Structural Biochemistry/RIG-I-like Receptors (RLR)

The RIG-I-like receptor family (RLRs) is comprised of RIG-I (retinoic acid inducible gene 1), MDA5 (melanoma differentiation associated gene 1), and LGP2 (laboratory of genetics and physiology 2) which functions as an intracellular PRR (pattern recognition receptor) sensor that act as the first line of defense against RNA viruses through the detection of viral replication by direct interaction with dsRNA. These RLRs are in most tissues that initiate immune activation in cell types such as myeloid cells, epitheal cells, and cells in the central nervous system. They are able to control infections by detecting RNA located in the cytoplasm through immunity and inflammation. RLRs usually exist in low levels, but increase when exposed to IFN and viral infections. The RLR family or SF2 subfamily is also related to mammalian Dicer and motor proteins involved in gene silencing.

Structure


RIG-I is a 925 residue, 106kDa protein composed of two N-terminal tandem caspase activation and recruitment domains (CARD), a Zn2+-containing regulatory C-terminal domain (CTD), and a central DECH-box RNA helicase. N- and C-terminal RecA-like domains (Hel1 and Hel2 respectively) contain conserved sequence motifs implicated in ATP/nucleic acid binding and ATP hydrolysis, indicating that RIG-I functions as a dsRNA-dependent ATPase. RIG-I and MDA-5 share a conserved helicase core as well as similar signaling pathways and adaptor molecules whereas LGP2 lacks N-terminal CARDs.

RIG-I surrounds viral RNA and encloses it within the central cavity of the protein. Hel1 domain contains seven α helices and seven β strands that face the minor groove of the RNA and binds the RNA backbone of the dsRNA. The insert domain of Hel2 (Hel2i) interacts with the minor groove in the RNA backbone and also plays an important role in RNA recognition through Q511 and 2’OH of G5 residue on RNA bottom strand.

The CTD is most notable for its 5’triphosphate (5’-ppp) electrostatic binding via RNA loop-binding groove. The helicase interacts with the 3’ strand while CTD interacts mainly with the 5’ extremity, which includes 5’ppp. CTD is flexibly linked to the helicase and without strong interactions with the remainder of the protein, it acts as a sensor for 5’ppp dsRNA due to its higher affinity and longer off-rate for 5’ppp dsRNA compared to 5’OH ds-RNA.



Since the RNA binding occurs via 3 separate protein domains, large conformational changes are induced upon binding to dsRNA. Free RIG-I has an extended, multipart shape that collapses into a compact variant upon binding. Oligomerization of RIG-I is thought to be essential for activation and is triggered by RNA binding and dependent on dsRNA length.

Autoinhibited RIG-I is held in closed configuration by casein kinase II phosphorylation and via interactions with repressor domain in the C-terminus, prevents CARD binding. Engagement of 5’ppp RNA and K63-linked polyubiquitination through TRIM25 and RING finger protein allows for RIG-I activation through helicase/CTD enclosure of RNA and the outward exposure of CARD.

Receptor-Ligand Interaction
RIG-I is activated by both positive-strand and negative-strand RNA viruses such as Rabdoviridae, Orthomysoviridae, Paramyxoviridae, and Hepacivirus genera. In vitro activation of RIG-I requires either blunt-ended, base-paired region of 18-20 nucleotides with a 5’ppp end or dsRNA of longer length (>200bp). RNA binding to RIG is thought to be mediated by both the CTD and helicase domain. The presence of 5’PPP end on RNA substrates acts as a non-self marker or pathogen-associated molecular patterns (PAMPs) that can be differentiated from autoantigens. The CTD binds blunt-end 5’ppp-dsRNA and induces conformational changes that exposes CARDs and allows polyubiquitination of Lys 172 via E3 ligase, which recruits IPS-1 adaptor and allows for induction of type I IFN production.

Sequence composition of RNA ligands may also contribute to activation of RIG-I-dependent signaling as well. Preferential IFN signaling has been noted in response to polyuridine motifs containing interspersed C nucleotides (poly-U/UC) such as that in hepatitis C viral genome. Also, RNA cleavage products generated by 2’, 5’-linked oligoadenylate-activated RNase L, which produce 3’ monophosphates instead of 5’ppp, can trigger RIG-dependent IFN production. It is thought that PAMP RNA sequence composition in tandem with 5’ppp are vital determinants for RIG-I binding.



Inflammatory Signaling
RLR signaling program relies on its recruitment of IPS-1 adaptor protein and assembly of IPS-1 signalosome that drives downstream activation of IFN transcriptional responses. IPS-1 associates with TNR-receptor-associated factor 3 (TRAF3) through its C-terminal TRAF domain. TRAF3 recruits and activates two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKε), which further phosphorylate IRF-3 and IRF-7. Phosphorylation of IRF-3 and IRF-7 induces formation of IRF homodimers/heterodimers which translocate to the nucleus and bind IFN-stimulated response elements (ISRE), resulting in expression of type I IFN genes and IFN-inducible genes. Also, FAS-associated death domain-containing protein (FADD) interacts with caspase-8, caspase-10, and IPS-1 which further activates NF-κB downstream and induces proinflammatory cytokines.

Regulation
Regulation of the inflammatory response is vital to prevent uncontrolled interferon production that may lead to autoimmunity or immune toxicity. RIG-I, an essential antiviral PRR, is regulated through various mechanism such as its C-terminal portion which contains a repressor domain and inhibits RIG-I signaling in steady state. LGP2, which may function as both a positive and negative regulator, encodes a functional repressor domain that can suppress RIG-I signaling through interactions with RIG-I and MDA5.

NLRX1, a member of the Nod-like receptor (NLR) family, acts as a negative regulator of RLR-induced antiviral response by disruption of IPS-1 interaction with RLR signaling. Furthermore, NLRC5, another member of the NLR family, interacts with RIG-I and MDA5 to inhibit IFN production by direct blockage of IKKα and IKKβ phosphorylation and its subsequent NF-κB transcriptional activation.

Posttranslational modifications such as ubiquitination and deubiquitination also control both positive and negative regulation events. TRIM25 mediates K63-linked polyubiquitination at Lys 172 and stabilizes interactions between RIG-I and IPS-1. K63-linked polyubiquitination binding is thought to act as a second ligand for RIG-signaling activation. However, phosphorylation at Ser 8 or Thr 170 residues inhibit the TRIM25-RIG-I interface. RNF125 is another ubquitin ligase that acts as a negative feedback loop in concert with E2 ligase HbcH5c to conjugate K48-linked ubiquitin to RIG-I for proteasomal destruction.