Antiviral Innate Response

Antiviral innate responses are initiated by the cell following sensing of viral infection. The cell responds by producing antiviral cytokines and enzymes to shut down viral replication and enhance adaptive immune response.

RIG-I-like Receptors

RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation associated factor 5), and LGP2 (laboratory of genetics and physiology 2) belong to the RIG-I-like receptor (RLR) family, which are involved in intracellular virus recognition. A discrepancy between RLR receptors and Toll-like Receptors (TLRs), is that TLRs identify extracellular viruses, whereas RLRs recognize viral DNA/RNA that has already entered the cell. The three receptors are DExD/H box helicases and exhibit high primary sequence conservation within their helicase domains. RLRs detect double-stranded (ds) viral DNA or RNA. The RIG-I and MDA5 proteins contain N-terminal CARD (Caspase Recruitment Domain) signaling domain, which when activated, lead to the triggering of the antiviral signaling pathway. While LGP2 contains an RNA binding domain, it does not contain a CARD-domain Instead, the protein negatively inhibits RIG-I and MDA5. Other suppressors of RIG-I/MDA5 include Dihydroxyacetone kinase (DAK), A20, ring-finger protein 125 (RNF125), suppressor of IKKε (SIKE), and peptidyl-propyl isomerase 1 (Pin1).

RLR Activation

RLRs are activated via specific sequence patterns. Conserved motifs found in pathogens including virus particles are called pathogen-associated molecular patterns (PAMPs).  RIG-I and MDA5 can detect dsRNA and 5’ -triphosphate short dsRNA. Specific sequence composition of the RNA ligand has been shown to activate RIG-I-dependent signaling. “Self RNA” species are immune to detection by RIG-I-like receptors.

Once activated, RIG-I and MDA5 induce dimerization of a protein called MAVS (Mitochondrial Antiviral-Signaling Protein). MAVS dimerization subsequently activates TRAF3 (TNF Receptor-associated Factor 3), which recruits adapter proteins TANK, NAP1, and SINTBAD. TANK connects upstream RLR signaling to the TANK-binding kinase 1 (TBK1), inducing phosphorylation of IRF-3 (Interferon regulatory factor 3). IRF3 is a transcription factor involved in the type I interferon (IFN)- dependent immune response. IRF3 phosphorylation and dimerization cause the protein to translocate to the nucleus where it binds to the Interferon Stimulates Response Element (ISRE).

dsDNA induces antiviral gene expression through a distinct pathway, although some crosstalk exists. Cyclic GMP-AMP synthase (cGAS) responds to DNA binding by producing cyclic dinucleotide c-GMP-AMP (cGAMP). cGAMP binds to STING (Stimulator of interferon genes), activating TBK1-IRF-3-mediated IFN expression.

Activation of the RLR receptors can also induce NF-κB. For viral RNA sensing, RLR induces polymerization of MAVS and promotes binding of TRAF2, TRAF5, and TRAF6. The activity of the TRAF proteins is controlled by NLRX1 (NLR Family Member X1). Deubiquitination of OTUB1/2 (Ubiquitin thioesterase) regulates ubiquitination of TRAF2/5/6 and is required for association with MAVS. TRAF2/5/6 recruit NEMO and the IκB kinases IKKα/IKKβ, which phosphorylate IκB, activating NF-κB signaling.

The role that RLRs play in regulating the adaptive immune response is not well studied and has been shown to vary from virus to virus. However, the induction of interferon and antiviral immune response genes following RLR signaling is clear.

RLR Dysregulation in Human Disease

Mutations or polymorphisms in RIG-I-like receptors have been implicated in a number of diseases. Increased expression of type I IFN has been shown in patients with systemic lupus erythematosus (SLE) and Aicardi–Goutières syndrome (AGS).On the other hand, deficiencies in the production of IFN, lead to an increase in susceptibility to viral infection. RIG-I is known to recognize RNA viruses such as Japanese encephalitis virus (JEV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), Sendai virus and influenza virus. On the other hand, MDA5 is able to sense picornavirus and encephalomyocarditis virus (EMCV).

Modulation of the innate immune response is a key driving force of cancer immunotherapy. Toll-like receptors (such as TRL-1, TRL-2, TRL-3 TRL-4, TRL-5, TRL-6, TRL-7, TLR-8 and TLR-9) are implicated in TRL signaling which, along with NOD-like receptors (NLRs), AIM-2-like receptors and RLRs are considered pattern recognition receptors (PRRs), which can detect not only invading pathogens, but intracellular ligands. This makes PRRs the predominant mode of defense against not only infectious pathogens but also cancer. PRRS trigger many signaling pathways such as NK- κB, type I IFN and inflammasome, leading to cytokine production and eventual DC (dendritic cell) maturation. RIG-I, in fact, may act as a tumor suppressor gene and has been shown a decreased expression in many cancer types including skin and colorectal cancer. PRRs represent an important potential means to systematically modulate immunotherapy as a treatment to cancer.

Latest Progress for RLRs

In a study performed by Zhou and colleagues (2018), a protein called MLL5, which is a lysine methyltransferase involved in cell cycle progression, was shown to repress the RIG-I-mediated antiviral immune response. MLL5 deficient (MLL5-/-) mutant mice were generated using CRISPR/Cas9. Both MLL5-/- and wild-type mice were exposed to different PAMP ligands. Quantitative reverse transcription PCR (qRT-PCR) was used to quantify type I IFN and cytokine mRNA expression. They saw that MLL5-/- mutants had a higher expression of Ifn-β, Tnf-α, and Il-6 in comparison to wild-type following exposure to certain PAMP ligands. Additionally, they showed that MLL5 defect led to an accumulation of the RIG-I protein in both human cells (in vitro) and mouse (in vivo). Evidence that epigenetics plays an important role in the immune and inflammatory response is becoming increasingly stronger. This evidence opens avenues into new therapeutics to modulate innate immune response to viral infection. 

References

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Y.-M., & Gale, M. (2011). Immune signaling by RIG-I-like receptors. Immunity, 34(5), 680–692. https://doi.org/10.1016/j.immuni.2011.05.003

Yoneyama, M., & Fujita, T. (2007). Function of RIG-I-like Receptors in Antiviral Innate Immunity. Journal of Biological Chemistry, 282(21), 15315–15318. https://doi.org/10.1074/jbc.R700007200

Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., … Fujita, T. (2005). Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. Journal of Immunology (Baltimore, Md.: 1950), 175(5), 2851–2858.

Zhou, P., Ding, X., Wan, X., Liu, L., Yuan, X., Zhang, W., … Zhang, Y. (2018). MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation. Nature Communications, 9(1), 1243. https://doi.org/10.1038/s41467-018-03563-8