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Abstract

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RIG-I like receptors and their ligands

RIG-I (retinoic-acid inducible gene I) is the best characterized receptor within the RLR (RIG-I-like receptor family) family. Together with MDA5 (melanoma differentiation-associated 5) and LGP2 (laboratory of genetics and physiology 2), this family of receptors are involved in the cellular front line defense against viral infections. As soluble pattern recognition receptors (PRRs) that are located in the cytoplasm of the cell, the RLRs are sentinels for intracellular viral RNA that is a product of viral infection. The RIG-I receptor prefers to bind single- or double-stranded RNA carrying an uncapped 5’ triphosphate and additional motifs such as poly-uridine rich RNA motifs (Saito nature 2008 523). RIG-I triggers an immune response to RNA viruses from various families including the Paramyxoviruses (e.g. measles), Rhabdoviruses (e.g. Vesicular Stomatitis virus) and Orthomyxoviruses (e.g. influenza A) (Baum et al, 2010; Gitlin et al, 2006; Hornung et al, 2006; Schlee et al, 2009; Wang & Ryu, 2010). The viral RNA preferred by MDA5 is poorly characterized, but MDA5 is essential in the detection of double stranded RNA from Picornaviruses (e.g. encephalomyelitis virus) (Kato et al, 2006). Although RIG-I and MDA5 respond to distinct viruses, there are cases where their specificity overlaps. For example, both RIG-I and MDA5 respond to dengue virus and West Nile virus, which are both Flaviviruses (Fredericksen et al, 2008; Loo et al, 2008; Nasirudeen et al, 2011), and their role, at least in West Nile virus infection is complementary (Errett et al, 2013). The ligands of LGP2 are also poorly characterized, but LGP2 prefers double-stranded RNA over single-stranded RNA (Murali et al, 2008).

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Structural features of the RLR receptors

The RLR receptors share a common domain architecture. All three receptors are members of the DExD/H box helicase family that is characterized by a core catalytic helicase domain made up of two RecA-like domains. The catalytic core contains at least 9 highly conserved sequence motifs that coordinate ATP and RNA binding and the hydrolysis of ATP to unwind RNA. A C-terminal domain (CTD) follows the helicase core and this domain also binds viral RNA. Distinct RNA-binding loops within the CTD of three RLRs confer dictate the type of RNA they can bind (Takahasi et al, 2009). In addition to the helicase core and CTD, RIG-I and MDA5 have two N-terminal CARD (caspase active recruitment domains) domains that are essential to the initiation of downstream signaling. LGP2 is dissimilar to RIG-I and MDA5 as it lacks the CARD signaling domains and this has implications for its function.

RIG-I antiviral signaling

In uninfected cells that are absent of viral RNA RIG-I exists in an inactive conformation in which the CARD domains are masked due to their interaction with the CTD (Luo et al, 2011). Upon binding RNA RIG-I changes into a conformation in which the CARD domains are exposed and ‘available’ for signaling. As an additional safeguard, these exposed CARDs can undergo modifications (e.g. ubiquitination, phosphorylation) that either positively or negatively regulate RIG-I signaling. In the activated state the exposed RIG-I CARD domains interact with the CARD domains of MAVS (mitochondrial anti-viral signaling protein, also known as IPS-1, VISA or Cardif) which sits on the outer surface of the mitochondria. This binding event is essential to signaling as it causes MAVS to form large functional aggregates which activate the transcription factors interferon regulatory factor (IRF)-3, IRF7 and NF-kb (nuclear factor kappa B) which leads to the production of proinflammatory cytokine and type I and type III interferons (IFN). Although secreted IFNs are in themselves antiviral agents, they prompt the production of more antiviral agents. The type I IFNs (IFNa and IFNb) accomplish this by binding cell surface type I IFN receptors to activate JAK-STAT (Janus kinase/signal transducers and activators of transcription) signaling. Overall this causes the death of infected cells, the protection of surrounding cells and the activation of the antigen-specific antiviral immune response. Collectively this coordinated antiviral immune response controls the viral infection.

Unlike RIG-I MDA5 is uninhibited in the absence of RNA and exists in an extended conformation (Berke & Modis, 2012) in which the CARDs are unhindered. However, as per RIG-I, the CARDs of MDA5 are modified as a means of regulating signaling via MAVS. Given LGP2 lacks CARD signaling domains it is unable to bind MAVS to initiate signaling. Instead, LGP2 is both a positive and negative regulator of RIG-I and MDA5, and consequently signaling (Bruns et al, 2014; Childs et al, 2013; Komuro & Horvath, 2006; Parisien et al, 2018; Saito et al, 2007; Satoh et al, 2010; Uchikawa et al, 2016)

Regulation of RLR signaling

As prolonged IFN production is linked to human disease, RLR signaling must be tightly regulated. One of various ways that this is achieved is by modifying or tagging host RLR signaling proteins with ubiquitin, a small 8 kDa protein. This process is called ubiquitination and is carried out by enzymes known as E3 ligases. These tags can also be removed, which adds an additional regulatory layer to RLR signaling. These modifications, and their removal, are prevalent in RLR signaling and even regulate the RIG-I receptor itself. Most famously the ubiquitination of the exposed RIG-I CARDs by the E3 ligase TRIM25 is essential to the induction of the RLR-mediated antiviral immune response (Gack et al, 2007). This modification is so pertinent to the success of RLR signaling that it is targeted by the influenza A virus.

Viral hijacking of RLR signaling

Viruses have evolved ways to subvert RLR signaling to enhance their survival. For example, influenza A uses its NS1 (nonstructural protein 1) protein to block TRIM25 from ubiquitinating RIG-I and this in turn inhibits IFN production (Gack et al, 2009). This outcome is also achieved by the hepatitis C NS3/4A protein by removing a part of MAVS (Li 2005 PNAS 17717) and the foot-and-mouth disease Leader protease (Lpro) which cleaves LGP2 (Rodriguez Pulido et al, 2018). Another prominent example is that of the Paramyxovirus V proteins, which directly bind various RLR signaling proteins including MDA5, LGP2, and STAT (Andrejeva et al, 2004; Childs et al, 2007; Rodriguez & Horvath, 2014)

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