Comparative Structure and Function Analysis of the RIG-I-Like Receptors: RIG-I and MDA5

Abstract

RIG-I (Retinoic acid-inducible gene I) and MDA5 (Melanoma Differentiation-Associated protein 5), collectively known as the RIG-I-like receptors (RLRs), are key protein sensors of the pathogen-associated molecular patterns (PAMPs) in the form of viral double-stranded RNA (dsRNA) motifs to induce expression of type 1 interferons (IFN1) (IFNα and IFNβ) and other pro-inflammatory cytokines during the early stage of viral infection. While RIG-I and MDA5 share many genetic, structural and functional similarities, there is increasing evidence that they can have significantly different strategies to recognize different pathogens, PAMPs, and in different host species. This review article discusses the similarities and differences between RIG-I and MDA5 from multiple perspectives, including their structures, evolution and functional relationships with other cellular proteins, their differential mechanisms of distinguishing between host and viral dsRNAs and interactions with host and viral protein factors, and their immunogenic signaling. A comprehensive comparative analysis can help inform future studies of RIG-I and MDA5 in order to fully understand their functions in order to optimize potential therapeutic approaches targeting them.

Keywords: CARD; MDA5; PAMP; PRRs; RIG-I; antiviral; inflammation; interferon.

Figure 1

RIG-I/MDA5 signaling pathway RIG-I and MDA5 are first activated by recognition of PAMP dsRNA, which causes them to interact with MAVS. Following the activation of MAVS by RIG-I/MDA5, a molecular cascade involves the interaction of IKKε and TBK1, which is followed by phosphorylation of the transcription factors IRF3 and IRF7, ensure to translocate the phosphorylated p-IRF3 and p-IRF7 into the nucleus, where they dimerize and bind to transcription factor binding sites of the IFNα and IFNβ genes to activate their transcriptions. Expression and exportation of these genes into the cellular milieu trigger the IFN1 signaling cascade in an autocrine or paracrine fashion to induce expression of hundreds of interferon stimulated genes (ISGs) and inflammatory genes to confer antiviral resistance. RIG-I and MDA5 also activate the NF-κB pathway. RIG-I appears to act upstream of the canonical pathway, which results in the translocation of the two functional NF-κB units (p50 and p65) into the nucleus, while MDA5 appears to affect NF-κB expression independently from this pathway. Figure created using BioRender software.

Figure 2

Venn diagram comparing the signaling and functional similarities and differences between RIG-I and MDA5.

Figure 3

Organization and known structures of RIG-I and MDA5. (A,B) Graphic representing the domain organization of RIG-I (A) and MDA5 (B). (C–E) Known structures of human RIG-I and MDA5, with X-ray crystallography structures of RIG-I Hel-CTD (C) and MDA5-Hel-CTD (D) interacting with dsRNA, and CARD1/2 of RIG-I interacting with the MAVS CARD domain (E). In (C–E), the helicase domains are shown in red, the CTD in purple, RNA in green, the CARD1 domain in blue, the CARD2 domain in aquamarine and the MAVS CARD domain in orange (C) was adapted from reference (61), (D) from reference (62), and (E) from reference (63).

Figure 4

Activation mechanisms of RIG-I and MDA5. RIG-I and MDA5 are activated by interacting with viral dsRNA at the C terminal domain. In their endogenous and inactivated state, RIG-I and MDA5 are phosphorylated at their N and C terminal domains (A,F). MDA5 may exist between open and close forms in its inactivated state (F). Upon recognizing PAMP dsRNA, the C terminal domain becomes dephosphorylated and ubiquitinated for RIG-I and dephosphorylated for MDA5 (B,G). RIG-I also dimerizes (B). Next, RIG-I oligomerizes (D) and MDA5 forms longer filaments on dsRNA (H), and the N terminal CARD domains of RIG-I becomes dephosphorylated (C) then ubiquitinated (D). Finally, the CARD domain of the RIG-I oligomers interacts with the mitochondrial protein MAVS (E), and the MDA5 dsRNA filaments also activate MDA5 (though it has a weaker CARD-CARD interaction with MAVS) (I). Figure created using BioRender software.

Figure 5

RNA species that interact with RIG-I and MDA5. Table summarizes the general structural features of RNA species, their source during experimental studies and their ability to activate the ATPase functions of RIG-I and MDA5. RNA constructs are shown in green, and DNA constructs in purple.

Figure 6

RIG-I/MDA5 interactions with host and cellular proteins. Host proteins (shown in purple) and viral proteins (shown in orange) that modulate RIG-I and MDA5 signaling are shown. Figure created using BioRender software.

Figure 7

Evolutionary timeline of RIG-I, MDA5, and other related DExD/H-box helicases. The evolutionary tract of RIG-I, MDA5, and related DExD/H-box helicases are shown as a phylogenetic tree, along with their lowest level of biological taxonomy that these proteins are found in present day. In short, the precursor of the MDA5 helicase-CTD likely originated from a common ancestor with the precursor for LGP2, which was then duplicated to create the helicase-CTD precursor of RIG-I in the common ancestor of vertebrates. CARD2 was then grafted onto the helicase-CTD protein, and this protein was duplicated to create the CARD2-helicase-CTD precursor of MDA5. Finally, CARD1 was grafted onto these proteins in separate events, forming the modern-day RIG-I and MDA5.