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Science 339 (6120): 690-693

Copyright © 2013 by the American Association for the Advancement of Science

Paramyxovirus V Proteins Disrupt the Fold of the RNA Sensor MDA5 to Inhibit Antiviral Signaling

Carina Motz1, Kerstin Monika Schuhmann2, Axel Kirchhofer1,*, Manuela Moldt1, Gregor Witte1, Karl-Klaus Conzelmann2, and Karl-Peter Hopfner1,3,{dagger}

1 Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
2 Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
3 Center for Integrated Protein Sciences, Munich, Germany.

Figure 1
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Fig. 1. The ssMDA5:PIV5 V complex. (A and B) Structure of the ssMDA5:PIV5 V core complex in ribbon representation with annotated secondary structures. The helical insertion domain 2B of ssMDA5 is shown in blue and the superfamily 2 (SF2) ATPase domain 2A in yellow. The CTD of PIV5 V protein is shown in red with Zn2+ ions in gray. PIV5 V protein and MDA5 form a 1:1 complex through interaction of core secondary-structure elements.


Figure 2
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Fig. 2. Structural details and analysis of the interaction interface. (A) Close-up views of the intermolecular ssMDA52A:PIV5 VCTD and the intramolecular PIV5 VNTD:VCTD interactions (indicated by dashed lines). Domain 2A and the NTD are shown in yellow, the CTD is shown in purple. (B) Structure-based alignments of the interface sequences of selected RIG-I–like receptors (left) and paramyxoviral V proteins (right) with highlighted conserved residues and motifs. Stars indicate residues involved in interface interactions; nonfilled stars indicate residues mutated in this study. Species abbreviations are as follows: Ap, Anas platyrhynchos; Gg, Gallus gallus; Hs, Homo sapiens, Mm, Mus musculus; Ss, sus scrofa. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (C) Western-blot analysis of coimmunoprecipitation experiments (Co-IP) performed with flag-tagged hsMDA5 or hsRIG-I and MV V protein (n = 3). EV, empty vector. Left: Mutations affecting the salt-bridge formation between the conserved glutamate of V protein and arginine of MDA5. Right: Mutation of RIG-I to mimic the critical MDA5 arginine. (D) Comparison of RIG-I2A (PDB code: 3TBK), MDA52A:PIV5 VCTD, and full-length PIV5 V (PDB code: 2HYE).


Figure 3
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Fig. 3. Impact of V protein on the ATPase- and the RNA-binding activity of MDA5. (A) The ATPase activity of hs{Delta}CARD-MDA5 (100 nM final) in response to MV V protein. Data represent the mean ATPase activity (±SEM) determined by analyzing the initial linear slopes of the ATPase reactions (n = 3). (B) RNA-binding affinities analyzed by electrophoretic mobility–shift assays of mmMDA5 in the presence and absence of PIV5 V protein (n = 3). The accumulation of a faster-migrating species is marked by a star. (C) mmMDA5 filament formation on poly(I:C), visualized by negative-stain EM in the presence and absence of ATP and PIV5 V protein. (D) Proposed model for inhibition of MDA5 signaling assemblies by paramyxoviral V proteins (29, 30). dsRNA-bound MDA5 filaments might interact with MAVS and induce the formation of MAVS fibrils postulated to be active signal entities (17). V proteins disrupt the MDA5-SF2 architecture by a β-strand–replacement mechanism accompanied by double-unfolding of both V protein and MDA5, resulting in disruption of MDA5:dsRNA filaments and hence of signal transmission of the antiviral response. The structure and interactions of the NTD in the MDA5 complex are only illustrated here and need to be addressed in future studies.


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