RIG-I (Retinoic Acid Inducible Gene-I)
RIG-I (retinoic acid-inducible gene-I, DDX58) is the founding member of the family of RIG-I like receptors (RLRs) that have been demonstrated to have a role in antiviral immunity. It was first reported in Pig by Zhang et al. as a helicase induced during reproductive and respiratory syndrome virus replication (Zhang et al. 2000). In 2004, Yoneyama et al. identified RIG-I as a sensor of viral RNA using a cDNA library screen of molecules that potently enhanced type-I IFN production in response to dsRNA molecules (Yoneyama et al. 2004). RIG-I is a cytoplasmic pattern recognition receptor that belongs to the class of ATP-dependent helicases. This class also includes MDA5 and LGP2. RIG-I is described to have three different domains each with different functions. It has a tandem CARD domain at the N-terminus, a central helicase/ATPase domain, and a C-terminal domain (CTD). The C-terminal domain or repressor domain (RD) recognizes uncapped 5′-tri-phosphorylated double-stranded RNA molecule produced during viral replication. The processes of capping and modifications of nucleotides in eukaryotes occur as a post-transcriptional modification in the nucleus, before export of mRNAs into the cytosol, which allows RIG-I to distinguish it from foreign RNA. Studies involving modification of RNA 5′ triphosphate groups showed the abrogation of RIG-I signaling, and attenuated signaling was also observed if 5′ tri-phosphate groups are replaced with mono- or diphosphate groups of RNA. Recognition of viral RNA in the cytoplasm by RIG-I initiates an antiviral immune signaling cascade that induces upregulation of interferon-stimulated genes (ISGs), production of type I interferons, and activation of transcription factors IRF3, IRF7, and NF-kβ.
The host requires both innate and adaptive immune responses to protect it against invading pathogens. The innate defense uses a set of evolutionarily conserved molecules called as pattern recognition receptors (PRRs) expressed on and inside of the host cell that recognizes conserved structures on pathogens (Takeuchi and Akira 2010). The conserved structures of pathogens called MAMPs (microbial-associated molecular patterns) are composed of protein, carbohydrates, nucleic acids, or mixture of lipids and protein. The recognition of these structures by innate immune system allows it to differentiate itself from adaptive immune system, which recognizes foreign or “non-self” molecules presented by antigen-presenting cells. The receptors of innate immune system are classified into various different groups based on the location (surface, endosomal, or cytoplasmic) and recognition of their cognate ligands. In the cytosol, specific receptors that recognize viral nucleic acids in the host cells are classified as RIG-I, MAVS, and LGP2 (Yoneyama et al. 2015). As mentioned before, RIG-I was the first RLR to be identified (Zhang et al. 2000). Later on, its role in antiviral innate immunity was described and became the prototype for our understanding of RLR signaling (Yoneyama et al. 2004).
RIG-I is expressed in cells of both hematopoietic and non-hematopoietic origin that includes epithelial cells, fibroblasts, dendritic cells (DCs), and lymphocytes. Very little or no expression of RIG-I was reported in the pluripotent stem cells. The recognition of viral RNA by RIG-I results in the induction of type-I interferons in all cell types. The only exception among the DCs is plasmacytoid DCs, which does not induce type-I interferon signaling upon recognition of viral RNA by RIG-I (Kato et al. 2005). The expression of RIG-I is upregulated in cells infected with RNA viruses than compared to the resting cells. RIG-I expression is also upregulated after interferon therapy (Yoneyama et al. 2004; Sumpter et al. 2005).
RIG-I recognizes RNA from both single- and double-stranded RNA viruses such as herpes virus, influenza virus, paramyxovirus, and flavivirus (Chan and Gack 2016). Many recent studies have demonstrated that recognition of dsRNA by RIG-I involves two phases; the initial interaction is described as nucleation, in which the protein monomer interacts with dsRNA, and is followed by propagation of a protein filament along the dsRNA (Patel et al. 2013; Peisley et al. 2013). Filament formation results in the alignment and interaction of CARDs with the CARD region of MAVS (Hou et al. 2011; Xu et al. 2014). Current information suggests that the initiation of MAVS prion-like assembly is a central outcome of RNA recognition by the RLR proteins and is critical for the efficient activation of downstream antiviral signaling cascades.
Structure of RIG-I
The recognition of microbial-associated molecular patterns by pattern recognition receptors present at various locations in the host cell results in the induction of signaling pathway involved in the clearance of pathogen. During viral infections, the two intracellular sensors viz RIG-I and MDA5 play an important role in controlling and clearing the virus by inducing interferon responses. RIG-I after recognition of viral RNA initiates downstream signaling by interacting through their N-terminal caspase activation and recruitment domains (CARDs) with downstream adaptor MAVS also known as IPS-1 or Cardif or VISA (Kawai et al. 2005; Meylan et al. 2005; Seth et al. 2005; Xu et al. 2005). Upon recognition of 5′ triphosphate, of/or RNA molecule by the repressor domain of RIG-I lead to its activation, which then allows ATP-containing domain of RIG-I to hydrolyze ATP and change its confirmation. The activated RIG-I then allows its CARD domains to be exposed and undergo ubiquitination by E3 ubiquitin ligase (TRIM25) at various sites on the CARD domain. TRIM25 belongs to TRIM family of proteins, which are characterized by a conserved RING domain at the N-terminus followed by two B-box domains, coiled coil dimerization domains, and C-terminus SPRY domain (Rajsbaum et al. 2014). RIG-I binding to C-terminus SPRY domain of TRIM25 occurs through its CARD domains. The process of poly-ubiquitination helps RIG-I to form higher-order oligomers that will allow it to part ways with TRIM25. Once separated from TRIM25, RIG-I can interact with MAVS leading to the activation of downstream signaling. In addition, CARD domains of RIG-I also bind free K63 poly-ubiquitin generated by TRIM25 (Gack et al. 2007).
Downstream of MAVS and TRAF6 also regulates NF-κβ signaling by interacting with TRAF2 and TNF receptor-associated death domain (TRADD). TRADD exists in complex with Fas-associated death domain-containing protein (FADD) and the death domain kinase (RIP1). TRADD-FADD-RIP1 complex then recruits NEMO/IKKγ, which then activates IKKs. Activated IKKs then phosphorylates IκB subunit attached to NF-κβ. Phosphorylation of IκB induces its degradation, which then allows the translocation of NF-κβ to the nucleus leading to the production of pro-inflammatory cytokines (Fig. 2).
Regulators of RIG-I Signaling Pathway
The RIG-I signaling is either positively or negatively regulated by several molecules present upstream or downstream of the adaptor molecule MAVS. Several complex factors involved in the TLR signaling also regulate RIG-I signaling as a part of signal transduction pathways connected to the activation of NF-κβ and interferon regulatory factors. Among the factors that control RIG-I signaling, ubiquitin ligases play an important role. Ubiquitin ligases are involved at various steps, for e.g. TRIM25 controls RIG-I signaling after the binding of RIG-I ligand by promoting K63 linked polyubiquitation of the CARD domains. Similarly, RIPLET or REUL an E3 ubiquitin ligase interacts specifically to RIG-I also promote ubiquitination of CARD domain at three different amin oacid positions (Lysine154, 164 and 172) (Gao et al. 2009). In addition to TRIM25 and RIPLET, another ubiquitin ligase, TRAF3 (TNF receptor associated factor 3) also reported to play an important role in controlling the downstream activation of RIG-I dependent IRF3 activation during virus infection. Other ubiquitin and deubiquitin ligases like cIAP1, cIAP2, TRIAD3, OTUB1 and OTUB2, DUBA also regulate RIG-I signaling by modulating the function of ubiquitin ligases TRAF3 and TRAF6 (Li et al. 2010; Mao et al. 2010). Deubiquitin enzymes OTUB1 and OTUB2 negatively regulate RIG-I signaling by interacting with TRAF3 and TRAF6. Overexpression of these molecules inhibited IRF3 and NF-kb signaling (Li et al. 2010).
The transcription factor fork box head protein, FOXO1 and FOXO3 regulate antiviral immune signaling downstream of RIG-I by inhibiting the transcriptional activity of IRF3 and IRF7 (Lei et al. 2013 and Liu et al. 2016). Cellular FLIP long isoform protein (cFLIP) also controls RIG-I signaling by inhibiting the interaction of IRF3 with IFNb promoter and its co-activator protein (CREB binding protein) (Gates and Shisler 2016). Several other molecules like SEC14L1, a member of SEC14 like protein family controls RIG-I signaling by interacting with its N-terminal domain upon activation of RIG-I and prevent its interaction with downstream adaptor MAVS thus blocking the downstream signaling leading to the production of cytokines (Li et al. 2013). Over expression and knockdown of SEC14L1 in cell lines showed that it inhibits transcriptional activity of IFNβ promoter. The molecules protein kinase C-α⁄β and casein kinase II are critical regulators of RIG-I signaling as they control phosphorylation of various domain of RIG-I (Maharaj et al. 2012; Sun et al. 2011). ARF-like protein also regulates RIG-I signaling by binding to its C-terminal domain in a GTP dependent manner and prevents the interaction of RIG-I with its ligand (Yang et al. 2011). Besides phosphorylation, acetylation and SUMOylation also controls RIG-I signaling pathways.
Role of RIG-I in Antiviral Immune Responses and Cancer
RIG-I as discussed above is known to have an important role in controlling antiviral immune responses by recruiting several adaptor molecules that involve kinases, ubiquitin ligases, and transcription factors. However, aberrant RIG-I signaling or mutation in RIG-I itself could lead to the development of autoimmune diseases and nonfunctional antiviral immune responses.
Recognition of viruses by innate immune receptors pose a unique challenge as viruses contain few unique features that can differ from the host. In Hepatitis B virus (HBV) infection, TRIM25, an ISG member, controls ubiquitination of RIG-I and thereby interferon production during the development of chronic hepatitis B, which may result in liver cirrhosis and cancer. During HBV infection TRIM25 is downregulated and thus prevents the generation of robust antiviral immune signaling resulting in increased viral titers [Ref]. During hepatitis E virus infection (HEV), RIG-I uses interferon-independent signaling pathway to control viremia as shown in an in vitro cell culture-based assay. Further, overexpression of RIG-I activates interferon-stimulated genes that result in anti-HEV activity leading to their clearance (Xu et al. 2017). Studies performed with Japanese encephalitis virus (JEV) showed a crosstalk between RIG-I and STING (a DNA sensor). Genetic ablation of STING inhibited the activation of RIG-I-dependent interferon signaling upon recognition of ssRNA of JEV, resulted in reduced cytokine and chemokine production, and increased viral load (Nazmi et al. 2012).
RIG-I is recently shown as a therapeutic target in pancreatic cancer. In vitro activation of RIG-I with its cognate ligand resulted in the induction of apoptosis in pancreatic cancer cell lines. Dyeing cells when co-cultured with DCs showed enhanced activation by upregulating co-stimulatory molecules. Further, DC-T-cell co-culture experiment showed strong activation of cytotoxic T cells resulting in the killing of tumor specific target cells (Duewell et al. 2014). In another study, use of ionizing radiations and chemotherapy for curtailing cancer showed promising result because of generation of RIG-I ligands that resulted in the death of tumor cells and promote strong adaptive immune responses. However, there are reports, which suggest that ionizing radiation has harmful effects during the treatment because of overactivation of RIG-I-like receptors resulted in cytokine storm. RIG-I is also considered as a therapeutic target for hepatocellular carcinoma, melanoma, and cervical cancers as it inhibits proliferation and cell cycle progression by blocking mitogenic MAPK activation and PI3K/AKT signaling pathway involved in cell survival. In a recent study, RIG-I deficiency is correlated with development of colitis-associated cancer due to disturbed microbial community, which is associated with reduced IgA production and Reg3γ expression (Houbao Zhu et al. 2017).
Conclusion and Future Perspectives
Recognition of foreign nucleic acids by intracellular sensor RIG-I represents an important aspect of intracellular receptors in controlling various pathological conditions. The importance of different adaptors of RIG-I signaling in antiviral immune response highlights a potential therapeutic target for controlling viral diseases. The crosstalk between RIG-I and the DNA sensor STING further allows for a better understanding of the function of RIG-I during cancer. Future studies on finding different interacting partners of RIG-I and an elaborate understanding of its involvement and regulation of the immune responses during viral and other diseases will allow the development of better therapeutic interventions.
- Duewell P, Steger A, Lohr H, Bourhis H, Hoelz H, Kirchleitner SV, Stieg MR, Grassmann S, Kobold S, Siveke JT, Endres S, Schnurr M. RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8+ T cells. Cell Death Differ. 2014;21(12):1825–37.PubMedPubMedCentralCrossRefGoogle Scholar
- Xu L, Wang W, Li Y, Zhou X, Yin Y, Wang Y, de Man RA, van der Laan LJ, Huang F, Kamar N, Peppelenbosch MP, Pan Q RIG-I is a key antiviral interferon-stimulated gene against hepatitis E virus dispensable of interferon production. Hepatology 2017. doi: 10.1002/hep.29105.