The mucosal immune system plays a critical role in defending the body against external pathogens and preserving homeostasis. The polymeric immunoglobulin receptor (PIGR) is a critical component of this system, responsible for facilitating the transport and secretion of soluble polymeric immunoglobulins across epithelial cells, thereby contributing to immune defense. In zebrafish, a pIgR-like (pIgRL) family exists, among which pIgRL4.2 is highly expressed in multiple immune organs, suggesting its potential role in immunity. In this study, spring viremia of carp virus (SVCV) infection significantly downregulates pIgRL4.2 expression. Conversely, overexpression of pIgRL4.2 in EPC cells markedly delays SVCV-induced cytopathic effects and effectively suppresses SVCV replication. Additionally, overexpression of pIgRL4.2 enhances IFN activation induced by both poly(I:C) treatment and SVCV infection. Furthermore, CRISPR/Cas9 was used to generate pIgRL4.2-null zebrafish, and disruption of pIgRL4.2 in zebrafish has been demonstrated to result in a reduction in survival rates following SVCV challenge. This is accompanied by a consistent downregulation of antiviral responsive genes, concomitant with an increase in SVCV replication in pIgRL4.2-deficient zebrafish. Therefore, this study demonstrates that pIgRL4.2 inhibits viral replication by positively regulating the IFN signaling pathway.

Methyltransferase VP39 is an important target for the treatment of monkeypox, and inhibition of VP39 can effectively suppresses the transcription and translation of early viral RNA. However, very few inhibitors have been designed against VP39 and other viral MTases. In this work, four inhibitors (SFG, TO507, TO427 and TO1119) were used to investigate the binding mechanism with VP39. Moreover, VP39 has different modes of existence, but we do not understand the interaction mechanism of the complex system formed by the inhibitors with different modes of VP39, so we performed 1000 ns molecular dynamics simulations of the complexes formed by four inhibitors with VP39 in mode 1 and mode 2, and performed energy calculation and conformational analysis. The results of binding free energy showed that in the inhibitors-VP39 (mode 1) systems, TO507 and TO427 had a strong inhibitory effect on VP39, and residues ASP95, ARG97, PHE115 and VAL139 played important roles in the binding process of all four systems. Surprisingly, in the inhibitors-VP39 (mode 2) systems, four inhibitors underwent a large conformational change, with the amino acid moieties of the inhibitors undergoing a nearly 90° folding. And this change reduced the inhibitory effect of the inhibitors on VP39. In addition, the inhibitor TO507 also had a good inhibition effect on nsp14 of SARS-CoV-2 and NS5 of Zika virus. Therefore, this study suggests new ideas for the design and improvement of pan-MTase inhibitors, which are important for the treatment of pandemic infectious diseases, such as monkeypox and SARS-CoV-2.

Human metapneumovirus (hMPV) is a seasonal respiratory virus that typically causes mild, flu-like symptoms. In some cases, it can lead to severe respiratory complications, such as pneumonia, bronchitis, and bronchiolitis, often requiring hospitalization. Recently, a surge in hMPV cases has been reported in China and other countries, raising concerns about a potential pandemic scenario reminiscent of COVID-19. This review explores the genomic structure, replication cycle, genetic diversity, and evolutionary trajectory of hMPV. It also discusses host immune responses and the available animal models to study pathogenesis and to screen for potential vaccines and antivirals. Additionally, we examine the shifting seasonal trends in hMPV circulation, evaluate the low pandemic risk posed by existing hMPV clades, and underscore the need for continued vaccine and antiviral development. Finally, we advocate for strengthened global surveillance, especially in low- and middle-income countries, as a critical strategy to mitigate the risks posed by emerging hMPV clades.

Retinoic-acid-inducible gene-I (RIG-I)-like receptors (RLRs) comprise a family of DExD/H-box RNA helicases that are pivotal in antiviral and inflammatory responses. Ubiquitination serves as a crucial regulatory mechanism for both RIG-I activation and the type I interferon (IFN) signaling pathway in mammals. Although RLRs have been found to be evolutionarily conserved in teleost fish, the functional characterization of RIG-I ubiquitination in this vertebrate group remains largely unexplored. Through the integration of computational prediction with experimental validation, six ubiquitination sites (K115, K118, K145, K163, K168, and K171) were identified on RIG-I in Epithelioma papulosum cyprini (EPC) cells. Among these, K163, K168, and K171 are evolutionarily conserved in mammalian RIG-I orthologs. Biochemical analyses confirmed K63-linked ubiquitination at residues K115, K118, and K163. Functional characterization revealed that mutant RIG-I-K163R and RIG-I-K118R significantly downregulated ifn expression along with three interferon-stimulated genes (ISGs: gig1, mx1, and viperin) in EPC RIG-I knockout (EPCrigi-/-) cells, demonstrating the essential role of these ubiquitination sites in RLR-mediated signaling activation. K118-mediated ubiquitination exerts a more pronounced regulatory effect on RIG-I activation compared to K163, as evidenced by enhanced spring viremia of carp virus (SVCV) proliferation in RIG-I-K118R-transfected EPCrigi-/- cells relative to mutant RIG-I-K163R or wild-type controls. Notably, the mutant RIG-I-K115R exhibited enhanced antiviral activity, characterized by increased type I IFN signaling and reduced viral replication in EPC cells. This unexpected outcome may partially result from the mutant's increased self-oligomerization compared to wild-type RIG-I. It is suggested that a tunable regulatory mechanism mediated by multisite ubiquitination of RIG-I may be conserved in teleosts. These findings provide novel insights into the molecular mechanisms governing the RLR signaling pathway in teleosts, highlighting how multisite ubiquitination of RIG-I can lead to divergent antiviral outcomes.

Excessive endothelial pro-inflammatory response is an early hallmark of sepsis-induced acute lung injury (ALI). Xuebijing (XBJ), a traditional Chinese medicine, is widely used in clinical practice to treat sepsis.

This study aims to investigate the molecular mechanisms underlying the beneficial effects of XBJ.

Plasma samples from septic patients treated with or without XBJ were collected and analyzed. The mouse model of sepsis was established by intraperitoneal injection of LPS (10 mg/kg). XBJ (10 ml/kg) was administrated intraperitoneally twice before LPS challenge and one time after LPS challenge. The severity of lung injury and the levels of inflammation and coagulation were evaluated. In vitro, HUVEC were used to explore the mechanisms of XBJ and its compounds in regulating the ACLY/MYB/RIG-I axis.

XBJ significantly reduced the plasma levels of endothelial cell (EC) damage-related markers in septic patients. The in vivo and in vitro data demonstrated that XBJ alleviated LPS-induced lung injury and reduced the levels of inflammation and coagulation activation in ECs. XBJ inhibited the phosphorylation-dependent activation of ATP citrate lyase (ACLY), thereby suppressing the acetylation-dependent nuclear translocation of the transcription factor MYB. The expression of retinoic acid inducible gene I (RIG-I) was downregulated, leading to the inhibition of NF-κB signaling and EC pro-inflammatory and coagulation activation, which further alleviated sepsis-associated ALI. Moreover, XBJ compounds Quercetin, Ferulic Acid, Kaempferol and Paeoniflorin all showed inhibitory effects on the activation of the downstream MYB/RIG-I signaling by binding to ACLY protein.

Our study revealed a novel regulatory mechanism of XBJ in sepsis-induced EC dysfunction and ALI. The compounds in XBJ inhibited the activity of ACLY, thereby inhibiting the expression of RIG-I by reducing the acetylation of transcription factor MYB, leading to the alleviation of EC activation and lung injury induced by sepsis. Our findings provide a theoretical basis for the clinical application of XBJ and shedding light on novel therapeutic targets for treating sepsis.

Vaccinology has revolutionized modern medicine, delivering groundbreaking solutions to prevent and control infectious diseases while pioneering innovative strategies to tackle non-infectious challenges, including cancer. Traditional vaccines faced inherent limitations, driving the evolution of next-generation vaccines such as subunit vaccines, peptide-based vaccines, and nucleic acid-based platforms. Among these, nucleic acid-based vaccines, including DNA and mRNA technologies, represent a major innovation. Pioneering studies in the 1990s demonstrated their ability to elicit immune responses by encoding specific antigens. Recent advancements in delivery systems and molecular engineering have overcome initial challenges, enabling their rapid development and clinical success. This review explores nucleic acid-based vaccines, including chemically modified variants, by examining their mechanisms, structural features, and therapeutic potential, while underscoring their pivotal role in modern immunization strategies and expanding applications across contemporary medicine.

Non-small cell lung cancer (NSCLC) represents a formidable challenge in oncology due to its molecular heterogeneity and the dynamic suppressive nature of its tumor microenvironment (TME). Despite the transformative impact of immune checkpoint inhibitors (ICIs) on cancer therapy, the majority of NSCLC patients experience resistance, necessitating novel approaches to overcome immune evasion. This review highlights shared and subtype-specific mechanisms of immune resistance within the TME, including metabolic reprogramming, immune cell dysfunction, and physical barriers. Beyond well-characterized components such as regulatory T cells, tumor-associated macrophages, and myeloid-derived suppressor cells, emerging players - neutrophil extracellular traps, tertiary lymphoid structures, and exosomal signaling networks - underscore the TME's complexity and adaptability. A multi-dimensional framework is proposed to transform cold, immune-excluded tumors into hot, immune-reactive ones. Key strategies include enhancing immune infiltration, modulating immunosuppressive networks, and activating dormant immune pathways. Cutting-edge technologies, such as single-cell sequencing, spatial transcriptomics, and nanomedicine, are identified as pivotal tools for decoding TME heterogeneity and personalizing therapeutic interventions. By bridging mechanistic insights with translational innovations, this review advocates for integrative approaches that combine ICIs with metabolic modulators, vascular normalizers, and emerging therapies such as STING agonists and tumor vaccines. The synergistic potential of these strategies is poised to overcome resistance and achieve durable antitumor immunity. Ultimately, this vision underscores the importance of interdisciplinary collaboration and real-time TME profiling in refining precision oncology for NSCLC, offering a blueprint for extending these advances to other malignancies.

Mucosal-mediated immune deficiency is associated with immune evasion and poor clinical outcomes in acute myeloid leukemia (AML). Here, we describe the elicitation of mucosal and systemic immune response by oral delivery of MDP-modified PEG-lipid (MDP-PEG-DSPE) and polylactic acid-polyhistidine (PLA-PHis) copolymer constructed nanosystem (mPOD) into Peyer's patches. To protect against gastrointestinal degradation, enteric-soluble capsules are utilized for encapsulating mPOD to promote penetration across intestinal mucus and engender robust Peyer's patch targeting initiated by MDP-PEG-DSPE. Compared with intravenous and intramuscular administration, the oral delivery of MDP-PEG-DSPE and 5'-triphosphate-modified RNA (ppp-RNA) into gut-associated lymphoid tissues reinforces dendritic cell maturation and migration, amplifies mucosal immune response, and boosts the production of secretory immunoglobulin A via retinoic acid-inducible gene I/nucleotide-binding oligomerization domain 2 (RIG-I/NOD2) signaling activation. In the AML murine model, the provoked mucosal immunity positively regulates the systemic cytotoxic immune reactions, which, in turn, eradicate disseminated malignant leukemic cells and provide defense against leukemia attacks.

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection.

Adeno-associated virus (AAV)-mediated gene therapy represents a promising approach for treating genetic disorders. However, challenges remain in achieving stable transgene expression and mitigating liver injury during long-term therapy. Previous studies have implicated the activation of RIG-I-like receptors (RLRs), which detect double-stranded RNA (dsRNA), as a potential inhibitor of transgene expression. In this study, we investigated the role of the RLR pathway in AAV-transduced cells, with a focus on the generation of sense and antisense RNA, as well as the formation of dsRNA. Our findings revealed that dsRNA is produced following AAV transduction, leading to the activation of the RLR pathway and the induction of innate immune responses. Prolonged AAV transduction in mice resulted in significant liver injury, which was independent of adaptive immune activation. Instead, mitochondrial antiviral signaling protein (MAVS) activation emerged as a critical mediator of these effects. Notably, downregulation of MAVS enhanced transgene expression, suggesting that modulating MAVS could enhance the efficacy of AAV-based gene therapy. This study elucidates the mechanisms underlying dsRNA formation and RLR pathway activation, highlighting their impact on the efficacy of AAV gene therapy. These findings suggest that strategies aimed at minimizing dsRNA production and targeting the RLR-MAVS pathway could reduce immune activation and enhance therapeutic transgene expression, thereby optimizing AAV-based interventions for genetic disorders.

Crimean-Congo hemorrhagic fever (CCHF) is a viral tick-borne disease with fatality rates of up to 30 %. Currently, there are no vaccines or specific antivirals available. The genome of the CCHF virus (CCHFV) encodes an ovarian tumor (OTU) protease with a deubiquitinating activity that is responsible for the evasion of the innate immune response. Therefore, the inhibition of the OTU protease could provide a strategy for the treatment of CCHFV infections. In this study, we screened for small-molecule inhibitors of CCHFV OTU using a fluorescent ubiquitin rhodamine 110 assay. We identified and validated a 2-aminothiazole hit compound (IC50 = 42.3 μM) followed by structure-activity relationships (SAR) studies resulting in a new inhibitor of the CCHFV OTU protease. The most active derivative is a competitive CCHFV OTU inhibitor with an IC50 value of 10.7 μM. Selectivity studies revealed that the ubiquitin-specific peptidase 7 (USP7), ubiquitin C-terminal hydrolase 5 (UCHL5), OTU deubiquitinase 1 (OTUD1), and Cezanne are also inhibited by this newly developed inhibitor indicating binding to conserved regions of the ubiquitin-binding site within the deubiquitinase superfamilies. Molecular docking into the active site of CCHFV OTU proposes starting points for further structural modifications to improve activity and selectivity. These structure-activity relationships are the first to our knowledge to be reported for the CCHFV OTU protease and will help guide further drug discovery efforts.

Small interfering RNAs (siRNAs), generated by Dicer proteins, play a pivotal role in antiviral immunity in eukaryotes. Dicer proteins also produce microRNAs (miRNAs), a class of endogenous small non-coding RNAs that regulate essential cellular functions through post-transcriptional mechanisms. In plants and insects, multiple Dicer proteins are produced and deployed to separately manage the biogenesis of antiviral siRNAs and miRNAs. This separation ensures that viral infections, especially the production of viral RNAi suppressors, do not severely compromise host growth or development. In contrast, nematode worms, such as Caenorhabditis elegans, rely on a single Dicer protein to produce both types of small RNAs. Probably as a strategy to mitigate the potential disruption of miRNA production by viral infections, nematodes have evolved distinct strategies for generating primary and secondary siRNAs for antiviral defense. This review explores the shared and unique features of siRNA-mediated antiviral immunity in Caenorhabditis elegans, shedding light on the specialized adaptations that enable robust antiviral defenses without compromising miRNA-mediated function.

Respiratory viral infections are a major global public health concern, and current antiviral therapies still have limitations. In recent years, research has revealed significant similarities between the immune systems of the gut and lungs, which interact through the complex physiological network known as the "gut-lung axis." As one of the largest immune organs, the gut, along with the lungs, forms an inter-organ immune network, with strong parallels in innate immune mechanisms, such as the activation of pattern recognition receptors (PRRs). Furthermore, the gut microbiota influences antiviral immune responses in the lungs through mechanisms such as systemic transport of gut microbiota-derived metabolites, immune cell migration, and cytokine regulation. Studies have shown that gut dysbiosis can exacerbate the severity of respiratory infections and may impact the efficacy of antiviral therapies. This review discusses the synergistic role of the gut-lung axis in antiviral immunity against respiratory viruses and explores potential strategies for modulating the gut microbiota to mitigate respiratory viral infections. Future research should focus on the immune mechanisms of the gut-lung axis to drive the development of novel clinical treatment strategies.

Lung cancer, one of the most lethal malignancies, has seen its therapeutic strategies become a focal point of significant scientific attention. Intrinsic immune signaling pathways play crucial roles in anti-tumor immunity but face clinical application challenges despite promising preclinical outcomes. Lactylation, an emerging research focus, may influences lung cancer progression by modulating the functions of histones and non-histone proteins. Recent findings have suggested that lactylation regulates key intrinsic immune molecules, including cGAS-STING, TLR, and RIG-I, thereby impacting interferon expression. However, the precise mechanisms by which lactylation governs intrinsic immune signaling in lung cancer remain unclear. This review presents a comprehensive and systematic analysis of the relationship between lactylation and intrinsic immune signaling pathways in lung cancer and emphasizes the innovative perspective of linking lactylation-mediated epigenetic modifications with immune regulation. By thoroughly examining current research findings, this review uncovers potential regulatory mechanisms and highlights the therapeutic implications of targeting lactylation in lung cancer. Future investigations into the intricate interactions between lactylation and intrinsic immunity are anticipated to unveil novel therapeutic targets and strategies, potentially improving patient survival outcomes.

Modernization of existing methods for the delivery of mRNA is vital in advanced therapeutics. Traditionally, mRNA has faced obstacles of poor stability due to enzymatic degradation. This work examines cutting-edge formulation and emerging techniques for safer delivery of mRNA vaccines. Inspired by the success of lipid nanoparticles (LNP) in delivering mRNA vaccines for COVID-19, a variety of other formulations have been developed to deliver mRNA vaccines for diverse infections. The meritorious features of nanoparticle-based mRNA delivery strategies, including LNP, polymeric, dendrimers, polysaccharide-based, peptide-derived, carbon and metal-based, DNA nanostructures, hybrid, and extracellular vesicles, have been examined. The impact of these delivery platforms on mRNA vaccine delivery efficacy, protection from enzymatic degradation, cellular uptake, controlled release, and immunogenicity has been discussed in detail. Even with significant developments, there are certain limitations to overcome, including toxicity concerns, limited information about immune pathways, the need to maintain a cold chain, and the necessity of optimizing administration methods. Continuous innovation is essential for improving delivery systems for mRNA vaccines. Future research directions have been proposed to address the existing challenges in mRNA delivery and to expand their potential prophylactic and therapeutic application.

Background: The bacterial gut microbiome has been the subject of many studies that have provided valuable scientific conclusions. However, many different populations of microorganisms that interact with each other to maintain homeostasis coexist inside the gut. The gut virome, especially, appears to play a key role in this interactive microenvironment. Intestinal viral communities, including bacteriophages, appear to influence health and disease, although their role has not yet been fully elucidated. In addition, bacteriophages or viruses that infect bacteria regulate bacterial growth, thus shaping the composition of the gut microbiome and affecting the immune system. Infant Gut Virome: The shaping of the gut microbiome during the first years of life has a significant role in the maturation of the infant's immune system. In contrast, early dysbiosis has been associated with chronic, including metabolic and autoimmune, disorders later in life. Purpose: Although viruses have been shown to be potential triggers of autoimmune diseases, there is a gap in the literature regarding the infant gut virome in autoimmunity development. Despite the lack of evidence, this review attempts to summarize and clarify what is known so far about this timely and important topic in the hope that its findings will contribute to future research.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutics with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here this challenge is addressed by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, it is found that covalent conjugation of 3pRNA improves loading efficiency, enhances immunostimulatory activity, protects against nuclease degradation, and improves serum stability. Additionally, it is found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter is only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increases type-I interferon levels relative to analogous carriers that use electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I.

Mammalian SUMO specific peptidase 2 (SENP2) plays vital roles in a variety of biological procedures including the immune response. However, the effects of teleost SENP2 are still mostly unexplored. In this study, the SENP2 of black carp (Mylopharyngodon piceus) was cloned and characterized. The open reading frame of black carp SENP2 (bcSENP2) consists of 1800 nucleotides, which encode 600 amino acids. The reporter assay results showed that over-expression of bcSENP2 alone had a weak effect on interferon (IFN) promoter transcription activity, whereas it significantly reduced bcMDA5/bcRIG-I mediated IFN promoter transcription activity. The interaction between bcSENP2 and bcMDA5 or bcRIG-I was detected by immunoprecipitation experiments. The plaque assay and qPCR results indicated that bcMDA5 or bcRIG-I mediated antiviral capacity was attenuated by bcSENP2, while knockdown of bcSENP2 led to enhanced antiviral resistance to SVCV in host cells. In addition, the expression level of bcMDA5/bcRIG-I protein was attenuated by co-expressed bcSENP2 and MG132 treatment rescued this attenuating effect. All of these data support the conclusion that bcSENP2 inhibits bcMDA5/bcRIG-I mediated antiviral signaling by enhancing ubiquitin-proteasome mediated degradation of bcMDA5/bcRIG-I in black carp.

In vitro transcription (IVT) is a technology of vital importance that facilitated the production of mRNA therapeutics and drove numerous breakthroughs in RNA biology. T7 polymerase-produced RNAs can begin with either 5'-triphosphate guanosine (5'-pppG) or 5'-triphosphate adenosine (5'-pppA), generating potential agonists for the RIG-I/type I interferon response. While it is established that IVT can yield highly immunogenic double-stranded RNA (dsRNA) via promoterless transcription, the specific contribution of initiating nucleosides to this process has not been previously reported. Our study shows that IVT-derived RNAs containing 5'-pppA are significantly more immunogenic compared with their 5'-pppG counterparts. We observed heightened levels of dsRNAs triggered by IVT with 5'-pppA RNA, activating the RIG-I signaling pathway in cultured cells, as well as in ex vivo and in vivo mouse models, where the IFN-β gene was substituted with the mKate2 fluorescent reporter. Elevated levels of dsRNA were found in both short and long 5'-pppA RNAs, including those of COVID-19 vaccines. These findings reveal the unexpected source of IVT RNA immunogenicity, offering valuable insights for both academic research and future medical applications of this technology.

Colorectal cancer (CRC) is one of the most prevalent malignant tumors in the world, and its occurrence and development are closely related to the complex immune regulatory mechanisms. As the first barrier of the body's defense, innate immunity plays a key role in tumor immune surveillance and anti-tumor response, in which type I/III interferon (IFN) is an important mediator with significant antiviral and anti-tumor functions. 5-methylcytosine (m5C) modification of RNA is a key epigenetic regulation that promotes the expression of CRC oncogenes and immune-related genes. It can enhance the proliferation, migration, and invasion of tumor cells by affecting mRNA stability, translation efficiency, and nuclear export. In addition, m5C modification modulates the activity of innate immune signaling pathways and inhibits interferon production and function, further helping tumor cells evade immune surveillance. However, there are insufficient elucidations on the interaction between m5C modification and innate immunity in CRC. In this study, the mechanism of interferon I/III in colorectal cancer was systematically reviewed and explored. This work focused on how m5C modification promotes tumor immune escape by affecting the interferon signaling pathway, thereby providing new diagnostic markers and therapeutic targets for clinical use, and enhancing the immunotherapy efficacy.

Heat shock cognate 70 (HSC70), a highly conserved molecular chaperone in the heat shock protein 70 (HSP70) family, plays an essential role in maintaining the homeostasis of the cellular environment. Furthermore, although previous studies have investigated potential function of HSC70 in innate antiviral immunity, further research is still required to fully elucidate its role. In this study, we cloned and characterized the HSC70 homolog gene from black carp (Mylopharyngodon piceus), which consists of 1950 nucleotides encoding 650 amino acids, migrates at approximately 71 kDa on SDS-PAGE, and is distributed in the cytoplasm. In response to different stimuli (SVCV, poly (I:C) and LPS), the transcription level of black carp HSC70 (bcHSC70) all increased to a certain extent. Luciferase reporter assay demonstrated that co-transfected bcHSC70 obviously reduced activity of interferon (IFN) promoters mediated by most factors in the RLRs pathway, and further qRT-PCR and plaque assay indicated that co-transfection of bcHSC70 with bcRIG-I decreased the bcRIG-I-mediated IFN transcription and antiviral ability resisting spring viremia of carp virus (SVCV), whereas knockdown of bcHSC70 improves the host cellular antiviral activity. Noteworthily, co-immunoprecipitation (co-IP) assay and immunofluorescence (IF) assay confirmed bcHSC70 interacts with bcRIG-I, and weaken K63-linked polyubiquitination of bcRIG-I. In summary, our study revealed that HSC70 negatively regulates IFN signaling pathway through impairing K63-linked ubiquitination of RIG-I in black carp, which provides an important basis for exploring innate immune regulatory mechanisms in teleost fish.

The viral protein mutations can modify virus-host interactions during virus evolution, and thus alter the extent of infection or pathogenicity. Studies indicate that nucleocapsid (N) protein of SARS-CoV-2 participates in viral genome assembly, intracellular signal regulation and immune interference. However, its biological function in viral evolution is not well understood. SARS-CoV-2 N protein mutations were analyzed in Delta, Omicron, and original strains. Two mutations with a methionine (M) residue at site 203 and a tyrosine (Y) residue at site 377 of the N protein were found in Delta strain but not in Omicron and original strains, and promoted SARS-CoV-2 infection therein. Those mutations, R203M and D377Y, enhanced the inhibitory impact of N protein on the impairment of RIG-I-mediated antiviral signaling, such as IRF3 phosphorylation and IFN-β activation. The viral RNA-binding activity of N protein was promoted by these mutations, effectively attenuating the recognition and interaction of RIG-I with viral RNA compared to the original or other variants. The R203M/D377Y mutations thus enhanced the suppressive activity of the N protein on RIG-I-mediated interferon induction both in vitro and in vivo, which in turn promoted viral replication. This study helps to understand the variability of SARS-CoV-2 in regulating host immunity.

Our experimental study showed that METTL3 was highly expressed in NSCLC cells and promoted the growth of tumor cells. METTL3 takes N6-methyladenosine (m6A) as the main means of mRNA modification to control the expression and function of RIG-I-MAVS signalling pathway. RIG-I-MAVS constitute the first line frontier in the innate immune defense of human cells. Activation of RIG-I-MAVS signaling can inhibit tumor cell growth and activate the immune microenvironment. Our experimental data reveal that lung cancer cells utilize METTL3-mediated methylation modifications to inhibit the activation of RIG-I-MAVS signaling pathway and immune responses. Our work provides new ideas for biotherapy and immunotherapy.

The sensing of nucleic acids by DEAD/H-box helicases, specifically retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), plays a critical role in inducing antiviral immunity following infection. However, this DEAD/H-box helicase family includes many additional proteins whose immune functions have not been investigated. While numerous DEAD/H-box helicases contribute to antiviral immunity, they employ diverse mechanisms beyond the direct sensing of nucleic acids. Some members have also been identified to play proviral (promoting virus replication/propagation) roles during infections, regulate other non-viral infections, and contribute to the regulation of autoimmunity and cancer. This review synthesizes the known and emerging functions of the broader DEAD/H-box helicase family in immune regulation and highlights ongoing efforts to target these proteins therapeutically.

The proteins encoded by Influenza A virus (IAV) evade the innate immune system through diverse strategies to facilitate their replication. However, the regulatory mechanisms remain not fully understood. In this study, we identified that the H9N2 PB1 protein suppressed the activities of the IFN-β, ISRE, and NF-κB promoters. Furthermore, H9N2 PB1 inhibited the phosphorylation of IRF3, IκBα, and TBK1 and the secretion of IFN-β. The results demonstrated H9N2 PB1 as a negative regulator of the RIG-I signaling pathway. Subsequent investigations revealed a specific interaction between H9N2 PB1 and MAVS, which disturbed the stability of MAVS. Notably, we discovered that H9N2 PB1 exploited the function of TRIM25, leading to the autophagic degradation of MAVS through K48-linked polyubiquitination. In conclusion, we uncovered a negative regulatory axis consisting of H9N2 PB1-TRIM25-MAVS-IFN-I. These findings provide valuable insights into the molecular interactions involved in the regulation of the host's innate immune antiviral response by IAV.

Type Ι interferon (IFN) production mediated by retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS) is essential for antiviral innate immune responses. Here, we report the identification of a novel co-sensor for cytosolic nucleic acids: DEAD/H-box helicase 11 (DDX11), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knockdown or knockout of DDX11 attenuated the ability of cells to increase IFN-β, IFN-stimulated gene 56, and C-X-C motif chemokine ligand 10 in response to SeV and poly (I:C) by blocking the activation of TANK-binding kinase 1 and IFN regulatory factor 3. Nucleic acid sensing by DDX11 was independent of the stimulator of IFN genes but was dependent on RIG-I and MAVS. DDX11 regulated RIG-I-MAVS-mediated IFN signaling by specifically interacting with nucleic acid, RIG-I, and MAVS to enhance RIG-I-double-strand RNA and RIG-I-MAVS binding affinity. Overall, our results identified a critical role for DDX11 in the innate immune response and provided molecular insights into the mechanisms by which DDX11 recognized cytosolic nucleic acid and interacted with RIG-Ι and MAVS for potent IFN signaling and antiviral immunity.

Innate immunity is the first and most rapid host defense against virus infection. Recognition of viral RNA by the retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) initiates innate antiviral immune responses. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified DEAD/H-box helicase 11 (DDX11) as a positive regulator of the RIG-I-mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathways. Mechanistically, we demonstrated that DDX11 bound to viral RNA, interacted with RIG-I, and promoted their binding to viral RNA. DDX11 also promoted the interaction between RIG-I and MAVS and activation of RIG-I-MAVS signaling. Overall, our results elucidate the role of DDX11 in RIG-I-MAVS-dependent signaling pathways and may shed light on innate immune gene regulation.

Background: FAT10 is a member of the ubiquitin-like modifier family. Similar to ubiquitin, FAT10 has a distinct enzyme cascade consisting of E1-activating, E2-conjugating, and possibly several E3-ligating enzymes, which will covalently link FAT10 to substrate proteins in order to target them directly for proteasomal degradation. FAT10 was reported to be phosphorylated by IKKβ during infection with influenza A virus. Methods: To assess the difference between the FAT10-dependent degradation of phosphorylated FAT10 and the non-phosphorylated FAT10 wild type (FAT10 WT), a mutated FAT10 that mimicked phosphorylation (FAT10 D) was constructed by replacing several serine residues and one threonine residue with aspartic or glutamic acid. The FAT10 degradation or conjugation was compared between the phospho-mimetic FAT10 and the wild-type FAT10 with respect to the dependence of the E3 ligase TRIM25, the UBL-UBA protein NUB1L, and the proteasomal ubiquitin receptor RPN10. Results: The phospho-mimetic FAT10 was more efficiently conjugated to substrate proteins as compared to the wild-type FAT10, particularly if TRIM25 was co-expressed. Additionally, the phospho-mimetic FAT10 was not bound by NUB1L. However, this did not affect FAT10 D or FAT10 WT degradation. No differences were found in the binding affinity of phospho-mimetic FAT10 to RPN10. Conclusions: In brief, the phospho-mimetic FAT10 shows enhanced conjugation efficiency, but phosphorylation does not alter its degradation by the proteasome. This reveals that phosphorylation may fine-tune FAT10's interactions with specific interaction partners without disrupting its core function of proteasomal degradation.

While certain members of ubiquitin-coupled enzymes (E2s) have garnered attention as potential therapeutic targets across diverse diseases, research progress on Ubiquitin-Conjugating Enzyme 5 (UBC5)-a pivotal member of the E2s family involved in crucial cellular processes such as apoptosis, DNA repair, and signal transduction-has been relatively sluggish. Previous findings suggest that UBC5 plays a vital role in the ubiquitination of various target proteins implicated in diseases and homeostasis, particularly in various cancer types. This review comprehensively introduces the structure and biological functions of UBC5, with a specific focus on its contributions to the onset and advancement of diverse diseases. It suggests that targeting UBC5 holds promise as a therapeutic approach for disease therapy. Recent discoveries highlighting the high homology between UBC5, UBC1, and UBC4 have provided insight into the mechanism of UBC5 in protein degradation and the regulation of cellular functions. As our comprehension of the structural distinctions among UBC5 and its homologues, namely UBC1 and UBC4, advances, our understanding of UBC5's functional significance also expands.

Pathogen-associated molecular patterns (PAMPs) are highly conserved motifs originating from microorganisms that act as ligands for pattern recognition receptors (PRRs), which are crucial for defense against pathogens. Thus, PAMP-mimicking vaccines may induce potent immune activation and provide broad-spectrum protection against microbes. Dextran encapsulation can regulate the surface characteristics of nanoparticles (NPs) and induces their surface modification. To determine whether dextran-encapsulated NPs can be used to develop antiviral vaccines by mimicking viral PAMPs, we synthesized NPs in a cyclohexane inverse miniemulsion (Basic-NPs) and further encapsulated them with dextran or tetramethylrhodamine isothiocyanate (TRITC)-dextran (Dex-NPs or TDex-NPs). We hypothesized that these dextran encapsulated NPs could activate innate immunity through cell surface or cytosolic PRRs. In vitro and in vivo experiments were performed using RAW 264.7 and C57BL/6 mice to test different concentrations and routes of administration. Only TDex-NPs rapidly increased retinoic acid-inducible gene I (RIG-I) at 8 h and directly bound to it, producing 120-300 pg/ml of IFN-α via the ERK/NF-κB signaling pathway in both in vitro and in vivo models. The effect of TDex-NPs in mice was observed exclusively with footpad injections. Our findings suggest that TRITC-dextran encapsulated NPs exhibit surface properties for RIG-I binding, offering potential development as a novel antiviral and anticancer RIG-I agonist.

RIG-I and MDA5 are members of RIG-I-like receptors (RLRs) that detect viral RNA within the cytoplasm and subsequently initiate antiviral immune responses. Necroptosis is a form of programmed cell death (PCD) executed by mixed lineage kinase domain-like (MLKL), which, upon phosphorylation by receptor-interacting protein kinase 3 (RIPK3), causes necrotic cell death. To date, no link between RLRs and necroptosis has been observed during bacterial infection. Edwardsiella tarda is a zoonotic bacterial pathogen that can thrive in host macrophages. In a previous study, we identified RIG-I and MDA5 as two hub factors of RAW264.7 cells responsive to E. tarda infection. The present study aimed to determine the specific form of cell death triggered by E. tarda and explore the association between RIG-I/MDA5 and PCD in the context of bacterial infection. Our results showed that E. tarda infection induced RIPK3-MLKL-mediated necroptosis, rather than pyroptosis or apoptosis, in RAW264.7 cells. Meanwhile, E. tarda promoted RIG-I/MDA5 production and activated the RIG-I/MDA5 pathways that led to IRF3 phosphorylation, IFN-β secretion, and interferon-stimulated gene (ISG) and cytokine expression. Both RIG-I and MDA5 were essential for E. tarda-triggered necroptosis and required for effective inhibition of intracellular bacterial replication. Furthermore, the regulatory effect of RIG-I/MDA5 on necroptosis was not affected by type I IFN or TNF-α signaling blockage. Together these results revealed that necroptosis could be triggered by intracellular bacterial infection through the RIG-I/MDA5 pathways, and that there existed intricate interplays between PCD and RLRs induced by bacterial pathogen.

Innate host defense mechanisms require posttranslational modifications (PTM) to protect against viral infection. Age-associated immunosenescence results in increased pathogenesis and mortality in the elderly, but the contribution of altered PTM regulation to immunosenescence is unknown. SUMOylation is a rapid and reversible post-translational modification that has been implicated in age-associated disease and plays conflicting roles in viral replication and antiviral defenses in mammals. We have discovered in Caenorhabditis elegans that induction of antiviral defense is regulated through SUMOylation of DRH-1, the ortholog of the DEAD/H-box helicase and cytosolic pattern recognition receptor RIG-I, and that this regulation breaks down during aging. We find the SUMO isopeptidase ULP-4 is essential for deSUMOylation of DRH-1 and activation of the intracellular pathogen response (IPR) after exposure to Orsay virus (OV), a natural enteric C. elegans pathogen. ULP-4 promotes stabilization of DRH-1, which translocates to the mitochondria to activate the IPR in young animals exposed to virus. Loss of either drh-1 or ulp-4 compromises antiviral defense resulting in a failure to clear the virus and signs of intestinal pathogenesis. During aging, expression of ulp-4 decreases, which results in increased proteosomal degradation of DRH-1 and loss of the IPR. Mutating the DRH-1 SUMOylated lysines resulted in the constitutive activation of the IPR in young animals and partially rescued the age-associated lost inducibility of the IPR. Our work establishes that aging results in dysregulated SUMOylation and loss of DRH-1, which compromises antiviral defense and creates a physiological shift to favor chronic pathological infection in older animals.

Hematopoietic stem cells (HSCs) and erythropoiesis are activated during pregnancy and after bleeding by the derepression of retrotransposons, including endogenous retroviruses and long interspersed nuclear elements. Retrotransposon transcription activates the innate immune sensors cyclic guanosine 3',5'-monophosphate-adenosine 5'-monophosphate synthase (cGAS) and stimulator of interferon (IFN) genes (STING), which induce IFN and IFN-regulated genes in HSCs, increasing HSC division and erythropoiesis. Inhibition of reverse transcriptase or deficiency for cGAS or STING had little or no effect on hematopoiesis in nonpregnant mice but depleted HSCs and erythroid progenitors in pregnant mice, reducing red blood cell counts. Retrotransposons and IFN-regulated genes were also induced in mouse HSCs after serial bleeding and, in human HSCs, during pregnancy. Reverse transcriptase inhibitor use was associated with anemia in pregnant but not in nonpregnant people, suggesting conservation of these mechanisms from mice to humans.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder characterized by fat accumulation in the liver. This leads to aggravated hepatocyte inflammation due to impaired mitochondrial function, mitochondrial double-stranded RNA (mt-dsRNA) release, elevated oxidative stress, and reactive oxygen species (ROS) production. MicroRNA-29a (miR-29a) is used to reduce hepatic fibrosis in cases of cholestatic liver damage and lessen the severity of non-alcoholic steatohepatitis in animal studies by influencing mitochondrial protein balance. However, the effectiveness of miR-29a in diminishing mt-dsRNA-induced exacerbation of NAFLD remains poorly understood, particularly in the context of a Western diet (WD). Our results have found that mice with increased miR-29a levels and fed a WD showed notably decreased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and low-density lipoprotein cholesterol levels. They also experienced less weight gain and lower final body and liver weights. In addition, overexpression of miR-29a reduced the severity of fibrosis, alleviated hepatic oxidative stress, misfolded protein aggregates, and the release of mt-dsRNA. Moreover, miR-29a attenuated the innate immune mitochondrial antiviral-signaling protein (MAVS) pathway response. In vitro, the research using HepG2 cells confirmed that miR-29a reduces MAVS expression and decreases the release of mt-dsRNA and superoxide initiated by palmitic acid (PA). Analysis of luciferase activity further established that the specific binding of miR-29a to the 3'UTR of MAVS led to a repression of its expression. In conclusion, these groundbreaking findings underscore the potential of miR-29a in improving the treatment of NAFLD and liver steatofibrosis by inhibiting the MAVS signaling pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important RNA virus that has caused huge economic losses to swine industry in the whole world. Ubiquitin specific protease 8 (USP8), a pivotal regulator of protein degradation, intricately contributes to orchestrating the delicate balance of various biological processes through its deubiquitinating activity. However, the role of USP8 in antiviral immune response to PRRSV remains elusive. In the study, by means of overexpressing USP8, we identified that USP8 suppressed the replication of PRRSV, while reducing USP8 expression using siRNA significantly led to the promotion of PRRSV replication. And USP8 facilitates the production of IFN-β and some IFN-stimulated genes (ISGs) during PRRSV infection. Mechanistically, USP8 promoted mitochondrial antiviral signaling protein (MAVS)-mediated IFN-β signaling. Moreover, USP8 interacted with MAVS and exerted anti-PRRSV effects in a MAVS-dependent manner. This study highlights the importance of USP8 in regulating PRRSV replication, which may enhance our comprehension of its role in innate immunity and its impact on viral replication.

Repeating sequences of DNA, or repetitive elements (REs), are common features across both prokaryotic and eukaryotic genomes. Unlike many of their protein-coding counterparts, the functions of REs in host cells remained largely unknown and have often been overlooked. While there is still more to learn about their functions, REs are now recognized to play significant roles in both beneficial and pathological processes in their hosts at the cellular and organismal levels. Therefore, in this review, we discuss the various types of REs and review what is known about their evolution. In addition, we aim to classify general mechanisms by which REs promote processes that are variously beneficial and harmful to host cells/organisms. Finally, we address the emerging role of REs in cancer, aging, and neurological disorders and provide insights into how RE modulation could provide new therapeutic benefits for these specific conditions.

Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with mitochondrial antiviral signaling proteins (MAVSs) to activate IRF3 and nuclear factor κB (NF-κB) signaling, initiating innate immune responses. Thus, RLR activation plays a vital role in the removal of invasive RNA viruses while maintaining immune homeostasis. However, inadequate or excessive activation of immunity can cause harm and can even lead to lethal consequences. In this study, we identify an E3 ligase, ankyrin repeat and IBR domain containing 1 (ANKIB1), which suppresses RLR signaling via MAVS. ANKIB1 binds to MAVS to enhance K48-linked polyubiquitination with K311R, causing proteasomal degradation of MAVS. Deficiency of ANKIB1 significantly increases the RLR-mediated production of type I interferon (IFN) along with pro-inflammatory factors. Consequently, ANKIB1 deficiency remarkably increases antiviral immunity and decreases viral replication in vivo. Therefore, we reveal that ANKIB1 restricts RLR-induced innate immune activation, indicating its potential role as a therapeutic target for viral infections.

Since 2011, the emergence of Pseudorabies virus (PRV) variants has led to significant vaccine failures, resulting in severe economic losses in China's swine industry. Conventional PRV vaccines have shown limited efficacy against these emergent variants, underscoring the urgent need for novel immunization strategies. This study aimed to develop and evaluate a novel recombinant PRV vaccine candidate with improved safety and immunogenicity profiles. Utilizing the homology-directed repair (HDR)-CRISPR/Cas9 system, we generated a recombinant PRV strain, designated PRV SX-10ΔgI/gE/TK/UL24, with deletions in the gI, gE, TK, and UL24 genes. In vitro analyses demonstrated that the recombinant virus exhibited similar replication kinetics and growth curves comparable to the parental strain. The immunological properties of the recombinant PRV were assessed in murine and porcine models. All animals inoculated with PRV SX-10ΔgI/gE/TK/UL24 survived without exhibiting significant clinical signs or pathological alterations. Immunological assays revealed that PRV SX-10ΔgI/gE/TK/UL24 elicited significantly higher levels of gB-specific antibodies, neutralizing antibodies, and cytokines (including IFN-γ, IL-2, and IL-4) compared to both the Bartha-K61 and PRV SX-10ΔgI/gE/TK strains. Notably, both murine and porcine subjects immunized with PRV SX-10ΔgI/gE/TK/UL24 demonstrated enhanced protection against challenges with the variant PRV SX-10 strain, compared to other vaccine strains. These findings suggest that PRV SX-10ΔgI/gE/TK/UL24 represents a promising PRV vaccine candidate strain, offering valuable insights for the prevention and control of PRV in clinical applications.

Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective.IMPORTANCEThe innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses.