MDA5 assembles into a polar helical filament on dsRNA

Abstract

Melanoma differentiation-associated protein 5 (MDA5) detects viral dsRNA in the cytoplasm. On binding of RNA, MDA5 forms polymers, which trigger assembly of the signaling adaptor mitochondrial antiviral-signaling protein (MAVS) into its active fibril form. The molecular mechanism of MDA5 signaling is not well understood, however. Here we show that MDA5 forms helical filaments on dsRNA and report the 3D structure of the filaments using electron microscopy (EM) and image reconstruction. MDA5 assembles into a polar, single-start helix around the RNA. Fitting of an MDA5 homology model into the structure suggests a key role for the MDA5 C-terminal domain in cooperative filament assembly. Our study supports a signal transduction mechanism in which the helical array of MDA5 within filaments nucleates the assembly of MAVS fibrils. We conclude that MDA5 is a polymerization-dependent signaling platform that uses the amyloid-like self-propagating properties of MAVS to amplify signaling.

Fig. 1.

Image reconstruction of MDA5-dsRNA filaments. (A and B) Negatively stained electron micrographs of MDA5 bound to viral genomic dsRNA with ATP (A) and ATP-γ-S (B). (C) ΔCARD-MDA5 with ATP-γ-S. (D) Power spectrum from 9,154 overlapping segments of the ATP-γ-S filaments in B showing a strong meridional layer line (blue arrow) and a weak near-meridional layer line (red arrow). (E) Improved power spectrum after sorting by twist (1,362 segments). (F and G) Reconstructions from this subset (F) and from ΔCARD-MDA5 filaments with ATP-γ-S (G) revealing an asymmetric unit of four domains (labeled 1–4 in F) arranged as a ring around a central RNA cavity (4-σ contour). (H) At the 1-σ contour level, two lobes of density extend from domain 1 in full-length MDA5 (gray) but not in ΔCARD-MDA5 (blue), identifying the approximate position of the CARDs or the CARD–Hel1 linker (black arrows). A difference map shows density at the same two positions (Fig. S2).

Fig. 2.

Atomic model of the MDA5-dsRNA filament with ATP-γ-S present. (A) Domain organization of ΔCARD-MDA5. (B) Atomic homology models fitted into the ΔCARD-MDA5 reconstruction. The C terminus of Hel1 (blue dot) may connect to CTD N termini in the same or adjacent rings (red dots), 20 Å and 38 Å away, respectively. (C and D) Ring contacts are formed by a Hel1–CTD interface (C) and a Hel1–Hel2 interface (with minor Hel1–Hel1 contacts) (D). Note that the C-terminal sequence of the CTD was modeled in an extended conformation (Materials and Methods). (E) The ring-invading Hel1-CTD model results in an intermolecular interface between the Hel2i domain (medium gray) and the CTD (cyan). One ΔCARD-MDA5 molecule is shown as a surface; adjacent molecules are shown as cartoons.