation events. Elongation ends abruptly upon encounter of dsRNA ends or neighboring filament termini. The insufficiency of a single nucleus to propagate and saturate the entire length of dsRNA suggests unidirectional elongation of MDA5 filaments. The direction of elongation likely is determined by asymmetric binding of a nucleus to symmetric dsRNA with an equal probability of facing either direction. (D) A representative electron micrograph of full-length MDA5 (0.3 μM) in complex with 2,012-bp dsRNA (1.2 μg/mL; i.e., 0.34 μM MDA5 binding site). Gaps (arrows) indicate the presence of multiple filaments propagated from independent nuclei on a single dsRNA. (E) Histogram of the elongation rates of MDA5hSNAP filaments calculated from the slope of the first linear elongation phase of ∼350 assembly traces. Fluorescence intensity was converted to base pair numbers by assuming 100% coverage of 512 bp at saturation. (F) Averaged traces of filament propagation of MDA5, MDA5h, and MDA5hSNAP synchronized by the initial nucleation event (n = 331, 257, and 542, respectively). (G) Histogram of the nucleation times of MDA5hSNAP (50 nM) on 512-bp dsRNA and the single-exponential fit. (H) Dependence of the nucleation time (mean value) on dsRNA length (n = 250–500).
Fig. 6.
Fig. 6.
MDA5 dissociates from individual filament ends. (A) Averaged traces of filament disassembly reaction. MDA5hSNAP filaments were preformed on 512-bp dsRNA and were immobilized on the flow cell surface, and unbound filaments were washed out. Disassembly reactions were initiated by the addition of 2 mM ATP, ADPCP, or ATP with 0.5 mM EDTA at t = 0. Single-molecule TIRF microscopy was performed as in Fig. 5. n = ∼500–700 for each sample. (B) Averaged traces of MDA5hSNAP dissociating from 512-, 1,012-, and 2,012-bp dsRNAs and their single-exponential fits with time constants (τ) (n = 763, 570, and 258, respectively). (C) Representative traces of Alexa 647-labeled MDA5hSNAP dissociating from surface-immobilized 2,012-bp dsRNA. (D) A representative trace of MDA5hSNAP dissociating from 112-bp dsRNA fitted with a step function (black). (E) Histogram of step intensities during disassembly of 112-bp filaments from 95 independent traces and fitted double Gaussian function (black line). Filled and blank circles indicate labeled and unlabeled MDA5 monomers, respectively. (F) Histogram of the number of step decrements during disassembly of 112-bp filaments from 95 independent traces. Predicted distribution curves (black lines) are derived from a probability theory (SI Materials and Methods). (G) Representative electron micrographs of MDA5 2,012-bp dsRNA filaments during disassembly. Disassembly was initiated by the addition of ATP and heparin and was quenched with ADP⋅AlF4 at the indicated time points before EM (Methods and Materials). Arrows at t = 20 s indicate filament gaps. (H) A model of MDA5 filament disassembly. We propose that individual filaments disassemble independently from their ends, generating apparent internal breaks from regions containing filament discontinuities. (I) Histogram of individual filament lengths at t = 20 s after addition of ATP (n = 415 and 230 for 512-bp and 2,012-bp dsRNAs, respectively). The reaction was performed as in F.
Fig. 7.
Fig. 7.
ATP hydrolysis repairs discontinuities in MDA5 filaments and promotes the formation of longer, continuous filaments. (A) Proposed mechanism by which ATP hydrolysis repairs discontinuities in MDA5 filaments. Multiple filaments propagated from independent nuclei individually undergo cycles of ATP-driven disassembly and elongation, which allow the rearrangement of MDA5 molecules to generate more continuous filaments. (B) Averaged assembly traces of MDA5hSNAP filament on 2,012-bp dsRNA in the presence of 2 mM ADPCP or 0.2 mM ATP using 100 nM MDA5hSNAP. Individual traces were synchronized by the nucleation time (n = 386 and 495 for ADPCP and ATP, respectively). (C) Averaged disassembly traces of MDA5hSNAP filaments initially assembled on 2,012-bp dsRNA with or without ATP (n = 375 and 223, respectively). Disassembly reactions were performed as in Fig. 6C. (D) A representative electron micrograph of disassembly intermediates of MDA5 filaments initially formed with ATP. MDA5 filaments were formed on 2,012-bp dsRNA in the presence of 0.25 mM ATP. Dissociation was initiated at t = 0 by the addition of ATP and heparin and was quenched with ADP⋅AlF4 at t = 20 s. Fewer gaps (arrows) were observed than when filaments were formed without ATP (Fig. 6G). (E) Histogram of individual filament lengths at t = 20 s after the addition of ATP. Filaments were formed initially with ATP (light blue bars, n = 260) as in D or without ATP (dark blue bars) (from Fig. 6I).
Fig. 8.
Fig. 8.
Discrimination between MDA5 and MDA5h in filament formation. (A) Fluorescence images of filaments formed by a mixture of MDA5hSNAP (labeled with Alexa Flour 546) and MDA5 (labeled with Hylite647) on 1,012-bp dsRNA. A 1:1 mixture of MDA5hSNAP and MDA5 (100 nM each) was incubated with 1,012-bp dsRNA (0.1 nM), and the complex was immobilized on the flow cell surface. Two-color imaging identified 462 filaments with Alexa 546 fluorescence (Left) and 362 filaments with Hylite 647 fluorescence (Right). Among these, 128 filaments showed common, overlapping positions (such as the one in yellow circle), indicating that these 128 filaments contain both MDA5 and MDA5hSNAP. (B) Venn diagram summarizing the results in A and plot of Alexa 546 vs. Hylite 647 fluorescence intensities of the 128 filaments containing both MDA5 and MDA5hSNAP. (C) Representative traces of MDA5 filament dynamics in the mixture of MDA5 and MDA5hSNAP. Preformed filaments of Hylite 647-labeled MDA5 on 512-bp dsRNA were immobilized on the flow cell surface, and unbound filaments were washed out. A 1:1 mixture of Hylite 647-labeled MDA5 and Alexa 546-labeled MDA5hSNAP (100 nM each) was injected into the flow cell with ATP or ADPCP (2 mM). The fluorescence intensities of MDA5 and MDA5hSNAP were normalized against those of the filaments formed by the respective protein alone. (D) Averaged traces of MDA5 filament dynamics in the mixture of MDA5 and MDA5hSNAP. Experiments were performed as in C. n = 325 and 396 for ATP and ADPCP reactions, respectively. (E) Proposed model for length discrimination by MDA5. With ATP, MDA5 filaments continuously switch between assembly and disassembly phases. Because disassembly occurs primarily from filament ends, filaments on short dsRNA more frequently undergo a complete disassembly, which requires slow de novo nucleation for rebinding. In contrast, filaments on long dsRNA alternate between partial disassembly and fast elongation, bypassing nucleation. Thus the slow nucleation kinetics amplifies the time constant (τ) in Fig. 6B.
Fig. P1.
Fig. P1.
Proposed model for length discrimination by MDA5. MDA5 filaments switch continuously between assembly and disassembly phases in the presence of ATP. Because disassembly occurs primarily from the filament ends, filaments on short dsRNA undergo complete disassembly more frequently, requiring slow de novo nucleation for rebinding. In contrast, filaments on long dsRNA alternate between partial disassembly and fast elongation, consequently bypassing nucleation. Thus, slow nucleation kinetics amplifies the effect of length-dependent dissociation kinetics of MDA5 and prevents the accumulation of MDA5 on short dsRNAs and aberrant activation of antiviral signaling.
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