Non-coding RNAs and RIG-I/MAVS signaling pathway: Insights for targeted therapy in influenza infection

Influenza viruses are a significant challenge to the health of the global population, and are responsible for millions of severe cases and hundreds of thousands of deaths each year. The outbreaks of pandemics can result from the rapid antigenic drift and shift of the virus, which can also affect the effectiveness of the existing vaccines. Despite the availability of antiviral drugs like neuraminidase inhibitors, the rise in resistant strains is indicative of the urgent need for novel therapeutic strategies with distinct mechanisms of action. Developing treatment approaches that target host-directed pathways essential for viral replication and the immune response could be a pivotal step in mitigating this issue. The key pathway central to this strategy is the innate immune-sensing pathway mediated by retinoic acid-inducible gene I (RIG-I) and its adaptor protein mitochondrial antiviral-signaling protein (MAVS).^[1] This axis serves as the first line of intracellular defense, recognizing viral RNA (vRNA) and triggering a potent antiviral state through the induction of type I interferons (IFN-I) and hundreds of interferon-stimulated genes (ISGs). The fundamental function of the RIG-I/MAVS pathway in regulating influenza A and B virus replication in the respiratory epithelium is well-established.^[2] However, the activation of this pathway is complex and not merely a simple on/off switch. To play its role in eradicating the virus without causing excessive inflammation and immunopathology, it must be succinctly and tightly regulated. Notably, a diverse array of non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), has been identified as key regulators of this delicate balance.^[3] This editorial highlights the complex regulatory networks orchestrated by ncRNAs on the RIG-I/MAVS signaling hub during influenza infection and discusses their translational potential as targets for next-generation, host-directed antiviral therapies.

The RIG-I/MAVS Pathway: A Central Hub and Antiviral Battleground

During infection, the vRNA of the influenza virus is recognized by the RIG-I receptor of the host cell. The RIG-I binds to the viral double-stranded RNA via its C-terminal helicase domain, thus triggering a conformational change that exposes its N-terminal caspase activation and recruitment domain (CARD). Subsequently, the E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) mediates K63-linked ubiquitination of the CARD domains. The ubiquitinated RIG-I activates the mitochondrial adaptor MAVS using its CARD domain, thus triggering a cascade that mobilizes interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to produce IFN-I and cytokines, mounting a crucial antiviral defense. In addition, the RIG-I/MAVS pathway participates in antiviral innate immunity, as well as influences the host’s acquired immunological response. RIG-I signaling through MAVS is vital for generating strong, multi-functional T-cell responses to influenza A viruses (IAVs), which helps control initial infection, promotes immune memory, and provides broad protection against different IAV strains.^[2] The significance of this pathway is highlighted by the presence of evolved advanced mechanisms that influenza viruses have developed to directly inhibit RIG-I and TRIM25, particularly through their non-structural protein 1 (NS1), which significantly reduces the host’s IFN response.^[4] The host defense continuously counters viral infection, and the RIG-I/MAVS axis is the core pathway facilitating this. Furthermore, the host utilizes complex feedback systems to regulate this pathway and modulate host antiviral response, and ncRNAs play an important role during this process.^[3]

Diverse ncRNAs as Fine-Tuners of the Antiviral Immune Response

ncRNAs regulate the RIG-I/MAVS axis at multiple levels to ensure a balanced antiviral immune response while mitigating immune-mediated pathology. Their actions can be broadly categorized into proviral (suppressing immunity) or antiviral (enhancing immunity), though some exhibit context-dependent functions.^[3]

miRNAs: Precision tools for immune modulation

These small (~22 nt) RNAs primarily function by binding to complementary sequences in the 3’ untranslated region (UTR) of target messenger RNA (mRNAs), leading to transcript degradation or translational repression. The influenza virus exploits host miRNA expression to undermine antiviral immunity. This is achieved through specific mechanisms, such as miR-218 directly targeting the 3′UTR of RIG-I (DExD/H-box helicase 58, DDX58) mRNA, and miR-202-5p inhibiting the expression of TRIM25, both of which are critical components of the RIG-I/MAVS signaling pathway.^[5] By disrupting these processes, the virus effectively reduces the activation of the signaling cascade and facilitates immune evasion [Supplementary Figure 1, http://links.lww.com/CM9/D6]. Similarly, miR-122 and miR-125a/b are upregulated during infection and target MAVS mRNA, attenuating the downstream antiviral signal.^[6]

In contrast, the host can use miRNAs to strengthen its defense. For instance, miR-136 can function as an endogenous ligand for RIG-I, directly increasing its activation.^[7] In addition, miR-483-3p is transported via exosomes from infected cells and functions as a signaling molecule to activate the innate immune response in nearby cells.^[8] Moreover, miR-200c improves the stability of MAVS protein by reducing the activity of its negative regulators, contactin 1 (CNTN1) and ubiquitin specific peptidase 25 (USP25), demonstrating a method to strengthen the central signaling node. Collectively, these findings reveal a bidirectional link between the influenza virus and host miRNAs. The virus utilizes miRNAs to inhibit RIG-I/MAVS signaling as a means of evading the immune response, whereas the host employs other miRNAs to strengthen antiviral defenses via direct receptor activation, paracrine priming, or the stabilization of essential signaling components.

lncRNAs: Complex regulators with multifaceted roles

These are longer RNA transcripts (>200 nt) that regulate gene expression through diverse mechanisms, including acting as decoys, scaffolds, or guides. Their participation in the RIG-I/MAVS pathway introduces an additional layer of complexity. For instance, during viral infection, the lncRNA antivirus and activate neutrophil (AVAN) is induced, and this promotes the TRIM25-mediated ubiquitination of RIG-I by serving as a molecular scaffold, thereby enhancing IFN production [Supplementary Figure 1, http://links.lww.com/CM9/D6].^[9] Another regulatory mechanism is exemplified by MAVS antiviral-related lncRNA (MARL), which acts as a competing endogenous RNA (ceRNA) or “sponge” for miR-122. By sequestering miR-122, MARL prevents it from degrading MAVS mRNA, thereby up-regulating MAVS protein levels and promoting the antiviral state. In contrast, lnc-Lsm3b plays a crucial role as a feedback inhibitor in the late stages of infection. It has a stronger affinity for RIG-I than vRNA, keeping it in an inactive conformation and preventing sustained IFN production, which shields the host against severe inflammation.^[10] Thus, lncRNAs dynamically balance antiviral defense and immune homeostasis by both activating and suppressing the RIG-I/MAVS pathway at different stages of infection.

circRNAs: Stable mediators with direct and indirect functions

These covalently closed, non-polyadenylated RNAs are highly stable and can function as miRNA sponges, protein decoys, or even templates for translation. Their involvement in antiviral immunity is a promising and rapidly evolving field of study. Some unmodified circRNAs can be directly recognized by RIG-I, leading to the activation of the innate immune response. Particularly, it has been demonstrated that circular RNA vesicle-associated membrane protein 3 (circVAMP3) directly binds to and sequesters the viral nucleoprotein (NP) and non‑structural protein 1 (NS1) proteins to prevent influenza virus replication [Supplementary Figure 1, http://links.lww.com/CM9/D6]. This sequestration impairs the activity of the viral ribonucleoprotein complex and, as a secondary effect, relieves RIG-I from NS1-mediated inhibition, thereby restoring IFN signaling.^[4] In addition, circular phosphoinositide kinase FYVE-type zinc finger containing RNA (circPIKfyve) enhances the MAVS-mediated antiviral response by sponging miR-21-3p, a miRNA that normally suppresses MAVS expression. By neutralizing miR-21-3p, circPIKfyve upregulates MAVS levels, thereby strengthening the host’s ability to combat viral infection.

Targeting ncRNAs for Therapeutic Intervention: Strategies, Challenges, and Prospects

The intricate interplay between the influenza virus, host ncRNAs, and the RIG-I/MAVS signaling pathway presents a novel target for antiviral therapy. ncRNAs can precisely regulate key stages of the antiviral immune response. They can, however, either promote RIG-I/MAVS signaling to increase IFN production or decrease it to aid in viral immune evasion. Consequently, the potential therapeutic strategy (summarized in the Supplementary Table 1, http://links.lww.com/CM9/D6) involves using RNA-based drugs (such as mimics or inhibitors) to modulate specific ncRNAs. This will enhance beneficial immune responses while simultaneously suppressing excessive and detrimental inflammation.

Nevertheless, implementing this strategy in practice encounters two primary challenges. The first challenge is related to delivery. RNA therapeutics require carriers to achieve targeted delivery, cellular uptake, and endosomal escape. Current mainstream carriers like lipid nanoparticles (LNPs) exhibit liver tropism, making repeated pulmonary dosing difficult. Alternative carriers such as exosomes and polymeric nanoparticles offer greater targeting potential but face their own challenges, including production scalability, drug loading efficiency, and batch-to-batch consistency. The second challenge is specificity and safety. Hepatotoxicity, which is primarily a side effect of non-specific binding of RNA interference (RNAi) sequences rather than chemical toxicity, is a key off-target effect of RNAi therapeutics. This puts a great deal of pressure on the development of the precise design of guide RNAs.

In the future, multidisciplinary and collaborative innovation is needed to advance ncRNA-based treatments, with the primary goal of developing next-generation, lung-optimized delivery systems. For instance, modifying the surface proteins of exosomes may improve their ability to target the lungs, although issues concerning scalable manufacturing need to be addressed. Furthermore, it is important to incorporate systems biology and artificial intelligence (AI). Utilizing multi-omics data to map ncRNA regulatory networks and employing AI models to accurately predict and circumvent off-target risks will enable the rational design of these therapeutics. Furthermore, determining the optimal therapeutic “window” is crucial. Future research should identify dynamic biomarkers and utilize AI analysis to predict individual patient immune response trajectories, which can then facilitate the development of personalized treatment regimens.

Ultimately, clinical translation will likely depend on combination strategies, such as pairing ncRNA-targeting agents with direct-acting antivirals or immunomodulators. Progress in this field also necessitates the development of more predictive preclinical models that better replicate human lung physiology and immune responses. In conclusion, by converging advances in nanotechnology, bioinformatics, and immunology, targeting ncRNAs holds the promise of pioneering a new class of precision medicines against influenza and other respiratory viral infections.

Funding

This study was funded by the National Natural Science Foundation of China (Nos. 32560885 and 32260908), Guizhou Science and Technology Plan Project (No. QKHJC-ZK[2023]-Key057), Scientific Research Program of Guizhou Provincial Department of Education (No. QJJ [2023]019), and Science & Technology Program of Guizhou Province (No. QKHPTRC-CXTD[2022]014).

Conflicts of interest

None.

Supplementary Material

cm9-139-2848-s001.docx ^{(351.6KB, docx)}