Antiviral Signaling Research Articles

Limited impact of hepatitis A virus 3C protease-mediated cleavage on the functions of NEMO in human hepatocytes.

NF-κB essential modulator (NEMO) is critically involved in the induction of interferons (IFNs) and pro-inflammatory cytokines. Hepatitis A virus (HAV) 3C protease was recently identified to cleave NEMO in non-hepatic cells. This study aimed at understanding efficiency and function of HAV 3C-mediated NEMO cleavage in hepatocytes. HAV 3C protease and its precursor 3CD strongly affected NEMO abundance in ectopic expression models, which was not observed in HAV replicon cells and upon HAV infection. Using a cleavage-resistant NEMO mutant, we found that specific cleavage by 3C only marginally contributed to NEMO degradation, whereas the magnitude of the effect was due to cytotoxic effects induced by 3C activity. Cleavage efficiency generally did not suffice to disrupt the type I IFN or NF-κB signaling pathways. Knockout of NEMO indeed abrogated both pathways, whereas efficient knockdown had limited the impact on NEMO-mediated signaling, suggesting that low levels of NEMO are sufficient to maintain antiviral responses in hepatocytes. NEMO cleavage was barely detectable in a cell line harboring a persistent HAV replicon or in HAV-infected cells. HAV infection induced a robust innate immune response, which was not affected by efficient knockdown of NEMO, arguing for a limited potential contribution of NEMO cleavage to innate immune counteraction. Overall, our data suggest that HAV 3C is capable of partially cleaving NEMO as reported. However, since minute expression levels of NEMO were sufficient for induction of innate immunity, inefficient NEMO cleavage by HAV is unlikely to contribute to dampening of innate immune responses in hepatocytes.IMPORTANCEHepatitis A virus (HAV) establishes acute infections of the liver, which are always cleared, while a number of mechanisms have been identified contributing to immune escape. Among those, proteolytic cleavage of NF-κB essential modulator (NEMO) by HAV has been suggested to counteract innate immune responses. This study demonstrates that the HAV 3C protease cleaves NEMO inefficiently and does not result in substantial disruption of antiviral signaling. Importantly, NEMO remains capable of inducing an effective immune response in hepatocytes even at low expression levels. Our findings suggest a limited role for NEMO cleavage in HAV's interaction with host immunity and call for a revision of our understanding of HAV counteraction mechanisms.

The R203M and D377Y mutations of the nucleocapsid protein promote SARS-CoV-2 infectivity by impairing RIG-I-mediated antiviral signaling.

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.

Proteasome inhibition enhances oncolytic reovirus therapy in multiple myeloma independently of its direct cytotoxic effects

BackgroundReovirus (RV) is an oncolytic virus with natural tropism for cancer cells. We previously showed that RV administration in multiple myeloma (MM) patients was safe, but disease control associated with viral replication in the cancer cells was not observed. The combination with proteasome inhibitors (PIs) has shown to enhance RV therapeutic activity, but the mechanisms of action have not been fully elucidated.MethodsElectron microscopy, q-RT-PCR, single-cell mass cytometry (CyTOF), flow cytometry, plaque assays, immunohistochemistry, and Western blot analysis were used to assess RV infection of both myeloma and immune cells. Immune fluorescence, flow cytometry, and luciferase reporter assays were used to assess NF-κB pathway activation upon RV treatments. Immune profiling changes, both ex vivo and in MM patients, were analyzed by flow cytometry and CyTOF analysis. T-cell receptor (TCR) sequencing was also conducted both in immune competent MM mice and in patients enrolled in a phase 1b trial per a standard 3 + 3 dose escalation schedule.ResultsHere we show ex vivo and in vivo that proteasome inhibitors (PIs) potentiate reovirus replication in circulating classical monocytes, increasing viral delivery to myeloma cells. We found that the anti-viral signals in monocytes primarily rely on NF-κB activation and that this effect is impaired by the addition of PIs. Conversely, the addition of PIs to RV therapy supports immune activation and killing of MM, independently of direct PI sensitivity. To validate the importance of PIs in enhancing oncolytic viral therapy independently of their killing activity on cancer cells, we then conducted a phase 1b trial of the reovirus Pelareorep together with the PI carfilzomib in 13 heavily pretreated PI-resistant MM patients. Objective responses, which were associated with active reovirus replication in MM cells, T cell activation, and monocytic expansion, were noted in 70% of patients.ConclusionsAlthough characterized as immunosuppressive drugs, PIs improved RV delivery to MM cells but also enhanced anti-MM efficacy through immune-mediated killing of myeloma cells, independently of their PI sensitivity. These results highlight a more generalizable use of PIs as therapeutic companions to support oncolytic-based therapies in cancers.Trial registrationclinicaltrials.gov, NCT 02101944.

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

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. Brain-derived host RNA transcript and miRNA expression was examined from persons with or without HAND using bioinformatics platforms. 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). 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. 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.

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.

The Strategies Used by Animal Viruses to Antagonize Host Antiviral Innate Immunity: New Clues for Developing Live Attenuated Vaccines (LAVs).

As an essential type of vaccine, live attenuated vaccines (LAVs) play a crucial role in animal disease prevention and control. Nevertheless, developing LAVs faces the challenge of balancing safety and efficacy. Understanding the mechanisms animal viruses use to antagonize host antiviral innate immunity may help to precisely regulate vaccine strains and maintain strong immunogenicity while reducing their pathogenicity. It may improve the safety and efficacy of LAVs, as well as provide a more reliable means for the prevention and control of infectious livestock diseases. Therefore, exploring viral antagonistic mechanisms is a significant clue for developing LAVs, which helps to explore more viral virulence factors (as new vaccine targets) and provides a vital theoretical basis and technical support for vaccine development. Among animal viruses, ASFV, PRRSV, PRV, CSFV, FMDV, PCV, PPV, and AIV are some typical representatives. It is crucial to conduct in-depth research and summarize the antagonistic strategies of these typical animal viruses. Studies have indicated that animal viruses may antagonize the antiviral innate immunity by directly or indirectly blocking the antiviral signaling pathways. In addition, viruses also do this by antagonizing host restriction factors targeting the viral replication cycle. Beyond that, viruses may antagonize via regulating apoptosis, metabolic pathways, and stress granule formation. A summary of viral antagonistic mechanisms might provide a new theoretical basis for understanding the pathogenic mechanism of animal viruses and developing LAVs based on antagonistic mechanisms and viral virulence factors.

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.

A bacterial NLR-related protein recognizes multiple unrelated phage triggers to sense infection.

Immune systems must rapidly sense viral infections to initiate antiviral signaling and protect the host. Bacteria encode >100 distinct viral (phage) defense systems and each has evolved to sense crucial components or activities associated with the viral lifecycle. Here we used a high-throughput AlphaFold-multimer screen to discover that a bacterial NLR-related protein directly senses multiple phage proteins, thereby limiting immune evasion. Phages encoded as many as 5 unrelated activators that were predicted to bind the same interface of a C-terminal sensor domain. Genetic and biochemical assays confirmed activators bound to the bacterial NLR-related protein at high affinity, induced oligomerization, and initiated signaling. This work highlights how in silico strategies can identify complex protein interaction networks that regulate immune signaling across the tree of life.

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.