Antiviral Signaling Research Articles

Brain RNA profiling highlights multiple disease pathways in persons with HAND: uncovering determinants of biotype diversity

Objective: To discover microRNA (miRNA)-RNA transcript interactions dysregulated in brains from persons with HIV-associated neurocognitive disorder (HAND), we investigated RNA expression using machine learning tools. Design: Brain-derived host RNA transcript and miRNA expression was examined from persons with or without HAND using bioinformatics platforms. Methods: By combining next generation sequencing, droplet digital (dd)PCR quantitation of HIV-1 genomes, with bioinformatics and statistical tools, we investigated differential RNA expression in frontal cortex from persons without HIV (HIV[-]), with HIV without brain disease (HIV[+]), with HIV-associated neurocognitive disorder (HAND), or HAND with encephalitis (HIVE). Results: Expression levels for 147 transcripts and 43 miRNAs showed a minimum 4-fold difference between clinical groups with a predominance of antiviral (Type I interferon) signaling-, neural cell maintenance-, and neurodevelopmental disorder-related genes that was validated by gene ontology and molecular pathway inferences. Scale of signal-to-noise ratio (SSNR) and biweight midcorrelation (bicor) analyses identified 14 miRNAs and 45 RNA transcripts, which were highly correlated and differentially expressed (p ≤ 0.05). Machine learning applications compared regression models predicated on HIV-1 DNA, or RNA viral quantities that disclosed miR-4683 and miR-154-5p were dominant variables associated with differential expression of host RNAs. These miRNAs were also associated with antiviral-, cell maintenance-, and neurodevelopmental disorder-related genes. Conclusions: Antiviral as well as neurodevelopmental disorder-related pathways in brain were associated with HAND, based on correlated RNA transcripts and miRNAs. Integrated molecular methods with machine learning offer insights into disease mechanisms, underpinning brain-related biotypes among persons with HIV that could direct clinical care.

Antiviral therapy for hepatitis B virus infection is beneficial for the prognosis hepatocellular carcinoma

In this editorial, we comment on the article by Mu et al , published in the recent issue of the World Journal of Gastrointestinal Oncology . We pay special attention to the immune tolerance mechanism caused by hepatitis B virus (HBV) infection, the pathogenesis of hepatocellular carcinoma (HCC), and the role of antiviral therapy in treating HCC related to HBV infection. HBV infection leads to systemic innate immune tolerance by directly inhibiting pattern recognition receptor recognition and antiviral signaling pathways, as well as by inhibiting the immune functions of macrophages, natural killer cells and dendritic cells. In addition, HBV leads to an immunosuppressive cascade by expressing inhibitory molecules to induce exhaustion of HBV-specific cluster of differentiation 8 + T cells, ultimately leading to long-term viral infection. The loss of immune cell function caused by HBV infection ultimately leads to HCC. Long-term antiviral therapy can improve the prognosis of patients with HCC and prevent tumor recurrence and metastasis.

Single-cell analysis of lung epithelial cells reveals age and cell population-specific responses to SARS-CoV-2 infection in ciliated cells.

The ability of SARS-CoV-2 to evade antiviral immune signaling in the airway contributes to the severity of COVID-19 disease. Additionally, COVID-19 is influenced by age and has more severe presentations in older individuals. This raises questions about innate immune signaling as a function of lung development and age. Therefore, we investigated the transcriptome of different cell populations of the airway epithelium using pediatric and adult lung tissue samples from the LungMAP Human Tissue Core Biorepository. Specifically, lung lobes were digested and cultured into a biomimetic model of the airway epithelium on an air-liquid interface. Cells were then infected with SARS-CoV-2 and subjected to single-cell RNA sequencing. Transcriptional profiling and differential expression analysis were carried out using Seurat. The clustering analysis identified several cell populations: club cells, proliferating epithelial cells, multiciliated precursor cells, ionocytes, and two biologically distinct clusters of ciliated cells (FOXJ1high and FOXJ1low). Interestingly, the two ciliated cell clusters showed different infection rates and enrichment of processes involved in ciliary biogenesis and function; we observed a cell-type-specific suppression of innate immunity in infected cells from the FOXJ1low subset. We also identified a significant number of genes that were differentially expressed in lung cells derived from children as compared to adults, suggesting the differential pathogenesis of SARS-CoV-2 infection in children versus adults. We discuss how this work can be used to identify drug targets to modulate molecular signaling cascades that mediate an innate immune response and begin to understand differences in COVID-19 outcomes for pediatric vs. adult populations.

RTP4 restricts influenza A virus infection by targeting the viral NS1 protein.

The influenza A virus evades the host innate immune response to establish infection by inhibiting RIG-I activation through its nonstructural protein 1 (NS1). Here, we reported that receptor-transporting protein 4 (RTP4), an interferon-stimulated gene (ISG), targets NS1 to inhibit influenza A virus infection. Depletion of RTP4 significantly increased influenza A virus multiplication, while NS1-deficient viruses were unaffected. Mechanistically, RTP4 interacts with NS1 in an RNA-dependent manner and sequesters it from the TRIM25-RIG-I complex, thereby restoring TRIM25-mediated RIG-I K63-linked ubiquitination and subsequent activation of IRF3. Antiviral activity of RTP4 requires the evolutionarily conserved CXXC motifs and an H149 residue in the zinc finger domain, mutations of which disrupted RTP4-NS1 interaction and abrogated the ability of RTP4 to rescue RIG-I-mediated signaling. Collectively, our findings provided insights into the mechanism by which an ISG restricts influenza A virus replication by reactivating host antiviral signaling.

A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense.

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 Å crystal structure of Acb4 in complex with 3'3'-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

Cellular RNA interacts with MAVS to promote antiviral signaling.

Antiviral signaling downstream of RIG-I-like receptors (RLRs) proceeds through a multi-protein complex organized around the adaptor protein mitochondrial antiviral signaling protein (MAVS). Protein complex function can be modulated by RNA molecules that provide allosteric regulation or act as molecular guides or scaffolds. We hypothesized that RNA plays a role in organizing MAVS signaling platforms. We found that MAVS, through its central intrinsically disordered domain, directly interacted with the 3' untranslated regions of cellular messenger RNAs. Elimination of RNA by ribonuclease treatment disrupted the MAVS signalosome, including RNA-modulated MAVS interactors that regulate RLR signaling and viral restriction, and inhibited phosphorylation of transcription factors that induce interferons. This work uncovered a function for cellular RNA in promoting signaling through MAVS and highlights generalizable principles of RNA regulatory control of immune signaling complexes.

Platform influencers-host RNA control of antiviral immunity.

Cellular RNAs directly regulate the activity of an antiviral immune signaling complex.

DHX15 and Rig-I Coordinate Apoptosis and Innate Immune Signaling by Antiviral RNase L.

During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2'-5'-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3.

Equine lentivirus Gag protein degrades mitochondrial antiviral signaling protein via the E3 ubiquitin ligase Smurf1.

Equine infectious anemia virus (EIAV) and HIV-1 are both members of the Lentivirus genus and are similar in virological characters. EIAV is of great concern in the equine industry. Lentiviruses establish a complex interaction with the host cell to counteract the antiviral responses. There are various pattern recognition receptors in the host, for instance, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent interferon (IFN) response. However, viruses also exploit multiple strategies to resist host immunity by targeting MAVS, but the mechanism by which lentiviruses are able to target MAVS has remained unclear. In this study, we found that EIAV infection induced MAVS degradation, and that EIAV Gag protein recruited the E3 ubiquitin ligase Smurf1 to polyubiquitinate and degrade MAVS. The CARD domain of MAVS and the WW domain of Smurf1 are responsible for the interaction with Gag. EIAV Gag is a precursor polyprotein of the membrane-interacting matrix p15, the capsid p26, and the RNA-binding nucleocapsid proteins p11 and p9. Therefore, we analyzed which protein domain of Gag could interact with MAVS and Smurf1. We found that p15 and p26, but not p11 or p9, target MAVS for degradation. Moreover, we identified the key amino acid residues that support the interactions between p15 or p26 and MAVS or Smurf1. The present study describes a novel role of the EIAV structural protein Gag in targeting MAVS to counteract innate immunity, and reveals the mechanism by which the equine lentivirus can antagonize against MAVS.IMPORTANCEHost anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), and subsequently initiates downstream signaling through interaction with mitochondrial antiviral signaling protein (MAVS). However, viruses have developed various strategies to counteract MAVS-mediated signaling, although the method of antagonism of MAVS by lentiviruses is still unknown. In this article, we demonstrate that the precursor (Pr55gag) polyprotein of EIAV and its protein domains p15 and p26 target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase Smurf1. MAVS degradation leads to the inhibition of the downstream IFN-β pathway. This is the first time that lentiviral structural protein has been found to have antagonistic effects on MAVS pathway. Overall, our study reveals a novel mechanism by which equine lentiviruses can evade host innate immunity, and provides insight into potential therapeutic strategies for the control of lentivirus infection.

RNA sensing induced by chromosome missegregation augments anti-tumor immunity.

Viral mimicry driven by endogenous double-stranded RNA (dsRNA) stimulates innate and adaptive immune responses. However, the mechanisms that regulate dsRNA-forming transcripts during cancer therapy remain unclear. Here, we demonstrate that dsRNA is significantly accumulated in cancer cells following pharmacologic induction of micronuclei, stimulating mitochondrial antiviral signaling (MAVS)-mediated dsRNA sensing in conjunction with the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Activation of cytosolic dsRNA sensing cooperates with double-stranded DNA (dsDNA) sensing to upregulate immune cell migration and antigen-presenting machinery. Tracing of dsRNA-sequences reveals that dsRNA-forming transcripts are predominantly generated from non-exonic regions, particularly in locations proximal to genes exhibiting high chromatin accessibility. Activation of this pathway by pulsed monopolar spindle 1 (MPS1) inhibitor treatment, which potently induces micronuclei formation, upregulates cytoplasmic dsRNA sensing and thus promotes anti-tumor immunity mediated by cytotoxic lymphocyte activation invivo. Collectively, our findings uncover a mechanism in which dsRNA sensing cooperates with dsDNA sensing to boost immune responses, offering an approach to enhance the efficacy of cancer therapies targeting genomic instability.

Sustained antiviral insulin signaling during West Nile virus infection results in viral mutations.

Arthropod-borne viruses or arboviruses, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV) pose significant threats to public health. It is imperative to develop novel methods to control these mosquito-borne viral infections. We previously showed that insulin/insulin-like growth factor-1 signaling (IIS)-dependent activation of ERK and JAK-STAT signaling has significant antiviral activity in insects and human cells. Continuous immune pressure can lead to adaptive mutations of viruses during infection. We aim to elucidate how IIS-signaling in mosquitoes selects for West Nile virus escape variants, to help formulate future transmission blocking strategies. We hypothesize that passage of WNV under activation of IIS will induce adaptive mutations or escape variants in the infecting virus. To test our hypothesis, WNV was serially passaged through Culex quinquefasciatus Hsu cells in the presence or absence of bovine insulin to activate IIS antiviral pressure. We sequenced WNV genes encoding for E, NS2B, NS3, and NS5 and identified variants in E and NS5 arising from IIS antiviral pressure. In parallel to the genetic analyses, we also report differences in the levels of virus replication and Akt activation in human cells and mosquitoes using virus passaged in the presence or absence of insulin. Finally, using adult Culex quinquefasciatus, we demonstrated the enhancement of immune response gene expression in virus-infected mosquitoes fed on insulin, compared to control. Notably, virus collected from insulin-fed mosquitoes contained a non-synonymous mutation in NS3. These results contribute towards achieving our long-term goal of manipulating mosquito IIS-dependent antiviral immunity to reduce WNV or other flavivirus transmission to mammalian hosts.

Avian TRIM13 attenuates antiviral innate immunity by targeting MAVS for autophagic degradation

ABSTRACT MAVS (mitochondrial antiviral signaling protein) is a crucial adaptor in antiviral innate immunity that must be tightly regulated to maintain immune homeostasis. In this study, we identified the duck Anas platyrhynchos domesticus TRIM13 (ApdTRIM13) as a novel negative regulator of duck MAVS (ApdMAVS) that mediates the antiviral innate immune response. Upon infection with RNA viruses, ApdTRIM13 expression increased, and it specifically binds to ApdMAVS through its TM domain, facilitating the degradation of ApdMAVS in a manner independent of E3 ligase activity. Furthermore, ApdTRIM13 recruits the autophagic cargo receptor duck SQSTM1 (ApdSQSTM1), which facilitates its interaction with ApdMAVS independent of ubiquitin signaling, and subsequently delivers ApdMAVS to phagophores for degradation. Depletion of ApdSQSTM1 reduces ApdTRIM13-mediated autophagic degradation of ApdMAVS, thereby enhancing the antiviral immune response. Collectively, our findings reveal a novel mechanism by which ApdTRIM13 regulates type I interferon production by targeting ApdMAVS for selective autophagic degradation mediated by ApdSQSTM1, providing insights into the crosstalk between selective autophagy and innate immune responses in avian species. Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CARD: caspase recruitment domain; co-IP: co-immunoprecipitation; DEFs: duck embryonic fibroblasts; DTMUV: duck Tembusu virus; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; IKBKE/IKKε: inhibitor of nuclear factor kappa B kinase subunit epsilon; IP: immunoprecipitation; IRF7: interferon regulatory factor 7; ISRE: interferon-stimulated response element; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB: nuclear factor kappa B; pAb: polyclonal antibody; poly(I:C): Polyriboinosinic polyribocytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like-receptor; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious dose; TM: tansmembrane; TOLLIP: toll interacting protein; TRIM: tripartite motif containing; UBA: ubiquitin-associated domain; Ub: ubiquitin; VSV: vesicular stomatitis virus; WT: wild type.

Targeting PRMT7-mediated monomethylation of MAVS enhances antiviral innate immune responses and inhibits RNA virus replication

RIG-I-like receptors (RLRs)-mitochondrial antiviral signaling protein (MAVS) are crucial for type I interferon (IFN) signaling pathway and innate immune responses triggered by RNA viruses. However, the regulatory molecular mechanisms underlying RNA virus-activated type I IFN signaling pathway remain incompletely understood. Here, we found that protein arginine methyltransferase 7 (PRMT7) serves as a negative regulator of the type I IFN signaling pathway by interacting with MAVS and catalyzing monomethylation of arginine 232 (R232me1) in MAVS. RNA virus infection leads to the downregulation and dissociation of PRMT7 from MAVS as well as the decrease of R232me1 methylation, enhancing MAVS/RIG-I interaction, MAVS aggregation, type I IFN signaling activation, and antiviral immune responses. Knock-in mice with MAVS R232 substituted with lysine (MavsR232K-KI) are more resistant to Vesicular Stomatitis Virus infection due to enhanced antiviral immune responses. PiPRMT7-MAVS, a short peptide inhibitor designed to interrupt the interaction between PRMT7 and MAVS, inhibits R232me1 methylation, thereby enhancing MAVS/RIG-I interaction, promoting MAVS aggregation, activating type I IFN signaling, and bolstering antiviral immune responses to suppress RNA virus replication. Moreover, the clinical relevance of PRMT7 is highlighted that it is significantly downregulated in RNA virus-infected clinical samples, such as blood samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus, as well as H1N1-infected bronchial epithelial cells. Our findings uncovered that PRMT7-mediated arginine methylation plays critical roles in regulating MAVS-mediated antiviral innate immune responses, and targeting arginine methylation might represent a therapeutic avenue for treating RNA viral infection.

IMMU-42. UNRAVELING THE ROLE OF NLRX1 IN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract •Gliomas are primary brain tumors that develop from glial cells within the central nervous system (CNS) and are among the deadliest human cancers. Glioblastoma (GBM) is the most aggressive glioma. Innate immune cells such as microglia and macrophages account for >50% of the cellular population within the GBM microenvironment. Innate immune cells express pattern recognition receptors (PRRs). NLRX1 is a PRR that regulates diverse signaling pathways and cellular processes including antiviral signaling, ROS production, apoptosis, autophagy, metabolic homeostasis, etc. Additionally, the tumor-suppressive and tumor-promoting functions of NLRX1 have been observed in many cancer types. For example, the tumor-suppressive role of NLRX1 has been observed in colitis-associated carcinogenesis, pancreatic cancer, and primary breast cancer. In contrast, the tumor-promoting role of NLRX1 was found in basal-like and metastatic breast carcinoma. Hence, the effect of NLRX1 on cancer may be context-dependent on cancer type or cell type aided by differences in the microenvironment. Further, how NLRX1 regulates GBM pathophysiology remains largely unexplored. This study aims to determine the expression pattern of NLRX1 in GBM cells and GBM-associated innate immune cells and investigate how NLRX1 expression regulates various cellular processes in GBM cell lines to regulate GBM pathophysiology. We report that NLRX1 is differentially expressed in GBM cell lines, GBM-associated innate immune cells, and glioma patient tissues. si-RNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229 to proliferate and migrate. Further, Nlrx1-/- GBM cells show increased tunneling nanotube (TNT) formation capabilities. TNTs help in metabolic homeostasis maintenance by enabling the exchange of subcellular organelles such as mitochondria and endoplasmic reticulum. Nlrx1-/- GBM cells exhibit attenuated ability to form 3D spheroids. This research will aid the understanding of GBM pathophysiology, leading to novel diagnostic/prognostic markers and the discovery of signaling pathways that, in turn, may help create better GBM therapy approaches and improve overall survival.

An NLR family member X1 mutation (p.Arg707Cys) suppresses hepatitis B virus infection in hepatocytes and favors the interaction of retinoic acid-inducible gene 1 with mitochondrial antiviral signaling protein

NLR family member X1 (NLRX1) is an important member of the NOD-like receptor (NLR) family and plays unique roles in immune system regulation. Patients with hepatitis B virus (HBV) infection are more likely to have the NLRX1 mutation p.Arg707Cys than healthy individuals. It has been reported that NLRX1 increases the infection rate of HBV in HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). However, the role of NLRX1 mutation (p.Arg707Cys) in hepatitis remains unclear. We constructed Huh7 cells that stably overexpressed NTCP, using LV003 lentivirus. First, wild-type (WT) and mutant (MT) NLRX1 overexpression plasmids were constructed. The MT plasmid contained a point mutation at position 707 of the WT overexpression plasmid. Then, Huh7-NTCP cells were transfected with the WT or MT NLRX1 overexpression plasmid, and subsequent NLRX1 expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. HBV RNA levels were determined using RT-qPCR. HBsAg and HBcAg levels were confirmed immunohistochemically. Interferon alpha (IFN-α), interleukin 6 (IL-6), and type I interferon beta (IFN-β) levels were determined using enzyme-linked immunosorbent assay kits. p-p65, p-interferon regulatory factor (IRF) 3, and p-IRF7 expression levels were examined using western blot. The interaction of NLRX1 and retinoic acid-inducible gene (RIG)-1/mitochondrial antiviral signaling (MAVS) protein was confirmed by coimmunoprecipitation. The interaction of NLRX1 with IFN-α, IL-6, or IFN-β was analyzed by dual luciferase reporter gene assay. The levels of HBV RNA, HBsAg, and HBcAg in infected cells transfected with the WT NLRX1 or MT NLRX1 expression plasmid were higher than those in the untransfected control group; and these levels were lower in the cells transfected with MT NLRX1 than in those transfected with WT NLRX1. The levels of IFN-α, IFN-β, IL-6, p-p65, p-IRF3, and p-IRF7 were lower in cells transfected with WT NLRX1 or MT NLRX1 than in control cells. The levels of IFN-β, p-p65, p-IRF3, and p-IRF7 were higher in cells transfected with MT NLRX1 than in those transfected with WT NLRX1. Moreover, NLRX1 competitively inhibited RIG1 binding to MAVS, but the mutation in MT NLRX1 reduced this inhibitory effect. In addition, NLRX1 decreased the promoter activity of IFN-α, IFN-β, and IL-6. Our findings revealed that NLRX1 is a regulatory factor that inhibits the anti-HBV ability of hepatocytes and that the mutation p.Arg707Cys in NLRX1 suppresses HBV infection and activates the IFN/nuclear factor κB pathway.

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.

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.

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.

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 homolog 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.