Antiviral Signaling Research Articles

A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

OAS-RNase L is a double-stranded RNA-induced antiviral pathway triggered in response to diverse viral infections. Upon activation, OAS-RNase L suppresses virus replication by promoting the decay of host and viral RNAs and inducing translational shutdown. However, whether OASs and RNase L are the only factors involved in this pathway remains unclear. Here, we develop CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identifies IRF2 as a key regulator of OAS3. Mechanistically, we demonstrate that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 works in concert with the interferon response through STAT2 to further enhance OAS3 expression. We propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense. This rapid response provides host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

Helicase protein DDX11 as a novel antiviral factor promoting RIG-I-MAVS-mediated signaling pathway.

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.

MDA5 Is a Major Determinant of Developing Symptoms in Critically Ill COVID-19 Patients.

Apart from the skin and mucosal immune barrier, the first line of defense of the human immune system includes MDA5 (ifih1 gene) which acts as a cellular sensor protein for certain viruses including SARS-CoV-2. Upon binding with viral RNA, MDA5 activates cell-intrinsic innate immunity, humoral responses, and MAVS (mitochondrial antiviral signaling). MAVS signaling induces type I and III interferon (IFN) expressions that further induce ISGs (interferon stimulatory genes) expressions to initiate human cell-mediated immune responses and attenuate viral replication. SARS-CoV-2 counteracts by producing NSP1, NSP2, NSP3, NSP5, NSP7, NSP12, ORF3A, ORF9, N, and M protein and directs anti-MDA5 antibody production presumably to antagonize IFN signaling. Furthermore, COVID-19 resembles several diseases that carry anti-MDA5 antibodies and the current COVID-19 vaccines induced anti-MDA5 phenotypes in healthy individuals. GWAS (genome-wide association studies) identified several polymorphisms (SNPs) in the ifih1-ifn pathway genes including rs1990760 in ifih1 that are strongly associated with COVID-19, and the associated risk allele is correlated with reduced IFN production. The genetic association of SNPs in ifih1 and ifih1-ifn pathway genes reinforces the molecular findings of the critical roles of MDA5 in sensing SARS-CoV-2 and subsequently the IFN responses to inhibit viral replication and host immune evasion. Thus, MDA5 or its pathway genes could be targeted for therapeutic development of COVID-19.

Pseudorabies virus infection triggers mitophagy to dampen the interferon response and promote viral replication.

Pseudorabies virus (PRV) utilizes multiple strategies to inhibit type I interferon (IFN-I) production and signaling to achieve innate immune evasion. Among several other functions, mitochondria serve as a crucial immune hub in the initiation of innate antiviral responses. It is currently unknown whether PRV inhibits innate immune responses by manipulating mitochondria. In this study, we found that PRV infection damages mitochondrial structure and function, as shown by mitochondrial membrane potential depolarization, reduction in mitochondrial numbers, and an imbalance in mitochondrial dynamics. In addition, PRV infection triggered PINK1-Parkin-mediated mitophagy to eliminate the impaired mitochondria, which resulted in a suppression of IFN-I production, thereby promoting viral replication. Furthermore, we found that mitophagy resulted in the degradation of the mitochondrial antiviral signaling protein, which is located on the mitochondrial outer membrane. In conclusion, the data of the current study indicate that PRV-induced mitophagy represents a previously uncharacterized PRV evasion mechanism of the IFN-I response, thereby promoting virus replication.IMPORTANCEPseudorabies virus (PRV), a pathogen that induces different disease symptoms and is often fatal in domestic animals and wildlife, has caused great economic losses to the swine industry. Since 2011, different PRV variant strains have emerged in Asia, against which current commercial vaccines may not always provide optimal protection in pigs. In addition, there are indications that some of these PRV variant strains may sporadically infect people. In the current study, we found that PRV infection causes mitochondria injury. This is associated with the induction of mitophagy to eliminate the damaged mitochondria, which results in suppressed antiviral interferon production and signaling. Hence, our study reveals a novel mechanism that is used by PRV to antagonize the antiviral host immune response, providing a theoretical basis that may contribute to the research toward and development of new vaccines and antiviral drugs.

Classical swine fever virus inhibits serine metabolism-mediated antiviral immunity by deacetylating modified PHGDH.

Classical swine fever (CSF) seriously restricts the healthy development of China's aquaculture industry, and the unclear pathogenic mechanism and pathogenesis of classical swine fever virus (CSFV) are the main obstacle to CSF prevention, control, and purification. Therefore, it is of great significance to explore the molecular mechanism of CSFV and host interplay, to search for the key signaling pathways and target molecules in the host that regulate the replication of CSFV infection, and to elucidate the mechanism of action of host immune dysfunction and immune escape due to CSFV infection for the development of novel CSFV vaccines and drugs. This study reveals the mechanism of serine metabolizing enzyme post-translational modifications and antiviral signaling proteins in the replication of CSFV and enriches the knowledge of CSFV infection and immune metabolism.

BcIRF5 activates bcTBK1 phosphorylation to enhance PANoptosis during GCRV infection

TBK1 is an important IFN antiviral signalling factor, and in previous work black carp TBK1 (bcTBK1) and black carp IRF5 (bcIRF5) together promoted cell death in GCRV-infected cells. In this research, bcTBK1 and bcIRF5 were investigated both in vivo and in vitro to delineate their individual and combined functions. This study demonstrated that both bcTBK1 and bcIRF5 expressions were modulated in response to GCRV infection across the intestine, gill, kidney and spleen. In bcgill cells, overexpression of bcTBK1 and bcIRF5 initially suppressed the expression of cell death-related genes, including RIPK1, caspase1, caspase3 and bax, but this suppression was negated upon GCRV infection. In vivo, mRNA expression levels of RIPK1 and related genes varied by tissue following bcTBK1 or bcIRF5 overexpression and GCRV infection. Notably, intracellular co-overexpression of bcTBK1 and bcIRF5 led to significant upregulation of caspase3, caspase1, bax, and IL1β, along with enhanced caspase3 activity post-GCRV infection. This co-expression correlated with higher survival rates in black carp during GCRV infection and increased caspase3 mRNA in the spleen and gills. Hematoxylin-eosin (HE) staining indicated disorganized spleen tissue and edematous, hyperplastic gill changes in co-transfected groups after infection. TUNEL staining of tissue sections showed that DNA breakage was significantly stronger in the co-transfected group than in the other groups during GCRV infection. Further phosphorylation experiments showed that bcIRF5 promoted phosphorylation modification of bcTBK1. Thus, these data suggest that bcIRF5 activates bcTBK1 by enhancing its phosphorylation and promotes PANoptosis in GCRV-infected cells.

Inflammatory profile of eosinophils in asthma-COPD overlap and eosinophilic COPD: a multi-omics study.

Elevated blood eosinophil levels in patients with chronic obstructive pulmonary disease (COPD) with or without asthma are linked to increased exacerbations and the effectiveness of inhaled corticosteroid treatment. This study aimed to delineate the inflammatory cellular properties of eosinophils in patients with asthma-COPD overlap (ACO) and eosinophilic COPD (eCOPD). Eosinophils were isolated from the peripheral blood of healthy volunteers, patients with non-eCOPD, and those with ACO/eCOPD. Multi-omics analysis involving transcriptomics, proteomics, and lipidomics was performed, followed by bioinformatic data analyses. In vitro experiments using eosinophils from healthy volunteers were conducted to investigate the molecular mechanisms underlying cellular alterations in eosinophils. Proteomics and transcriptomics analyses revealed cellular characteristics in overall COPD patients represented by viral infection (elevated expression of sterol regulatory element-binding protein-1) and inflammatory responses (elevated levels of IL1 receptor-like 1, Fc epsilon receptor Ig, and transmembrane protein 176B). Cholesterol metabolism enzymes were identified as ACO/eCOPD-related factors. Gene Ontology and pathway enrichment analyses demonstrated the key roles of antiviral responses, cholesterol metabolism, and inflammatory molecules-related signaling pathways in ACO/eCOPD. Lipidomics showed the impaired synthesis of cyclooxygenase-derived mediators including prostaglandin E2 (PGE2) in ACO/eCOPD. In vitro assessment confirmed that IL-33 or TNF-α stimulation combined with IL-5 and IFN-γ stimulation induced cellular signatures in eosinophils in ACO/eCOPD. Atorvastatin, dexamethasone, and PGE2 differentially modulated these inflammatory changes. ACO/eCOPD is associated with viral infection and an inflammatory milieu. Therapeutic strategies using statins and inhaled corticosteroids are recommended to control these pathogenic changes.

E3 Ubiquitin Ligase Smurf1 Regulates the Inflammatory Response in Macrophages and Attenuates Hepatic Damage during Betacoronavirus Infection.

The E3 ubiquitin ligase Smurf1 catalyzes the ubiquitination and proteasomal degradation of several protein substrates related to inflammatory responses and antiviral signaling. This study investigated the role of Smurf1 in modulating inflammation induced by Betacoronavirus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 (wild-type) or Smurf1-deficient (Smurf1-/-) mice were infected with MHV-A59 to evaluate the inflammatory response in vitro. Smurf1 was found to be required to downregulate the macrophage production of pro-inflammatory mediators, including TNF, and CXCL1; to control viral release from infected cells; and to increase cell viability. To assess the impact of Smurf 1 in vivo, we evaluated the infection of mice with MHV-A59 through the intranasal route. Smurf1-/- mice infected with a lethal inoculum of MHV-A59 succumbed earlier to infection. Intranasal inoculation with a 10-fold lower dose of MHV-A59 resulted in hematological parameter alterations in Smurf1-/- mice suggestive of exacerbated systemic inflammation. In the lung parenchyma, Smurf1 expression was essential to promote viral clearance, downregulating IFN-β mRNA and controlling the inflammatory profile of macrophages and neutrophils. Conversely, Smurf1 did not affect IFN-β mRNA regulation in the liver, but it was required to increase TNF and iNOS expression in neutrophils and decrease TNF expression in macrophages. In addition, Smurf1-/- mice exhibited augmented liver injuries, accompanied by high serum levels of alanine aminotransferase (ALT). These findings suggest that Smurf1 plays a critical role in regulating the inflammatory response in macrophages and attenuating systemic inflammation during Betacoronavirus infection.

Black carp RNF135 enhances RIG-I-mediated antiviral signaling by facilitating its oligomerization

RNF135, also known as RIPLET, plays a crucial role in facilitating RIG-I signaling in mammals. However, the function and regulatory mechanism of RNF135 in teleosts remain much to be elucidated. In this study, RNF135 homologue of black carp (bcRNF135) has been cloned and identified. The coding sequence (CDS) of bcRNF135 gene comprises 1221 nucleotides, encoding a protein of 407 amino acids. Immunoblotting (IB) and immunofluorescence (IF) assays identified that bcRNF135 is approximately 50 kDa and localized in the cytoplasm. qRT-PCR demonstrated that bcRNF135 mRNA levels were increased in host cells following SVCV infection and poly (I:C) stimulation. Co-expressed bcRNF135 obviously enhanced the induced transcription of IFN promoters by bcRIG-I in reporter assay, as well as improved bcRIG-I triggered antiviral response. Notably, bcRNF135 knockdown reduced the antiviral ability of host cells and increased virus replication. Co-immunoprecipitation (Co-IP) assays and IF assays confirmed that bcRNF135 interacted with bcRIG-I. Moreover, SDD-AGE revealed that bcRNF135 promotes the oligomerization of bcRIG-I, a process critical for RIG-I activation. Overall, our data conclude that bcRNF135 enhances bcRIG-I-mediated antiviral signaling by facilitating its ubiquitination and oligomerization, enriching our understanding of RIG-I regulation in teleost innate immunity.

EP.02F.06 Suppression of Chemotherapy Induced Antiviral Signaling by YAP/TAZ in Lung Cancer

EP.02F.06 Suppression of Chemotherapy Induced Antiviral Signaling by YAP/TAZ in Lung Cancer

Type I IFN drives unconventional IL-1β secretion in lupus monocytes.

Opsonization of red blood cells that retain mitochondria (Mito+ RBCs), a feature of systemic lupus erythematosus (SLE), triggers type I interferon (IFN) production in macrophages. We report that monocytes (Mos) co-produce IFN and mature interleukin-1β (mIL-1β) upon Mito+ RBC opsonization. IFN expression depended on cyclic GMP-AMP synthase (cGAS) and RIG-I-like receptors' (RLRs) sensing of Mito+ RBC-derived mitochondrial DNA (mtDNA) and mtRNA, respectively. Interleukin-1β (IL-1β) production was initiated by the RLR antiviral signaling adaptor (MAVS) pathway recognition of Mito+ RBC-derived mtRNA. This led to the cytosolic release of Mo mtDNA, which activated the inflammasome. Importantly, mIL-1β secretion was independent of gasdermin D (GSDMD) and pyroptosis but relied on IFN-inducible myxovirus-resistant protein 1 (MxA), which facilitated the incorporation of mIL-1β into a trans-Golgi network (TGN)-mediated secretory pathway. RBC internalization identified a subset of blood Mo expressing IFN-stimulated genes (ISGs) that released mIL-1β and expanded in SLE patients with active disease.

FBXL19 in endothelial cells protects the heart from influenza A infection by enhancing antiviral immunity and reducing cellular senescence programs.

Influenza A virus (IAV) infection while primarily affecting the lungs, is often associated with cardiovascular complications. However, the mechanisms underlying this association are not fully understood. Here, we investigated the potential role of FBXL19, a member of the Skp1-Cullin-1-F-box family of E3 ubiquitin ligase, in IAV-induced cardiac inflammation. We demonstrated that FBXL19 overexpression in endothelial cells (ECs) reduced viral titers and IAV matrix protein 1 (M1) levels while increasing antiviral gene expression, including interferon (IFN)-α, -β, and -γ and RANTES (regulated on activation normal T cell expressed and secreted) in the cardiac tissue of IAV-infected mice. Moreover, EC-specific overexpression of FBXL19 attenuated the IAV infection-reduced interferon regulatory factor 3 (IRF3) level without altering its mRNA level and suppressed cardiac inflammation. Furthermore, IAV infection triggered cellular senescence programs in the heart as indicated by the upregulation of p16 and p21 mRNA levels and the downregulation of lamin-B1 levels, which were partially reversed by FBXL19 overexpression in ECs. Our findings indicate that EC-specific overexpression of FBXL19 protects against IAV-induced cardiac damage by enhancing interferon-mediated antiviral signaling, reducing cardiac inflammation, and suppressing cellular senescence programs.NEW & NOTEWORTHY Our study reveals a novel facet of IAV infection, demonstrating that it can trigger cellular senescence within the heart. Intriguingly, upregulation of endothelial FBXL19 promotes host innate immunity, reduces cardiac senescence, and diminishes inflammation. These findings highlight the therapeutic potential of targeting FBXL19 to mitigate IAV-induced cardiovascular complications.

Molecular depiction and functional delineation of E3 ubiquitin ligase MARCH5 in yellowtail clownfish (Amphiprion clarkii).

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi, AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H2O2-treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii.

The Mitochondrial Ubiquitin Ligase MARCHF5 Cooperates with MCL1 to Inhibit Apoptosis in KSHV-Transformed Primary Effusion Lymphoma Cell Lines.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people with HIV including Kaposi's sarcoma and primary effusion lymphoma (PEL). We have previously shown that PEL cell lines require myeloid cell leukemia-1 (MCL1) to inhibit apoptosis. MCL1 is an oncogene that is amplified in cancers and causes resistance to chemotherapy regimens. MCL1 is thus an attractive target for drug development. The emerging clinical relevance and therapeutic potential of MCL1 motivated us to study the roles of this oncogene in PEL in depth. Using a systems biology approach, we uncovered an unexpected genetic interaction between MCL1 and MARCHF5 indicating that they function in the same pathway. MARCHF5 is an E3 ubiquitin ligase most known for regulating mitochondrial homeostasis and antiviral signaling, but not apoptosis. We thus investigated how MCL1 and MARCHF5 cooperate to promote PEL cell survival. CRISPR knockout (KO) of MARCHF5 in PEL cell lines resulted in a significant increase in apoptosis despite the presence of MCL1. The anti-apoptotic function of MARCHF5 was dependent on its E3 ligase and dimerization activities. Loss of MARCHF5 or inhibition of the 26S proteasome furthermore stabilized the MCL1 antagonist NOXA without affecting levels of MCL1. Interestingly, NOXA KO provides a fitness advantage to PEL cells suggesting that NOXA is the pro-apoptotic signal that necessitates the anti-apoptotic activities of MCL1 and MARCHF5. Finally, endogenous reciprocal co-immunoprecipitation experiments show that MARCHF5 and NOXA are found in the same protein complex. Our findings thus provide the mechanistic link that underlies the genetic interaction between MCL1 and MARCHF5. We propose that MARCHF5 induces the degradation of the MCL1 antagonist NOXA thus reinforcing the pro-survival role of MCL1 in these tumor cells. This newly appreciated interaction of the MCL1 and MARCHF5 oncogenes may be useful to improve the design of combination therapies for KSHV malignancies.

Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response

Mitochondrial morphology and function change dynamically in response to intracellular signaling and the surrounding environment. The mitochondrial fission factor Mff, which localizes to the outer mitochondrial membrane, mediates not only mitochondrial fission by recruiting the dynamin-related GTPase Drp1 to mitochondrial fission sites but also the double-stranded RNA-induced antiviral response on mitochondria through mitochondrial antiviral signaling (MAVS). Mff is reported to be regulated by AMP-activated protein kinase (AMPK)-mediated protein phosphorylation and alternative pre-mRNA splicing; however, the relationships among RNA splicing, phosphorylation, and multiple functions of Mff have not been fully understood. Here, we showed that mouse Mff has a tissue-specific splicing pattern, and at least eight Mff splice isoforms were expressed in mouse embryonic fibroblasts (MEFs). We introduced single Mff isoforms into Mff knockout MEFs and found that insertion of exon 6 just after the phosphorylation site, by the alternative splicing, reduced its phosphorylation by AMPK and its functions in mitochondrial fission and the antiviral response. In addition, the underlying mechanism repressing these functions was independent of phosphorylation. These results indicate that multiple functions of Mff on mitochondria are regulated by AMPK-mediated phosphorylation and alternative splicing, under the control of energy metabolism and cellular differentiation.

Bunyavirus SFTSV nucleoprotein exploits TUFM-mediated mitophagy to impair antiviral innate immunity

ABSTRACT Severe fever with thrombocytopenia syndrome is an emerging viral hemorrhagic fever caused by a tick-borne bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), with a high case fatality. We previously found that SFTSV nucleoprotein (NP) induces macroautophagy/autophagy to facilitate virus replication. However, the role of NP in antagonizing host innate immunity remains unclear. Mitophagy, a selected form of autophagy, eliminates damaged mitochondria to maintain mitochondrial homeostasis. Here, we demonstrate that SFTSV NP triggers mitophagy to degrade MAVS (mitochondrial antiviral signaling protein), thereby blocking MAVS-mediated antiviral signaling to escape the host immune response. Mechanistically, SFTSV NP translocates to mitochondria by interacting with TUFM (Tu translation elongation factor, mitochondrial), and mediates mitochondrial sequestration into phagophores through interacting with LC3, thus inducing mitophagy. Notably, the N-terminal LC3-interacting region (LIR) motif of NP is essential for mitophagy induction. Collectively, our results demonstrated that SFTSV NP serves as a novel virulence factor, inducing TUFM-mediated mitophagy to degrade MAVS and evade the host immune response. Abbreviation: 3-MA: 3-methyladenine; ACTB: actin beta; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DMSO: dimethyl sulfoxide; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; HTNV: Hantan virus; IAV: influenza A virus; IFN: interferon; LAMP1: lysosomal associated membraneprotein 1; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associatedprotein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MT-CO2/COXII: mitochondrially encoded cytochrome C oxidase II; NP: nucleoprotein; NSs: nonstructural proteins; poly(I:C): polyinosinic:polycytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SFTSV: severe fever withthrombocytopenia syndrome virus; TCID50: 50% tissue culture infectiousdose; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20:translocase of outer mitochondrial membrane 20; TUFM: Tu translation elongationfactor, mitochondrial.

The E3 ligase ASB3 downregulates antiviral innate immunity by targeting MAVS for ubiquitin-proteasomal degradation.

E3 ubiquitin ligases are very important for regulating antiviral immunity during viral infection. Here, we discovered that Ankyrin repeat and SOCS box-containing protein 3 (ASB3), an E3 ligase, are upregulated in the presence of RNA viruses, particularly influenza A virus (IAV). Notably, overexpression of ASB3 inhibits type I IFN (IFN-I) responses induced by Sendai virus (SeV) and IAV, and ablation of ASB3 restores SeV and H9N2 infection-mediated transcription of IFN-β and its downstream interferon-stimulated genes (ISGs). Interestingly, animals lacking ASB3 presented decreased susceptibility to H9N2 and H1N1 infections. Mechanistically, ASB3 interacts with MAVS and directly mediates K48-linked polyubiquitination and degradation of MAVS at K297, thereby inhibiting the phosphorylation of TBK1 and IRF3 and downregulating downstream antiviral signaling. These findings establish ASB3 as a critical negative regulator that controls the activation of antiviral signaling and describe a novel function of ASB3 that has not been previously reported.

TGFβ primes alveolar-like macrophages to induce type I IFN following TLR2 activation.

Alveolar macrophages (AMs) are key mediators of lung function and are potential targets for therapies during respiratory infections. TGFβ is an important regulator of AM differentiation and maintenance, but how TGFβ directly modulates the innate immune responses of AMs remains unclear. This shortcoming prevents effective targeting of AMs to improve lung function in health and disease. Here we leveraged an optimized ex vivo AM model system, fetal-liver derived alveolar-like macrophages (FLAMs), to dissect the role of TGFβ in AMs. Using transcriptional analysis, we first globally defined how TGFβ regulates gene expression of resting FLAMs. We found that TGFβ maintains the baseline metabolic state of AMs by driving lipid metabolism through oxidative phosphorylation and restricting inflammation. To better understand inflammatory regulation in FLAMs, we next directly tested how TGFβ alters the response to TLR2 agonists. While both TGFβ (+) and TGFβ (-) FLAMs robustly responded to TLR2 agonists, we found an unexpected activation of type I interferon (IFN) responses in FLAMs and primary AMs in a TGFβ-dependent manner. Surprisingly, mitochondrial antiviral signaling protein and the interferon regulator factors 3 and 7 were required for IFN production by TLR2 agonists. Together, these data suggest that TGFβ modulates AM metabolic networks and innate immune signaling cascades to control inflammatory pathways in AMs.

Targeting APT2 improves MAVS palmitoylation and antiviral innate immunity

Innate immunity serves as the primary defense against viral and microbial infections in humans. The precise influence of cellular metabolites, especially fatty acids, on antiviral innate immunity remains largely elusive. Here, through screening a metabolite library, palmitic acid (PA) has been identified as a key modulator of antiviral infections in human cells. Mechanistically, PA induces mitochondrial antiviral signaling protein (MAVS) palmitoylation, aggregation, and subsequent activation, thereby enhancing the innate immune response. The palmitoyl-transferase ZDHHC24 catalyzes MAVS palmitoylation, thereby boosting the TBK1-IRF3-interferon (IFN) pathway, particularly under conditions of PA stimulation or high-fat-diet-fed mouse models, leading to antiviral immune responses. Additionally, APT2 de-palmitoylates MAVS, thus inhibiting antiviral signaling, suggesting that its inhibitors, such as ML349, effectively reverse MAVS activation in response to antiviral infections. These findings underscore the critical role of PA in regulating antiviral innate immunity through MAVS palmitoylation and provide strategies for enhancing PA intake or targeting APT2 for combating viral infections.

E3 ubiquitin ligase ANKIB1 attenuates antiviral immune responses by promoting K48-linked polyubiquitination of MAVS

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 invivo. Therefore, we reveal that ANKIB1 restricts RLR-induced innate immune activation, indicating its potential role as a therapeutic target for viral infections.