The RLR Signaling Pathway

The RIG-I-like receptor (RLR) signaling pathway is a crucial component of the innate immune system that plays a central role in recognizing viral infections. RLRs are a family of pattern recognition receptors (PRRs) that are responsible for detecting viral RNA in the cytoplasm of infected cells. The primary members of the RLR family include Retinoic acid-inducible gene I (RIG-I), Melanoma differentiation-associated protein 5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2). Activation of RLRs initiates a signaling cascade that leads to the production of interferons and other antiviral molecules, contributing to the host defense against viral infections.

1. Recognition of Viral RNA: The RLR signaling pathway is activated by the recognition of viral RNA in the cytoplasm. RIG-I primarily recognizes short double-stranded RNA with 5′-triphosphate (5′-ppp) or 5′-diphosphate (5′-pp) ends, typically found in the genomes of RNA viruses. MDA5, on the other hand, detects long double-stranded RNA, a common feature in the replicative intermediates of RNA viruses.

LGP2, while lacking a signaling domain, plays a regulatory role by enhancing or inhibiting the signaling activity of RIG-I and MDA5. The binding of RIG-I or MDA5 to viral RNA induces a conformational change in the receptors, exposing their N-terminal caspase activation and recruitment domains (CARDs).

2. Activation of RLRs: Once activated by viral RNA, RIG-I and MDA5 undergo a conformational change that exposes their CARDs. The activated RLRs then interact with the mitochondrial antiviral signaling protein (MAVS), also known as IPS-1, Cardif, or VISA. MAVS is localized on the outer mitochondrial membrane and serves as a critical adaptor protein in the RLR signaling pathway.

RIG-I interacts with MAVS through its CARD domain, while MDA5 utilizes its CARDs and helicase domain for MAVS binding. LGP2, without a CARD domain, modulates the activity of RIG-I and MDA5 by competing for viral RNA binding.

3. MAVS-Mediated Signal Transduction: The interaction between activated RLRs and MAVS triggers the formation of a signaling complex on the mitochondrial membrane, initiating downstream signaling events. MAVS contains a C-terminal domain that recruits various signaling molecules to propagate the antiviral response.

• TRAF3 Activation: MAVS recruits TNF receptor-associated factor 3 (TRAF3), which becomes activated through ubiquitination. Activated TRAF3 then associates with and activates TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε).
• Activation of TBK1 and IKKε: TBK1 and IKKε are serine/threonine kinases that phosphorylate the transcription factors interferon regulatory factor 3 (IRF3) and IRF7. Phosphorylated IRF3 and IRF7 then form homo- and heterodimers and translocate to the nucleus, where they induce the transcription of type I interferons (IFN-α and IFN-β).
• NF-κB Activation: Simultaneously, MAVS activates another pathway involving the transcription factor NF-κB. TRAF3 also associates with and activates IKKα and IKKβ, leading to the phosphorylation and degradation of the inhibitor of NF-κB (IκB). This results in the release and translocation of NF-κB to the nucleus, where it induces the expression of pro-inflammatory genes, including cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α).

4. Interferon Response and Antiviral Defense: The primary outcome of the RLR signaling pathway is the induction of type I interferons (IFN-α and IFN-β). These interferons are crucial for establishing an antiviral state in infected and neighboring cells. Type I interferons activate the JAK-STAT signaling pathway, leading to the expression of interferon-stimulated genes (ISGs). ISGs encode proteins with various antiviral functions, including inhibition of viral replication, enhancement of antigen presentation, and modulation of immune responses.

The antiviral state induced by type I interferons is essential for limiting viral spread and coordinating adaptive immune responses. Additionally, the RLR signaling pathway contributes to the activation of natural killer (NK) cells and the recruitment of other immune cells to the site of infection.

5. Negative Regulation of RLR Signaling: To prevent excessive immune activation and maintain immune homeostasis, the RLR signaling pathway is subject to negative regulation. Several mechanisms exist to modulate RLR activity, including the action of ubiquitin-editing enzymes and the induction of negative regulators.

• Ubiquitin Editing: Ubiquitination of signaling molecules, such as TRAF3 and MAVS, is a reversible process controlled by ubiquitin-editing enzymes. Deubiquitinating enzymes, like CYLD, play a role in removing ubiquitin chains, thereby inhibiting excessive activation of the RLR signaling pathway.
• Negative Regulators: Several negative regulators, including cellular RNA helicases and ubiquitin ligases, regulate RLR signaling at various steps. For instance, the E3 ubiquitin ligase RNF125 targets RIG-I for ubiquitination and degradation, providing a mechanism for downregulating RLR activity.

Understanding the RLR signaling pathway is crucial for developing therapeutic strategies against viral infections and related immune disorders. Dysregulation of this pathway can contribute to chronic viral infections, autoimmune diseases, and inflammatory conditions. Ongoing research continues to uncover the intricate details of RLR signaling, offering potential targets for the development of antiviral therapies and immunomodulatory drugs.