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. 2011 Dec 27;108(52):21010-5.
doi: 10.1073/pnas.1113651108. Epub 2011 Dec 12.

Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition

Alys Peisley  1 , Cecilie Lin, Bin Wu, McGhee Orme-Johnson, Mengyuan Liu, Thomas Walz, Sun Hur
Affiliations

Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition

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

Abstract

MDA5, an RIG-I-like helicase, is a conserved cytoplasmic viral RNA sensor, which recognizes dsRNA from a wide-range of viruses in a length-dependent manner. It has been proposed that MDA5 forms higher-order structures upon viral dsRNA recognition or during antiviral signaling, however the organization and nature of this proposed oligomeric state is unknown. We report here that MDA5 cooperatively assembles into a filamentous oligomer composed of a repeating segmental arrangement of MDA5 dimers along the length of dsRNA. Binding of MDA5 to dsRNA stimulates its ATP hydrolysis activity with little coordination between neighboring molecules within a filament. Individual ATP hydrolysis in turn renders an intrinsic kinetic instability to the MDA5 filament, triggering dissociation of MDA5 from dsRNA at a rate inversely proportional to the filament length. These results suggest a previously unrecognized role of ATP hydrolysis in control of filament assembly and disassembly processes, thereby autoregulating the interaction of MDA5 with dsRNA, and provides a potential basis for dsRNA length-dependent antiviral signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Only binding of MDA5 to dsRNA is cooperative and stimulates ATP hydrolysis. (A) EMSA with 112 bp/nt dsRNA, ssRNA, and dsDNA. A lane in which approximately 50% of MDA5 is bound to the respective nucleic acid (red box) was chosen for each sample, and the distribution of the complexes is displayed in the left-hand panel. (B) The EMSA results in A were fit to the Hill equation to obtain dissociation constant (Kd) and Hill coefficient (Nh). Estimated Kd and Nh are 22 nM and 4.0 for dsRNA, 33 nM and 1.5 for ssRNA, and 57 nM and 2.7 for dsDNA, respectively. Plotted values are mean ± SD (n = 2). (C) ATP hydrolysis rates (mean ± SD, n = 4) of MDA5 (0.3 μM) free of nucleic acids, MDA5 bound to 112 bp/nt dsRNA, ssRNA, dsDNA, DNA–RNA hybrid (▪ sequence 1, □ sequence 2, see Table S1), IRES (1 kb), and genomic RNA (gRNA) (∼7 kb) of EMCV and MV. Equivalent mass concentration (4.8 μg/mL) was used for all nucleic acids.
Fig. 2.
Fig. 2.
MDA5 forms filamentous oligomers along the length of dsRNA. (A) Representative electron micrographs of negatively stained MDA5 in complex with 112 and 512 bp dsRNA in the presence of ADP•AlF4. Representative particles used for averaging are circled in red. See Fig. S2C for electron micrographs of MDA5 in complex with ssRNA and dsDNA. (B) Representative class averages of MDA5 in complex with 112 bp dsRNA. The red box in the magnified view corresponds to a minimum binding unit of MDA5 in EMSA (C). The number of binding units per complex (nb) is displayed at the bottom left for each average. (See also Fig. S2D.) (C) EMSA of 62, 112, and 162 bp dsRNAs with MDA5 (0.3 μM) visualized with SybrGold stain to enhace the intermediate complexes that are otherwise difficult to detect. Enumeration of intermediate complexes reveals that the full complex contains 4, 7–8, and 11 binding units of MDA5 for 62, 112, and 162 bp dsRNAs, respectively. (D) Multiangle light scattering (-OD280 and ▪ Mr estimation) suggests that the molecular mass of the full complexes formed on 62 and 112 bp dsRNAs are 854 ( ± 43) kDa and 1,878 ( ± 13) kDa, respectively.
Fig. 3.
Fig. 3.
Intrinsic ATP hydrolysis activity of MDA5 is independent of filament length or catalytic activity of neighboring molecules. (A) Relative rates of ATP hydrolysis (mean ± SD, n = 3) of MDA5 (4 or 0.3 μM) with and without 15, 112, and 512 bp dsRNAs (0.6 or 40 μg/mL, equivalent to 0.16 or 10.7 μM MDA5 binding site), with respect to 15 bp rates. (B) Relative rates of ATP hydrolysis of mixtures of WT MDA5 and K335A or D443A at indicated ratios with total MDA5 concentration fixed at 0.3 μM. The protein mixture was incubated with 40 μg/mL (10.7 μM MDA5 binding site) of 15 or 512 bp dsRNA. Rates were normalized against that with 100% WT.
Fig. 4.
Fig. 4.
Disassembly of MDA5 filament is both ATP and RNA length-dependent. (A) Time-dependent EMSA monitoring of MDA5∶RNA complex dissociation. The reaction was initiated by adding a mixture of 2 mM ATP (or ADPCP or no ligand) and 200 μg/mL heparin to MDA5 (1.35 μM) in complex with 512 bp dsRNA (1.2 μg/mL, 0.32 μM MDA5 binding site), quenched on ice at indicated time points and analyzed on native gel. (B) Single-round ATP hydrolysis kinetics (mean ± SD, n = 3) of WT MDA5 (0.1 μM) with and without K335A or D443A (0.1 μM) bound to 512 bp dsRNA (1.2 μg/mL) with and without heparin (hep, 200 μg/mL). (C) Time evolution of the ATP hydrolysis rate derived from the single-round kinetics (Fig. S5F) of WT MDA5 (0.2 μM) bound to 512, 1,012, and 2,012 bp dsRNAs (1.2 μg/mL) using the finite difference method. The rates were normalized against the initial rate during the first 15 s. (D) Single-round ATP hydrolysis rate of WT MDA5 (0.1 μM) with K335A or D443A (0.1 μM) bound to 512, 1,012, and 2,012 bp dsRNAs (1.2 μg/mL) in the presence of heparin (200 μg/mL) during 4–10 min. No ATP hydrolysis was observed without the mutants during this time period.
Fig. 5.
Fig. 5.
SNP I923V forms shorter filaments and exhibits decreased kinetic stability. (A) Cartoon representation of MDA5 CTD [Protein Data Bank (PDB) ID code 2RQB]. Residue I923, distal to the Zn coordination site but proximal to the C terminus, is partially solvent-exposed. (B) EMSA binding curves (mean ± SD, n = 2) of WT and I923V MDA5 with 112 bp dsRNA. (C) Representative class averages of I923V in complex with 112 bp dsRNA. The number of binding units (nb) present for each complex is displayed at the bottom right. See also Fig. S6D. (D) Histogram of the length distribution of complexes formed by WT and I923V with 112 bp dsRNA. Each length represents the median value of each interval. The number of binding units per complex was estimated from the complex length. (E) Single-round ATP hydrolysis kinetics (mean ± SD, n = 3) of WT and I923V (0.2 μM) bound to 2,012 bp dsRNAs (1.2 μg/mL) in the presence of heparin (200 μg/mL). (F) Time evolution of the ATP hydrolysis rate derived from E using the finite difference method. Rates were normalized against the initial rate during the first 15 s.
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References

    1. Wilkins C, Gale M., Jr. Recognition of viruses by cytoplasmic sensors. Curr Opin Immunol. 2010;22:41–47. - PMC - PubMed
    1. Rehwinkel J, Reis e Sousa C. RIGorous detection: Exposing virus through RNA sensing. Science. 2010;327:284–286. - PubMed
    1. Loo MY, et al. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol. 2008;82:335–345. - PMC - PubMed
    1. Kato H, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 2006;441:101–105. - PubMed
    1. Baum A, Sachidanandam R, Garcia-Sastre A. Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing. Proc Natl Acad Sci USA. 2010;107:16303–16308. and erratum (2010) 108:3092. - PMC - PubMed

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