Abstract: Melanoma differentiation-associated gene 5 (MDA5) initiates type I interferon (IFN) production by detecting cytosolic viral RNA. Mammalian MDA5 is an IFN-inducible gene and controlled by IFN regulatory factor 1 (IRF1). Teleost MDA5 also induces type I IFN production in response to viruses, yet its regulation remains largely unexplored. This study used the large yellow croaker Larimichthys crocea (Lc) as a model organism and revealed that a type I IFN (LcIFNi) triggers the expression of LcMDA5 through the JAK-STAT signaling pathway, which involves phosphorylation of LcIRF11. LcMDA5 was transcriptionally regulated by LcIRF11. Mechanistically, LcIRF11 interacts with the IFN-stimulated response element (ISRE) within the LcMDA5 promoter, via α3 helix and loop1, and loop2 and loop3 in its DNA binding domain (DBD). Overexpression of LcIRF11 recruits p300 and RNA polymerase II (Pol II) to the LcMDA5 promoter region. Pull-down analysis further confirmed the interaction of LcIRF11 with these two proteins. This recruitment was accompanied by increased levels of histone H3K27 acetylation (H3K27ac) and histone H3K4 trimethylation (H3K4me3), both of which are strongly associated with active transcription. Conversely, silencing LcIRF11 reduced p300 and Pol II recruitments and hindered the enrichment of H3K27ac/H3K4me3 modifications at the LcMDA5 promoter. Thus, here we present the first report of IRF11 orchestrating the activation of MDA5 transcription by binding to the ISRE of MDA5 promoter and forming a transcriptional complex with p300 and Pol II. Our results revealed an ancient regulatory mechanism of MDA5 in lower vertebrates, providing insights into its function and evolution.

Abstract: Background Anti-MDA5+ dermatomyositis (DM), also called anti-MDA5+ syndrome, or clinically amyopathic dermatomyositis (CADM), is characterized by extra-muscular DM manifestations such as skin rash, arthralgia, and rapid progressive-interstitial lung disease. Between 2020 and 2024, an increase in serum titer of anti-MDA5+ autoantibodies (AABs) and MDA5+ DM cases was registered among the general population. Given the role of MDA5+ as a viral-RNA sensor, it is considered a key molecule in rheumatological disorders, as studies show its activity is triggered by viral infection. Here, we conducted a systematic review of studies reporting an unambiguous temporal link between SARS-CoV-2 infections and development of MDA5+ DM. The aim was to clarify our understanding of this idiopathic rheumatic nature. Methods This review meets Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines (PRISMA). The Google Scholar, PubMed, Scopus and ScienceDirect were searched using appropriate keywords to identify relevant studies published from 2020–2025. Twenty-nine studies concerning the development of MDA5+ DM in COVID-19 patients, as well as molecular pathogenetic mechanisms and pharmaceutical treatments were included. Results Anti-MDA5+ antibodies have been detected in patients with COVID-19, as well as in sera from post-COVID patients, and their presence correlates positively with disease severity. The onset of MDA5+ DM, in different phenotypic variants, increased during the COVID-19 pandemic, paralleled by an increase in the incidence of juvenile idiopathic inflammatory myopathies (JIIM). The literature here reported shows that MDA5+ DM arises after primary SARS-CoV-2 infection, which could stimulate an antiviral pathway overactivation, leading to innate and adaptive immune cells recruiting, cytokine storm, and synthesis of autoantibodies. Conclusion This review provides evidence for a link between primary SARS-CoV-2 infections, anti-MDA5+ AABs synthesis and emergence of MDA5+ DM in phenotypically different variants such as MIP-C, driven by the virus’s inclination to trigger type-I interferonopathy in genetically predisposed individuals. Systematic review registration https://www.crd.york.ac.uk/prospero/ , identifier 1129317.

Abstract: Type I interferons (IFNs), mainly IFN-α and IFN-β, play an essential role in defending against viral invasion by inducing the host’s innate antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to impair the IFN responses of infected hosts through the antibody-dependent enhancement (ADE) infection pathway, but the precise mechanisms employed are poorly understood. In this study, we showed that PRRSV alone induced a strong secretion of IFN-α and IFN-β in infected porcine alveolar macrophages (PAMs) by activating the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) signaling pathway. By contrast, ADE infection of PRRSV significantly down-regulated the production levels of IFN-α and IFN-β in PAMs by negatively regulating the RIG-I/MDA5 signaling pathway and considerably enhancing the replication level of PRRSV in PAMs. Next, small interfering RNA (siRNA) experiments revealed that Fc gamma receptor I (FcγRI) was responsible for the ADE infection of PRRSV in PAMs. In addition, we observed that FcγRI mediated the potent inhibition of IFN-α and IFN-β production through blocking the activation of the RIG-I/MDA5 signaling pathway in PAMs. Further, we found that FcγRI effectively inhibited PRRSV-induced synthesis of IFN-α and IFN-β by negatively regulating PRRSV-induced activation of the RIG-I/MDA5 signaling pathway in PAMs and significantly increased the viral production of PRRSV in PAMs. In conclusion, these results suggest that ADE infection of PRRSV may antagonize the secretion of type I IFNs (IFN-α/β) by interfering with the RIG-I/MDA5 pathway via FcγRI in PAMs, thereby facilitating the proliferation level of PRRSV in PAMs.

Abstract: Ebinur Lake virus (EBIV) is a recently identified orthobunyavirus with broad host range and zoonotic potential, posing a public health risk. However, the mechanisms underlying EBIV pathogenesis and host innate immune responses remain unclear. Here, we investigated the pattern recognition receptors (PRRs) responsible for sensing EBIV infection and subsequent pathogenesis. EBIV infects diverse cell types and exhibits broad tissue tropism in vivo. In vitro, RIG-I was essential for type I interferon (IFN-I) and inflammatory responses in HEK293 and A549 cells. In contrast, both RIG-I and MDA5 contributed to IFN-I induction in Huh-7 and HCT116 cells, correlating with the specific accumulation of viral dsRNA intermediates in these cell types. Both RIG-I and MDA5 preferentially recognize RNA derived from the viral S segment; however, they have different abilities in sensing incoming viral genomic RNA bearing a 5'-phosphate motif and the replication intermediates. In vivo, RIG-I deficiency severely impairs host defense, while MDA5 deficiency has a more restricted effect in the spleen and liver. In addition to RIG-I and MDA5, TLR7, which is predominantly expressed in dendritic cells, also plays a crucial role for host defense by mediating systemic inflammatory cytokine production without significantly impacting IFN-I response. Our findings suggest that multiple innate sensing receptors, including RIG-I, MDA5, and TLR7, are differentially involved in host defense against EBIV by mediating IFN-I and inflammatory responses, respectively, in a cell-specific manner.IMPORTANCEThis study elucidates the complex mechanisms by which host RIG-I, MDA5, and TLR7 sense the emerging EBIV and trigger cell-specific immune responses. These findings not only clarify crucial aspects of EBIV-host interactions, particularly the differential sensing of viral RNA by distinct PRRs, but also underscore how this differential sensing dictates cell-specific innate immune activation (IFN-I vs. inflammatory responses) and viral pathogenesis, providing critical insights for understanding and combating EBIV and related emerging bunyaviruses.

Abstract: MDA5 is an innate immune RNA sensor that senses infection with a range of viruses and other pathogens. MDA5’s RNA agonists are not well defined. We used single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to study its ligands. Surprisingly, upon infection with SARS-CoV-2 or encephalomyocarditis virus, MDA5 bound overwhelmingly to cellular RNAs. Many binding sites were intronic and proximal to Alu elements and to potentially base-paired structures. Concomitantly, cytoplasmic levels of intron-containing unspliced transcripts increased in infected cells and displayed enrichment of MDA5 iCLIP peaks. Moreover, overexpression of a splicing factor abrogated MDA5 activation. Finally, when depleted of viral sequences, RNA extracted from infected cells still stimulated MDA5. Taken together, MDA5 surveys RNA processing fidelity and detects infections by sensing perturbations of posttranscriptional events such as splicing, establishing a paradigm of innate immune ‘guarding’ for RNA sensors.

Abstract: The cGAS-STING cytosolic DNA-sensing pathway is a key mediator of the innate immune response and plays a crucial role in antitumor immunity. The expression of cGAS and STING is often suppressed in tumor cells, and reduced expression is associated with poor prognosis and inferior response to immunotherapy. In this study we systematically investigated the expression pattern of cGAS-STING pathway in tumors and its correlation with immunotherapy response. We showed that the expression of cGAS and STING was significantly decreased or undetectable in most breast cancer and murine tumor cell lines, while high cGAS and STING expression was associated with increased T cell infiltration, elevated PD-L1 and PD-1 levels, improved immunotherapy response and prolonged survival. In cGAS-STING-deficient MDA-MB-453 cells, DNMT inhibitor decitabine (DAC, 0.05-1 μM) dose-dependently restored the impaired pathway by reversing DNA methylation-mediated silencing. Furthermore, DAC combined with a chemotherapeutic agent cisplatin significantly enhanced the antitumor effect in MDA-MB-453 and MDA-MB-231 cells by activating the cGAS-STING pathway through cytoplasmic DNA accumulation. In addition, DNMT inhibition elevated intracellular dsRNA levels and activated the RIG-I/MDA5-MAVS pathway. These results suggest that DNMT inhibitors can epigenetically reprogram the cGAS-STING pathway, activate the RIG-I/MDA5-MAVS pathway, and in combination with chemotherapeutic agents, synergistically promote antitumor immunity. Together, this study identifies cGAS-STING as a potential predictor of immunotherapy response and highlights a novel therapeutic strategy for restoring innate immune function in cancer. Loss of cGAS-STING signaling in tumors impairs antitumor immunity and correlates with poor immunotherapy response. DNMT inhibition restores cGAS-STING pathway and concurrently activates the RIG-I/MDA5-MAVS signaling, synergistically enhancing immune infiltration and antitumor efficacy.

Abstract: ABSTRACT Problem Aging alters immune function in women and can lead increased risk of infections, particularly in the female reproductive tract (FRT). Method of Study To determine how aging affects innate immune responses in the cervical stroma of the FRT, we isolated endocervical (CX) and ectocervical (ECX) stromal fibroblasts and determine if their expression of multiple pattern recognition receptors (PRRs) and responses to viral stimulation varied with menopause and age. Results Constitutive expression of most PRRs did not vary with age or menopausal status in either cell type. However, the expression of TLR7, MDA5, and NOD2 by ECX stromal fibroblasts significantly increased in post‐menopausal women, while the expression of NOD1 by CX stromal fibroblast also significantly increased in post‐menopausal women. When stratified by age, the expression of TLR6 by CX stromal fibroblasts, and MDA5 and NOD2 by ECX stromal fibroblasts increased significantly with increasing age. Stimulation with the dsRNA viral mimic HMW poly (I:C), a ligand for MDA5, resulted in significantly increased expression of the Type I interferons (IFN) IFNβ and IFNε, the Type III interferon IFNλ1, and interferon‐stimulated genes (ISGs) MxA, OAS2, and ISG15 in both cell populations. However, upregulation of IFNβ, IFNλ1, MxA, OAS2, and ISG15 in response to poly (I:C) significantly declined with increasing post‐menopausal age in ECX stromal fibroblasts. There was no effect of age or menopause on either IFN or ISG expression in CX stromal fibroblasts. Conclusion Overall, these studies demonstrate that ECX and CX fibroblasts are phenotypically distinct populations and that increasing post‐menopausal age reduces IFN and ISG upregulation in ECX stromal fibroblasts in response to viral stimulation, potentially leading to decreased protection against incoming viral pathogens in older post‐menopausal women.

Abstract: Type I interferon (IFN) signaling is central to antiviral immunity and immune homeostasis, yet the mechanisms that sustain and amplify this response remain unclear. Here, we identify two IFN-inducible Alu-free lncRNAs, lnc-CCDC122 and CATIP-AS2, that amplify IFN responses through the cytosolic RNA sensor MDA5. These lncRNAs are induced downstream of IFNβ but exhibit delayed kinetics relative to canonical ISGs, suggesting a distinct regulatory role in sustaining signaling. Under prolonged type I IFN exposures, both transcripts were accumulated in senescent fibroblasts prior to the increase of Alu-containing RNA transcripts and were correlated with IFN signature intensity in PBMCs from systemic lupus erythematosus (SLE) patients. Mechanistically, lnc-CCDC122-2:1 and CATIP-AS2:2 prime MDA5 activation and promote antiviral gene expression kinetics, a function dependent on their cytoplasmic localization and RNA modification status. We found that both lncRNAs were m5C-modified and bound by the m5C reader ALY/REF, which facilitated their export and cytoplasmic accumulation. Disruption of ALY/REF or mutation of conserved m5C sites abolished their immunostimulatory function. Elevated levels of m5C-modified RNA in PBMCs from severe SLE patients further support a functional link. These findings uncover a novel class of regulatory lncRNAs whose m5C-dependent localization and signaling activity potentiate innate immune responses, highlighting an RNA modification–based mechanism for positive feedback in type I IFN signaling and offering potential targets for modulating IFN-driven pathology.

Abstract: Additional file 3. Virus-induced lncRNA-up4 expression is regulated by PRR-dependent innateimmune signalling. (A) Poly(I:C) stimulation led to dose-dependent upregulation of lncRNA-up4expression, as validated by RT‒PCR. MX1 expression confirmed the activation of the antiviral responseby poly(I:C) treatment. (B–C) TLR3 and MDA5 knockdown impaired virus-induced lncRNA-up4expression, as determined by RT‒PCR. All RT‒PCR experiments are representative of three independentexperiments with similar results.

Abstract: Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the host innate immune response to promote its replication. The ubiquitin–proteasome system (UPS) and ISGylation both play roles in modulating host innate immunity. Within this process, ISG15-conjugating enzyme E2L6 (UBE2L6) functions as an E2 ubiquitin/ISG15-conjugating enzyme, which is crucial for the enzymatic cascades of UPS and ISGylation. However, the role of UBE2L6 during PRRSV infection remains unclear. Here, we report that UBE2L6 was up-regulated at both the transcript and protein levels during PRRSV infection. Overexpression of UBE2L6 facilitated PRRSV replication, whereas knockdown of UBE2L6 reduced viral replication. Mechanistically, UBE2L6 promoted the degradation of RIG-I and MDA5 protein expression via the ubiquitin–proteasome pathway and decreased ISGylation levels during PRRSV infection, thereby inhibiting the expression of type I interferons and interferon-stimulated genes (ISGs). In addition, UBE2L6 interacted with PRRSV NSP5 and stabilised the NSP5 protein. Together, PRRSV NSP5 and UBE2L6 further facilitated the degradation of RIG-I and MDA5 via the K48-linked ubiquitination pathway, ultimately facilitating PRRSV replication. Notably, UBE2L6 had minimal impact on RIG-I and MDA5 expression in the absence of PRRSV infection. In summary, UBE2L6 regulated host innate immunity and viral replication through its ubiquitination and ubiquitination-like functions. These findings provide novel insights into how PRRSV NSP5 exploits the host UPS to inhibit the innate immune response and deepen our understanding of the mechanism of host-virus interaction.

Abstract: ABSTRACT Multigene family (MGF) 360 genes, which are African swine fever virus (ASFV) virulence genes, primarily target key host immune molecules to suppress host interferon (IFN) production and interferon-stimulated gene (ISG) transcription, impairing host innate immune responses for efficient viral replication. However, the interactions between MGF 360 virulence genes and host molecules, as well as the mechanisms through which MGF 360 genes regulate host immune responses and IFN signaling, require further elucidation. In this study, we discovered that ASFV MGF_360-4L interacts with MDA5 and recruits the mitochondrial selective autophagy receptor SQSTM1 to degrade MDA5, thus impairing IFN signaling and compromising host innate immune responses. Furthermore, MGF_360-4L inhibits the interaction between MDA5 and MAVS, blocking ISG15-mediated ISGylation of MDA5. MGF_360-4L deficiency significantly attenuated virus-induced mitochondrial autophagy in vitro . Additionally, OAS1 ubiquitinates MGF_360-4L at residues K290, K295, and K327. Finally, a recombinant ASFV lacking the MGF_360-4L gene (ASFV-∆MGF_360-4L) was generated using ASFV-CN/SC/2019 as the backbone, which demonstrated that the replication kinetics of ASFV-∆MGF_360-4L in PAM cells were like those of the highly virulent parental ASFV-WT in vitro . Domestic pigs infected with ASFV-∆MGF_360-4L exhibited milder symptoms than those infected with parental ASFV-WT, and ASFV-∆MGF_360-4L-infected pigs presented with enhanced host innate antiviral immune response, confirming that the deletion of the MGF_360-4L gene from the ASFV genome highly attenuated virulence in pigs and provided effective protection against parental ASFV challenge. In conclusion, we identified a novel ASFV virulence gene, MGF_360-4L, further elucidating ASFV infection mechanisms and providing a new candidate for vaccine development. IMPORTANCE African swine fever virus (ASFV) infection causes acute death in pigs, and there is currently no effective vaccine available for prevention. Multigene family (MGF) virulence genes have been shown to be crucial for ASFV’s ability to evade host innate immune responses. However, the functions of most MGF genes remain unknown, which poses significant challenges for the development of ASFV vaccines and antiviral drugs. In this study, we identified a virulence gene of ASFV, MGF_360-4L, that targets and recruits the selective autophagy receptor p62 to mediate the degradation of the dsRNA sensor MDA5, thereby blocking interferon signaling. Additionally, it inhibits the ISG15-mediated ISGylation activation of MDA5. ASFV lacking MGF_360-4L showed reduced virulence and provided protection in pigs. Our data identify a novel virulence gene and provide new insights for ASFV vaccine development.

Abstract: We report a rare case of overlap syndrome between anti-MDA5-positive dermatomyositis (DM) and rheumatoid arthritis (RA) in a 65-year-old woman. Initially diagnosed with RA, the patient later developed characteristic DM manifestations including characteristic DM rash, generalized weakness, poor appetite, and unintentional weight loss. Laboratory findings revealed elevated muscle enzymes, positive anti-melanoma differentiation-associated gene 5 (MDA5) antibody and anti-Ro52 antibodies, and increased anti-citrullinated protein antibodies. The patient showed significant improvement following combination therapy with prednisone and mycophenolate mofetil, highlighting the importance of early recognition and appropriate management of this rare overlap syndrome.

Abstract: Heterogenous transcription start site (TSS) usage dictates the structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is strictly determined by TSS, 5′ leader structure, and RNA function. We show that cap-sequestered HIV-1 usRNAs (cap1G) destined for viral genome packaging are specifically targeted by MDA5, while translation-destined (cap3G) usRNAs are remarkably immunologically silent. Using mutant viruses which express usRNA with altered 5′ cap-exposed leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that cap exposure and nuclear export pathway choice are major determinants of both lentiviral RNA immunogenicity and function. In total, we identify innate immune system evasion as a possible rationale for the universal conservation of heterogenous TSS usage among ancestral and extant HIV-1 isolates and shed light on how MDA5 fundamentally discriminates between self and non-self RNAs.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is recognized by pattern recognition receptors (PRRs), which play a vital role in triggering innate immune responses such as the production of type I and III interferons (IFNs). While modest PRR activation helps to defend against SARS-CoV-2, excessive or sustained activation can cause harmful inflammation and contribute to severe Coronavirus Disease 2019 (COVID-19). Altered expression of Toll-like receptors (TLRs), which are among the most important members of the PRR family members, particularly TLRs 2, 3, 4, 7, 8 and 9, has been strongly linked to COVID-19 severity. Furthermore, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), collectively known as RLRs (RIG-I-like receptors), act as sensors that detect SARS-CoV-2 RNA. The expression of these receptors, as well as that of different DNA sensors, varies in patients infected with SARS-CoV-2. Changes in PRR expression, particularly that of TLRs, cyclic GMP-AMP synthase (cGAS), and the stimulator of interferon genes (STING), have also been shown to play a role in the development and persistence of long COVID (LC). However, SARS-CoV-2 has evolved strategies to evade PRR recognition and subsequent signaling pathway activation, contributing to the IFN response dysregulation observed in SARS-CoV-2-infected patients. Nevertheless, PRR agonists and antagonists remain promising therapeutic targets for SARS-CoV-2 infection. This review aims to describe the PRRs involved in recognizing SARS-CoV-2, explore their expression during SARS-CoV-2 infection, and examine their role in determining the severity of both COVID-19 and long-term manifestations of the disease. It also describes the strategies developed by SARS-CoV-2 to evade PRR recognition and activation. Moreover, given the considerable interest in modulating PRR activity as a novel immunotherapy approach, this review will provide a description of PRR agonists and antagonists that have been investigated as antiviral strategies against SARS-CoV-2. This review aims to explore the complex interplay between PRRs and SARS-CoV-2 in depth, considering its implications for prognostic biomarkers, targeted therapeutic strategies and the mechanistic understanding of long LC. Additionally, it outlines future perspectives that could help to address knowledge gaps in PRR-mediated responses during SARS-CoV-2 infection.

Abstract: Abstract MDA5 is a RIG-I-like receptor (RLR) that recognizes viral double-stranded RNA (dsRNA) to initiate the innate immune response. Its activation requires filament formation along the dsRNA, which triggers the oligomerization of N-terminal caspase activation and recruitment domains. The ATPase activity of MDA5 is critical for immune homeostasis, likely by regulating filament assembly. However, the molecular basis underlying this process remains poorly understood. Here, we show that MDA5 operates as an ATP-hydrolysis-driven motor that translocates along dsRNA in a one-dimensional (1D) manner. Multiple MDA5 motors can cooperatively load onto a single dsRNA, but their movements rarely synchronize, inhibiting spontaneous filament formation and activation. LGP2, a key regulator of MDA5 signaling, recognizes MDA5 motors and blocks their movement, thereby promoting filament assembly through a translocation-directed mechanism. This unique assembly strategy underscores the role of 1D motion in higher-order protein oligomerization and reveals a novel mechanism for maintaining immune homeostasis.

Abstract: The African swine fever virus (ASFV) employs sophisticated strategies to promote viral replication in the host; however,the underlying mechanisms remain incompletely understood. Here, we demonstrate that the ASFV-encoded pE199L protein acts as a viral mitophagy receptor that disrupts innate immune through structural mimicry. The pE199L localizes to mitochondria via its C-terminal hydrophobic domain (aa 155-199) and induces mitochondrial fission by promoting Drp1 phosphorylation.Importantly, pE199L contains three LC3-interacting regions (LIRs: W³⁵/I³⁸, F¹⁵⁷/L¹⁶⁰, F¹⁹³/L¹⁹⁶) that direct autophagic degradation of key immune adaptors.Specifically, Specifically, pE199L mediates mitophagic clearance of TBK1 (cGAS-STING pathway) and MAVS (RLR-MAVS pathway), thereby inhibiting type I interferon production and enhancing viral replication. This dual degradation mechanism was confirmed through rescue experiments using autophagy inhibitors and functional assays with LIR mutants. We identify pE199L as the first canonical mitophagy receptor encoded by ASFV, unveiling a novel immune evasion strategy and a potential targets for antiviral vaccine development.

Abstract: Additional file 3. pS183L interacts with RIG-I. (A) A co-immunoprecipitation assay was performed with whole cell lysates prepared with 293T cells co-transfected with Flag-RIG-I and HA-S183L for 24 h with Flag antibodies or control IgG. The immunocomplexes were analysed by immunoblotting with the indicated antibodies. (B) The experiment was performed as for panel A, except that Flag-S183L and HA-RIG-I were transfected.

Abstract: Additional file 2. Both LMW poly(I:C) and HMW poly(I:C) up-regulate MDA5 and RIG-I transcription. PK15 cells were transfected with LMW poly(I:C) or HMW poly(I:C) (10 μg/mL) for the indicated times. The cells were then harvested for RNA extraction. Semiquantitative PCR was carried out to detect MDA5 and RIG-I.