Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting

Abstract

Type I Interferons (IFNs) are requisite components in antiviral innate immunity. Classically, a Toll-like receptor-dependent pathway induces type I interferons. However, recent recognition of melanoma differentiation associated gene-5 (MDA-5) and retinoic acid inducible gene-I (RIG-I) as primary sensors of RNA viruses for type I interferon induction highlights a potentially unique pathway for innate immunity. Our present investigation tracing the phylogenetic origin of MDA-5 and RIG-I domain arrangement (CARD1-CARD2-helicase-DEAD/DEAH) indicates that these proteins originated specifically in mammals, firmly linking this family of proteins with interferons in a highly derived evolutionary development of innate immunity. MDA-5, but not RIG-I, orthologs are found in fish, indicating that MDA-5 might have evolved before RIG-I. Our analyses also reveal that the MDA-5 and RIG-I domain arrangement evolved independently by domain grafting and not by a simple gene-duplication event of the entire four-domain arrangement, which may have been initiated by differential sensitivity of these proteins to viral infection.

Fig. 1.

Diagrammatic representation of two possible pathways for the creation of linked but divergent CARD domains. (A) Domain structure of MDA-5 and RIG-I. Red box, CARD1; blue box, CARD2; green box, helicase domain; purple box, DEAD/DEAH domain. The numbers represent amino acid positions. (B) (Left) A simple domain duplication event to produce two linked domains. In this scenario, the two domains would be expected to have similar evolutionary histories. (Right) A more complicated pathway where linked domains, produced by independent and divergent evolution (dotted lines) of duplicated domains originally not in linkage, fuse to form linked domains. This second scenario would produce domains with differing evolutionary histories.

Fig. 2.

Relationships of the helicase-DEAD/DEAH domains. (A) Phylogenetic tree showing the relationships of helicases. See text for details. Accession numbers for all sequences used in this tree are in Table S1. See Fig. S2 for a magnification of this tree to observe the gene names. (B) A magnification of the blue clade from (A). This is the clade circumscribing the MDA-5 and RIG-I helicase domain families. Accession numbers for all sequences used in this tree are in Table S1. (C) Diagram showing the best-supported pathway for generating the helicase/DEAD domain structure in MDA-5 and RIG-I. The colors refer to colors of clades in A. LRM stands for LGP2/RIG-I/MDA-5. Eukaryotic ancestor1 indicates an ancestor where a duplication produced the helicase/DEAD/DEAH structure and a common ancestor of all other helicases. Eukaryotic ancestor2 indicates an ancestor where a duplication occurred that produced the DEAD RNA helicases and all other helicases. Solid black lines indicate duplication events. Colors of major hel-DEAD families correspond to color labels of genes in the trees in A.

Fig. 3.

Phylogenetic tree showing the relationships of the CARD boxes examined in this study. Accession numbers of genes used to generate this phylogeny are available in Table S2. The numbered stars refer to nodes in the tree where jackknife and Bayes probabilities were calculated and are summarized in Table 2.

Fig. 4.

Diagram showing the best-supported pathway for generating the CARD1 and CARD2 domains and ultimately the MDA-5 and RIG-I proteins. Abbreviations: csp, caspase; cdf, CARDif. Solid black lines indicate duplication events. Dotted lines indicate fusion events that we infer from the CARD phylogenetic analysis in Fig. 3. Colors correspond to domain family labels in Fig. 3. Alternative scenarios with fewer fusion events are less likely to represent the evolutionary history of MDA-5 and RIG-I, as explained in Fig. 5.

Fig. 5.

Cartoon showing two alternative pathways for generating the CARD1 and CARD2 domains and ultimately the MDA-5 and RIG-I proteins. (A) Dotted lines indicate fusion events that we infer from the CARD phylogenetic analysis in Fig. 3. Colors correspond to domain family labels in Fig. 4. This scenario suggests four fusion events, one each for the MDA-5 CARD1 and CARD2 boxes and the RIG-I CARD1 and CARD2 boxes. This scenario is less preferred because it infers one extra fusion event that cannot be supported by the phylogeny in Fig. 3. (B) This scenario suggests that MDA-5 and RIG-I CARD2 boxes have coevolved tightly after their fusion and before the duplication of MDA-5 and RIG-I whole genes. This scenario is less preferred because it suggests tight coevolution of CARD1 and CARD2 domains, which are clearly refuted by Table 1. Abbreviations: cdf, CARDif; csp, caspase. Solid black lines indicate duplication events.