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Viruses are obligate intracellular pathogens that hijack a myriad of host cell processes to facilitate replication and suppress host antiviral defenses. In its essence, a virus is a segment of foreign nucleic acid that engages host cell machinery to drive viral genome replication, gene transcription, and protein synthesis to generate progeny virions. Because of this, host organisms have developed sophisticated detection systems that activate antiviral defenses following recognition of aberrant nucleic acids. For example, recognition of viral nucleic acids by host DNA repair proteins results in compromised viral genome integrity, induction of antiviral inflammatory programs, cell cycle arrest, and apoptosis. Unsurprisingly, diverse viral families have evolved multiple strategies that fine-tune host DNA repair responses to suppress activation of antiviral defenses while simultaneously hijacking DNA repair proteins to facilitate virus replication. This review summarizes common molecular strategies viruses deploy to exploit host DNA repair mechanisms. [ABSTRACT FROM AUTHOR]

Viruses are obligate intracellular pathogens that hijack a myriad of host cell processes to facilitate replication and suppress host antiviral defenses. In its essence, a virus is a segment of foreign nucleic acid that engages host cell machinery to drive viral genome replication, gene transcription, and protein synthesis to generate progeny virions. Because of this, host organisms have developed sophisticated detection systems that activate antiviral defenses following recognition of aberrant nucleic acids. For example, recognition of viral nucleic acids by host DNA repair proteins results in compromised viral genome integrity, induction of antiviral inflammatory programs, cell cycle arrest, and apoptosis. Unsurprisingly, diverse viral families have evolved multiple strategies that fine-tune host DNA repair responses to suppress activation of antiviral defenses while simultaneously hijacking DNA repair proteins to facilitate virus replication. This review summarizes common molecular strategies viruses deploy to exploit host DNA repair mechanisms.

Throughout their life cycle, viruses interact with cellular host factors, thereby influencing propagation, host range, cell tropism and pathogenesis. The hepatitis E virus (HEV) is an underestimated RNA virus in which knowledge of the virus–host interaction network to date is limited. Here, two related high-throughput mammalian two-hybrid approaches (MAPPIT and KISS) were used to screen for HEV-interacting host proteins. Promising hits were examined on protein function, involved pathway(s), and their relation to other viruses. We identified 37 ORF2 hits, 187 for ORF3 and 91 for ORF4. Several hits had functions in the life cycle of distinct viruses. We focused on SHARPIN and RNF5 as candidate hits for ORF3, as they are involved in the RLR-MAVS pathway and interferon (IFN) induction during viral infections. Knocking out (KO) SHARPIN and RNF5 resulted in a different IFN response upon ORF3 transfection, compared to wild-type cells. Moreover, infection was increased in SHARPIN KO cells and decreased in RNF5 KO cells. In conclusion, MAPPIT and KISS are valuable tools to study virus–host interactions, providing insights into the poorly understood HEV life cycle. We further provide evidence for two identified hits as new host factors in the HEV life cycle. [ABSTRACT FROM AUTHOR]

Individuals with chronic obstructive pulmonary disease (COPD) are more susceptible to exacerbation crisis triggered by secondary lung infections due to the dysfunction of antiviral signaling, principally via suppression of IFN-γ. Although the probiotic is known for controlling pulmonary inflammation in COPD, the influence of the Lactobacillus rhamnosus (Lr) on antiviral signaling in bronchial epithelium exposed to cigarette smoke extract (CSE) and viruses, remains unknown. Thus, the present study investigated the Lr effect on the antiviral signaling and the secretion of inflammatory mediators from bronchial epithelial cells (16HBE cells) exposed to CSE and SARS-CoV-2. The 16HBE cells were cultured, treated with Lr, stimulated with CSE, and infected with SARS-CoV-2. The cellular viability was evaluated using the MTT assay and cytotoxicity measured by lactate dehydrogenase (LDH) activity. The viral load, TLR2, TLR3, TLR4, TLR7, TLR8, MAVS, MyD88, and TRIF were quantified using specific PCR. The pro-inflammatory mediators were measured by a multiplex biometric immunoassay, and angiotensin converting enzyme 2 (ACE2) activity, NF-κB, RIG-I, MAD5, and IRF3 were measured using specific ELISA kits. Lr decreased viral load, ACE2, pro-inflammatory mediators, TLR2, TLR4, NF-κB, TLR3, TLR7, and TLR8 as well as TRIF and MyD88 expression in CSE and SARS-CoV-2 -exposed 16HBE cells. Otherwise, RIG-I, MAD5, IRF3, IFN-γ, and the MAVS expression were restored in 16HBE cells exposed to CSE and SARS-CoV-2 and treated with Lr. Lr induces antiviral signaling associated to IFN-γ secreting viral sensors and attenuates cytokine storm associated to NF-κB in bronchial epithelial cells, supporting its emerging role in prevention of COPD exacerbation. [ABSTRACT FROM AUTHOR]

Type I IFN (IFN‐I) is the body's first line of defense against pathogen infection. IFN‐I can induce cellular antiviral responses and therefore plays a key role in driving antiviral innate and adaptive immunity. Canonical IFN‐I signaling activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which induces the expression of IFN‐stimulated genes and eventually establishes a complex antiviral state in the cells. Ubiquitin is a ubiquitous cellular molecule for protein modifications, and the ubiquitination modifications of protein have been recognized as one of the key modifications that regulate protein levels and/or signaling activation. Despite great advances in understanding the ubiquitination regulation of many signaling pathways, the mechanisms by which protein ubiquitination regulates IFN‐I‐induced antiviral signaling have not been explored until very recently. This review details the current understanding of the regulatory network of ubiquitination that critically controls the IFN‐I‐induced antiviral signaling pathway from three main levels, including IFN‐I receptors, IFN‐I‐induced cascade signals, and effector IFN‐stimulated genes. [ABSTRACT FROM AUTHOR]

ABSTRACT Extracellular vesicles (EVs) are produced by most known cell types as a form of intercellular communication to influence the physiological function of neighboring cells. During respiratory viral-bacterial coinfection, the preceding antiviral response can lead to an impaired antibacterial response, driven by miscommunication between cells responding to viruses and cells responding to bacteria. Previous studies have shown that antiviral signaling can influence EV cargo and promote antiviral defense in the recipient cell; however, how antiviral EVs may influence host defense against coinfecting microorganisms, specifically bacteria, is not known. Herein, we demonstrated that EVs released from the respiratory epithelium during antiviral signaling alter macrophage inflammatory signaling, induce anti-inflammatory metabolic reprogramming, and impair antibacterial activity against Staphylococcus aureus, a common coinfecting bacterial pathogen. Further proteomic analysis revealed that antiviral EVs are preferentially loaded with pyruvate kinase M2 (PKM2), a metabolic enzyme with immunomodulatory effects, and treatment with antiviral EVs leads to increased PKM2 in macrophages. Moreover, we showed that antiviral EV-treated macrophages displayed enhanced oxidative phosphorylation, a metabolic profile consistent with impaired S. aureus clearance, and that this metabolic state is phenocopied in macrophages treated with a PKM2 activator. Taken together, our findings identify EVs as a component of the epithelial antiviral response that contributes to impaired bacterial clearance through epithelial-macrophage crosstalk and suggest a role for EVs in driving disease progression during respiratory coinfection. IMPORTANCE Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2a (eIF2a) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-ß expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-ß mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-ß, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-ß expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-ß protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-ß in RIG-I-mediated antiviral immune responses. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) mediates innate immune response upon binding to cyclic GMP-AMP (cGAMP). It recruits tank-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3) through its C-terminal tail and facilitates TBK1-dependent phosphorylation of IRF3 via forming STING polymers in mammalian cells. However, the mechanism behind STING-mediated activation of NF-κB transcription factor, Relish, in insect cells is unknown. Our study revealed that insect STING formed oligomers and the cryptic RIP homotypic interaction motif (cRHIM) was required for its oligomerization and its anti-viral functions. Cells expressing cRHIM-deficient mutants exhibited lower levels of anti-viral molecules, higher viral load after viral infection and weak activation of Relish. Moreover, we observed that under cGAMP stimulation, insect STING interacted with IMD, and deletion of the cRHIM motif on either protein prevented this interaction. Finally, we demonstrated that cGAMP enhanced the amyloid-like property of insect STING aggregates by ThT staining. In summary, our research showed that insect STING employed a homotypic motif to form intermolecular interactions that are essential for its antiviral signaling. [Display omitted] • A cryptic RHIM (cRHIM) is required for the oligomerization and antiviral signaling of insect STING. • Insect STING interacts with IMD via their cRHIMs under cGAMP stimulation. • CGAMP enhances the amyloid-like property of insect STING aggregates. [ABSTRACT FROM AUTHOR]

The abnormal innate immune response is a prominent feature underlying autoimmune diseases. One emerging factor that can trigger dysregulated immune activation is cytosolic mitochondrial double-stranded RNAs (mt-dsRNAs). However, the mechanism by which mt-dsRNAs stimulate immune responses remains poorly understood. Here, we discover SRA stem-loop interacting RNA binding protein (SLIRP) as a key amplifier of mt-dsRNA-triggered antiviral signals. In autoimmune diseases, SLIRP is commonly upregulated, and targeted knockdown of SLIRP dampens the interferon response. We find that the activation of melanoma differentiation-associated gene 5 (MDA5) by exogenous dsRNAs upregulates SLIRP, which then stabilizes mt-dsRNAs and promotes their cytosolic release to activate MDA5 further, augmenting the interferon response. Furthermore, the downregulation of SLIRP partially rescues the abnormal interferon-stimulated gene expression in autoimmune patients' primary cells and makes cells vulnerable to certain viral infections. Our study unveils SLIRP as a pivotal mediator of interferon response through positive feedback amplification of antiviral signaling., Competing Interests: DECLARATION OF INTERESTS Jaeseon Lee, Soojin Jang, and Kyung Jin Lee are employees of ORGANOIDSCIENCES Ltd. Other authors have no conflict of interest to declare.

Calcium ions (Ca2+) act as secondary messengers in a plethora of cellular processes and play crucial role in cellular organelle function and homeostasis. The average resting concentration of Ca2+ is nearly 100 nM and in certain cells it can reach up to 1 µM. The high range of Ca2+ concentration across the plasma membrane and intracellular Ca2+ stores demands a well-coordinated maintenance of free Ca2+ via influx, efflux, buffering and storage. Endoplasmic Reticulum (ER) and Mitochondria depend on Ca2+ for their function and also serve as major players in intracellular Ca2+ homeostasis. The ER-mitochondria interplay helps in orchestrating cellular calcium homeostasis to avoid any detrimental effect resulting from Ca2+ overload or depletion. Since Ca2+ plays a central role in many biological processes it is an essential component of the virus-host interactions. The large gradient across membranes enable the viruses to easily modulate this buffered environment to meet their needs. Viruses exploit Ca2+ signaling to establish productive infection and evade the host immune defense. In this review we will detail the interplay between the viruses and cellular & ER-mitochondrial calcium signaling and the significance of these events on viral life cycle and disease pathogenesis. [ABSTRACT FROM AUTHOR]

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor‐induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV‐TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN‐based antiviral responses upon EHDV‐TAU challenge, and with potency of EHDV‐TAU‐induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV‐TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT‐based responses upon viral infection, EHDV‐TAU infected B16F10 cells secreted immune‐stimulatory chemokines. Accordingly, in vivo, EHDV‐TAU enhanced intratumoral infiltration of cytotoxic T‐cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV‐TAU have the potential to exploit the cellular context of low‐STAT1 tumors. What's new? The ability of oncolytic viruses to exploit tumor‐induced defects in antiviral responses suggests their potential to efficiently target definite subsets of melanomas, yet these should be defined. Here, the authors show that STAT1 is at the center of a tumor signature involving genes that mediate antiviral and immune activation responses. Using a murine melanoma model and a novel oncolytic virus, EHDV‐TAU, they identify epigenetic silencing as contributing to the underexpression of antiviral genes. Moreover, they demonstrate the potential of EHDV‐TAU to exploit this molecular landscape for effective oncolysis and immune stimulation. [ABSTRACT FROM AUTHOR]

• G5 (DUB inhibitor) affected Type I IFNs and NF-□B signaling induced by poly(I:C). • G5 (DUB inhibitor) affected NEMO ubiquitination induced by poly(I:C). • DUB gene differential expression induced poly(I:C) was analyzed in THP-1 cell. • New DUBs were screened as regulators in antiviral signaling. Type I interferons (IFNs) play a central role in host defense against viral infection. Multiple posttranslational modifications including ubiquitination and deubiquitination regulate the function of diverse molecules in type I IFN signaling. Many ubiquitin ligase enzymes, such as those of the TRAF and TRIM families, have been shown to participate in the production of type I IFNs and inflammatory cytokines. However, the function of deubiquitinating enzymes (DUBs), a protein family that counteracts the action of protein ubiquitination, on the regulation of antiviral immune responses is not well understood. In this study, we used the broad-spectrum DUB inhibitor G5 to reveal their function in antiviral signaling, and then systematically analyzed mRNA expression of the DUB genes upon poly (I:C) treatment in THP-1 cells. Based on this analysis, we cloned some DUB genes whose expression changed and determined their function in antiviral signaling. Taken together, we present a comprehensive DUB gene expression analysis in THP-1 cells, and suggest the involvement of this family of proteins in the regulation of host antiviral activities. [ABSTRACT FROM AUTHOR]

Cells express multiple molecules aimed at detecting incoming virus and infection. Recognition of virus infection leads to the production of cytokines, chemokines and restriction factors that limit virus replication and activate an adaptive immune response offering long-term protection. Recognition of cytosolic DNA has become a central immune sensing mechanism involved in infection, autoinflammation, and cancer immunotherapy. Vaccinia virus (VACV) is the prototypic member of the family Poxviridae and the vaccine used to eradicate smallpox. VACV harbors enormous potential as a vaccine vector and several attenuated strains are currently being developed against infectious diseases. In addition, VACV has emerged as a popular oncolytic agent due to its cytotoxic capacity even in hypoxic environments. As a poxvirus, VACV is an unusual virus that replicates its large DNA genome exclusively in the cytoplasm of infected cells. Despite producing large amounts of cytosolic DNA, VACV efficiently suppresses the subsequent innate immune response by deploying an arsenal of proteins with capacity to disable host antiviral signaling, some of which specifically target cytosolic DNA sensing pathways. Some of these strategies are conserved amongst orthopoxviruses, whereas others are seemingly unique to VACV. In this review we provide an overview of the VACV replicative cycle and discuss the recent advances on our understanding of how VACV induces and antagonizes innate immune activation via cytosolic DNA sensing pathways. The implications of these findings in the rational design of vaccines and oncolytics based on VACV are also discussed.

The innate antiviral response to RNA viruses is initiated by sensing of viral RNAs by RIG-I-like receptors and elicits type I interferon (IFN) production, which stimulates the expression of IFN-stimulated genes that orchestrate the antiviral response to prevent systemic infection. Negative regulation of type I IFN and its master regulator, transcription factor IRF7, is essential to maintain immune homeostasis. We previously demonstrated that AIP (aryl hydrocarbon receptor interacting protein) functions as a negative regulator of the innate antiviral immune response by binding to and sequestering IRF7 in the cytoplasm, thereby preventing IRF7 transcriptional activation and type I IFN production. However, it remains unknown how AIP inhibition of IRF7 is regulated. We show here that the kinase TBK1 phosphorylates AIP and Thr40 serves as the primary target for TBK1 phosphorylation. AIP Thr40 plays critical roles in regulating AIP stability and mediating its interaction with IRF7. The AIP phosphomimetic T40E exhibited increased proteasomal degradation and enhanced interaction with IRF7 compared with wildtype AIP. AIP T40E also blocked IRF7 nuclear translocation, which resulted in reduced type I IFN production and increased viral replication. In sharp contrast, AIP phosphonull mutant T40A had impaired IRF7 binding, and stable expression of AIP T40A in AIP-deficient mouse embryonic fibroblasts elicited a heightened type I IFN response and diminished RNA virus replication. Taken together, these results demonstrate that TBK1-mediated phosphorylation of AIP at Thr40 functions as a molecular switch that enables AIP to interact with and inhibit IRF7, thus preventing overactivation of type I IFN genes by IRF7., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells express multiple molecules aimed at detecting incoming virus and infection. Recognition of virus infection leads to the production of cytokines, chemokines and restriction factors that limit virus replication and activate an adaptive immune response offering long-term protection. Recognition of cytosolic DNA has become a central immune sensing mechanism involved in infection, autoinflammation, and cancer immunotherapy. Vaccinia virus (VACV) is the prototypic member of the family Poxviridae and the vaccine used to eradicate smallpox. VACV harbors enormous potential as a vaccine vector and several attenuated strains are currently being developed against infectious diseases. In addition, VACV has emerged as a popular oncolytic agent due to its cytotoxic capacity even in hypoxic environments. As a poxvirus, VACV is an unusual virus that replicates its large DNA genome exclusively in the cytoplasm of infected cells. Despite producing large amounts of cytosolic DNA, VACV efficiently suppresses the subsequent innate immune response by deploying an arsenal of proteins with capacity to disable host antiviral signaling, some of which specifically target cytosolic DNA sensing pathways. Some of these strategies are conserved amongst orthopoxviruses, whereas others are seemingly unique to VACV. In this review we provide an overview of the VACV replicative cycle and discuss the recent advances on our understanding of how VACV induces and antagonizes innate immune activation via cytosolic DNA sensing pathways. The implications of these findings in the rational design of vaccines and oncolytics based on VACV are also discussed. [ABSTRACT FROM AUTHOR]

It is well established that polyubiquitin chains, in particular those linked through K48 and K63, play a key role in the regulation of the antiviral innate immune response. However, the role of the atypical chains linked via any of the other lysine residues (K6, K11, K27, K29, and K33) and the M1-linked linear chains have not been investigated very well yet in this context. This is partially due to a lack of tools to study these linkages in their biological context. Interestingly though, recent findings underscore the importance of the atypical chains in the regulation of the antiviral immune response. This review will highlight the most important advances in the study of the role of atypical ubiquitin chains, particularly in the regulation of intracellular antiviral innate immune signaling pathways. We will also discuss the development of new tools and how these can increase our knowledge of the role of atypical ubiquitin chains.

The mammalian STE20-like (MST) protein kinases are composed of MST1, MST2, MST3, MST4 and YSK1. They play crucial roles in cell growth, migration, polarity and apoptosis. Dysfunction of these kinases often leads to diseases. MST kinases are extensively involved in development and function of immune system. Here, we review recent progresses on the regulatory function of MST kinases in innate immune signaling.

Importance: Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections., Competing Interests: The authors declare no conflict of interest.

Peroxisomes, in concert with mitochondria, have been established as platforms for the establishment of a rapid and stable antiviral immune response, due to the presence of the mitochondrial antiviral signaling protein (MAVS) at their membranes. Upon intracellular recognition of viral RNA, retinoic acid inducible gene-I (RIG-I)-like proteins interact with MAVS, inducing its oligomerization and the establishment of a signaling cascade that culminates with the production of direct antiviral effectors, preventing important steps in viral propagation. We and others have demonstrated that different viruses have developed specific mechanisms to counteract peroxisome-dependent antiviral signaling. We have shown that the human cytomegalovirus (HCMV) protein vMIA hijacks the peroxisome transport machinery to travel to the organelle, interact with MAVS, and inhibit the immune response. Here, we further unravel the mechanisms by which HCMV is able to evade peroxisome-dependent antiviral signaling. We demonstrate that vMIA localizes at the peroxisomes in a complex with MAVS and the stimulator of interferon genes (STING) protein. Furthermore, vMia interacts with mitochondrial fission factor (MFF) at the peroxisomal membrane, which we show to be essential for vMia-dependent inhibition of the antiviral immune response. Importantly, we demonstrate that vMIA’s interaction with MAVS impedes its oligomerization and the consequent activation of the downstream signaling cascade. Interestingly, our results underline important differences between vMIA’s mechanisms of action at the peroxisomes and the mitochondria. Our results unravel novel mechanisms involving the interplay between the HCMV and peroxisomes that may ultimately contribute to the discovery of novel targets for antiviral combat.

Hepatitis C virus (HCV) infections constitute a major public health problem and are the main cause of chronic hepatitis and liver disease worldwide. The existing drugs, while effective, are expensive and associated with undesirable secondary effects. There is, hence, an urgent need to develop novel therapeutics, as well as an effective vaccine to prevent HCV infection. Understanding the interplay between HCV and the host cells will certainly contribute to better comprehend disease progression and may unravel possible new cellular targets for the development of novel antiviral therapeutics. Here, we review and discuss the interplay between HCV and the host cell innate immunity. We focus on the different cellular pathways that respond to, and counteract, HCV infection and highlight the evasion strategies developed by the virus to escape this intracellular response.

RNF11 (RING finger protein 11) has been indicated to be related to numerous immunological diseases. As a neo-regulator in innate immunity, RNF11 was proved to inhibit antiviral signaling and interferon-β (IFN-β) production in human cell line. Chicken antiviral signaling pathway is different with other species. However, there is little study about the role of RNF11 in chicken antiviral signaling pathway and inflammatory response. This study was designed to identify the function of RNF11 in chicken antiviral signaling pathway and inflammatory response. We overexpressed and interfered RNF11 in chicken embryo fibroblasts (CEFs) and then used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to investigate the poly (I:C)-induced production of IFN-β and inflammatory response factors IL2 and IL10. The results indicated that RNF11 extremely significantly decreased the production of IFN-β and highly significantly increased the messenger RNA (mRNA) levels of IL2 and IL10, but depletion of RNF11 obtained inverse results. Thus, we concluded that RNF11 not only can inhibit antiviral signaling but also plays an important role in inflammatory response in chicken. [ABSTRACT FROM AUTHOR]

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication. [Display omitted] • SGs prevent excessive activation of dsRNA-induced innate immune signaling • dsRNA triggers MAVS-dependent immune-mediated apoptosis in SG-deficient cells • SGs regulate viral replication in RLR-dependent and -independent manners • SGs protect cells from self-derived dsRNA-mediated immunopathology Paget et al. report that stress granules, condensates that form on cellular stresses, are involved in antiviral innate immunity. Stress granules prevent excessive innate immune activation to protect cells from immune-mediated cell death in viral infections and auto-immunopathology diseases. This suggests a function for SGs to maintain cellular homeostasis. [ABSTRACT FROM AUTHOR]

Background: Mast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections., Objective: We studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways., Methods: Mast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca 2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient Kit W-sh/W-sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis., Results: Unexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis., Conclusion: This study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Kawakami, Kimura, Widjaja, Kasakura, Ando, Kawakami, Obar and Kawakami.)

Immune responses against HSV-1 and HSV-2 are complex and involve a delicate interplay between innate signaling pathways and adaptive immune responses. The innate response to HSV involves the induction of type I IFN, whose role in protection against disease is well characterized in vitro and in vivo. Cell types such as NK cells and pDCs contribute to innate anti-HSV responses in vivo. Finally, the adaptive response includes both humoral and cellular components that play important roles in antiviral control and latency. This review summarizes the innate and adaptive effectors that contribute to susceptibility, immune control and pathogenesis of HSV, and highlights the delicate interplay between these two important arms of immunity.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-β expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-β mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-β, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-β expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-β protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-β in RIG-I-mediated antiviral immune responses., (© 2023 Federation of American Societies for Experimental Biology.)

The importance of peroxisomes in the context of viral infections has been increasingly demonstrated in recent years. The discovery that MAVS localizes at peroxisomes and that peroxisomal and mitochondrial MAVS perform complementing functions within the antiviral response has raised the interest in studying the peroxisome-dependent signaling in the context of infection by different viruses. To that end, specific experimental procedures should be applied, taking into consideration the endogenous localization of MAVS at both organelles. The analysis of peroxisomal MAVS activation requires, hence, the preliminar generation and validation of cell lines where MAVS localizes solely at peroxisomes, as well as other specific cellular tools. Here, we present a detailed protocol to analyse the peroxisome-dependent antiviral response, using virus-specific and virus-unspecific stimuli., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Retinoic acid-inducible gene I (RIG-I) initiates a rapid innate immune response upon detection and binding to viral ribonucleic acid (RNA). This signal activation occurs only when pathogenic RNA is identified, despite the ability of RIG-I to bind endogenous RNA while surveying the cytoplasm. Here we show that ATP binding and hydrolysis by RIG-I play a key role in the identification of viral targets and the activation of signaling. Using biochemical and cell-based assays together with mutagenesis, we show that ATP binding, and not hydrolysis, is required for RIG-I signaling on viral RNA. However, we show that ATP hydrolysis does provide an important function by recycling RIG-I and promoting its dissociation from non-pathogenic RNA. This activity provides a valuable proof-reading mechanism that enhances specificity and prevents an antiviral response upon encounter with host RNA molecules.

RIG-I is a cytosolic receptor recognizing virus-specific RNA structures and initiates an antiviral signaling that induces the production of interferons and proinflammatory cytokines. Because inappropriate RIG-I signaling affects either viral clearance or immune toxicity, multiple regulations of RIG-I have been investigated since its discovery as the viral RNA detector. In this review, we describe the recent progress in research on the regulation of RIG-I activity or abundance. Specifically, we focus on the mechanism that modulates RIG-I-dependent antiviral response through post-translational modifications of or protein-protein interactions with RIG-I. [ABSTRACT FROM AUTHOR]

RIG-I is a well-studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4-Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG-I. The p97 complex is able to directly bind both non-ubiquitinated RIG-I and the E3 ligase RNF125, promoting K48-linked ubiquitination of RIG-I at residue K181. Viral infection significantly strengthens the interaction between RIG-I and the p97 complex by a conformational change of RIG-I that exposes the CARDs and through K63-linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG-I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy. [ABSTRACT FROM AUTHOR]

Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling. [ABSTRACT FROM AUTHOR]

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that plays a critical role in regulating antiviral signaling. cGAS binds to DNA and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which is essential for downstream signal transduction. The antiviral response is a rapid biological process; however, cGAS itself has relatively low DNA binding affinity, implying that formation of the cGAS-DNA complex requires an additional factor(s) that promotes cGAS-DNA binding, allowing efficient antiviral signal transduction. Here, we report that the Ku proteins (Ku80 and Ku70) directly interact with cGAS and positively regulate cGAS-mediated antiviral signaling. Mechanistically, we find that the interaction of the Ku proteins with cGAS significantly increases the DNA-binding affinity of cGAS and promotes cGAS condensation in the cytosol, thereby enhancing cGAS catalytic activity. Our results show that the Ku proteins are critical partners of cGAS in sensing DNA virus infection and ensuring efficient innate immune signal transduction. [Display omitted] • The Ku proteins, Ku80 and Ku70, interact with cGAS and augment cGAS-mediated signaling • Ku proteins increase the DNA binding affinity of cGAS and promote cGAS condensation • The cGAS-Ku protein interaction increases the catalytic activity of cGAS • Ku proteins function as co-sensors of cGAS to regulate cGAS-STING signaling Tao et al. show that Ku proteins act as co-sensors of cGAS. Ku proteins augment cGAS catalytic activity by increasing the DNA binding affinity of cGAS and promoting cGAS condensation, thus ensuring an efficient innate immune response to DNA virus infection. [ABSTRACT FROM AUTHOR]

The physiological function of the immune system and the response to therapeutic immunomodulators may be sensitive to combinatorial cytokine micro-environments that shape the responses of specific immune cells. Previous work shows that paracrine cytokines released by virus-infected human dendritic cells (DC) can dictate the maturation state of naïve DCs. To understand the effects of paracrine signaling, we systematically studied the effects of combinations cytokines in this complex mixture in generating an antiviral state. After naïve DCs were exposed to either IFNβ or to paracrine signaling released by DCs infected by Newcastle Disease Virus (NDV), microarray analysis revealed a large number of genes that were differently regulated by the DC-secreted paracrine signaling. In order to identify the cytokine mechanisms involved, we identified 20 cytokines secreted by NDV infected DCs for which the corresponding receptor gene is expressed in naïve DCs. By exposing cells to all combinations of 19 cytokines (leave-one-out studies) we identified 5 cytokines (IFNβ, TNFα, IL-1β, TNFSF15 and IL28) as candidates for regulating DC maturation markers. Subsequent experiments identified IFNβ, TNFα and IL1β as the major contributors to this antiviral state. This finding was supported by infection studies in vitro, by T cell activation studies and by in vivo infection studies in mouse. Combination of cytokines can cause response states in DCs that differ from those achieved by the individual cytokines alone. These results suggest that the cytokine microenvironment may act via a combinatorial code to direct the response state of specific immune cells. Further elucidation of this code may provide insight into responses to infection and neoplasia as well as guide the development of combinatorial cytokine immunomodulation for infectious, autoimmune and immunosurveillance-related diseases.

Virus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. [•] TFG negatively regulates RIG-I mediated antiviral signaling. [•] TFG depletion leads to enhanced viral replication. [•] TFG act downstream of MAVS. [Copyright &y& Elsevier]

Sterol metabolites are known to be associated with immune responses. 25-Hydroxycholesterol (25HC) is a cholesterol metabolite that is produced by macrophages. The production of 25HC was reported to be TLR-dependent, indicating the involvement of 25HC in innate immunity. Now Blanc et al. show that macrophages synthesize 25HC to exert an antiviral effect. STAT1 was shown to be the most essential transcriptional factor involved in the induction of cholesterol 25-hydroxylase (Ch25h), the enzyme required to produce 25HC, indicating the importance of STAT1 in oxysterol-mediated innate immunity. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS. [ABSTRACT FROM AUTHOR]

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses. The PB1-F2 protein has been shown to contribute to viral pathogenicity, but the molecular mechanisms for mediating virulence have been unclear. Previous reports demonstrate that PB1-F2 promotes cell death, causes immunopathology and increases pro-inflammatory responses. Our group has identified a single point mutation from asparagine (N) to serine (S) at position 66 in the PB1-F2 protein that dramatically increases the virulence of highly pathogenic avian H5N1 influenza viruses and of the 1918 pandemic strain. In search for the mechanism by which PB1-F2 N66S increases pathogenicity, we have identified and characterized a novel function of PB1-F2, i.e., interferon antagonism, both in vitro and in the mouse model. Here, we discuss a hypothesis for a possible molecular link between the pro-apoptotic and anti-interferon functions of PB1-F2. [ABSTRACT FROM AUTHOR]

Retinoic acid inducible gene-I (RIG-I) is a caspase recruitment domain (CARD) containing protein that acts as an intracellular RNA receptor and senses virus infection. After binding to double stranded RNA (dsRNA) or 5′-triphosphate single stranded RNA (ssRNA), RIG-I transforms into an open conformation, translocates onto mitochondria, and interacts with the downstream adaptor mitochondrial antiviral signaling (MAVS) to induce the production of type I interferon and inflammatory factors via IRF3/7 and NF-κB pathways, respectively. Recently, accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events, such as inflammation and inflammation related diseases, cell proliferation, apoptosis and even senescence. Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields. [ABSTRACT FROM AUTHOR]

Abstract: The deubiquitinating enzyme A20 (also known as TNFAIP3) is essential for maintaining immune homeostasis. A20 is a key regulator of inflammatory, antiviral and apoptotic signaling pathways. Here, we review recent advances illustrating the role of A20 as an essential negative regulator of inflammatory and antiviral signaling. [Copyright &y& Elsevier]

Immune responses against HSV-1 and HSV-2 are complex and involve a delicate interplay between innate signaling pathways and adaptive immune responses. The innate response to HSV involves the induction of type I IFN, whose role in protection against disease is well characterized in vitro and in vivo. Cell types such as NK cells and pDCs contribute to innate anti-HSV responses in vivo. Finally, the adaptive response includes both humoral and cellular components that play important roles in antiviral control and latency. This review summarizes the innate and adaptive effectors that contribute to susceptibility, immune control and pathogenesis of HSV, and highlights the delicate interplay between these two important arms of immunity. [ABSTRACT FROM AUTHOR]

Abstract: The NIK (NSP-interacting kinase)-mediated antiviral signaling pathway was identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). Here, we further characterized this layer of plant innate defense by identifying the ribosomal protein L10 (rpL10), a QM-like protein, as a downstream effector of the antiviral signaling. Although both ribosomal proteins rpL10 and rpL18 were found to associate with NIK1 through yeast two-hybrid screening, the NIK receptors specifically phosphorylated rpL10 in vitro. Furthermore, loss of rpL10 function significantly increased susceptibility to begomovirus infection, recapitulating the phenotype of nik knockout lines. Our results genetically linked rpL10 to the NIK-mediated antiviral signaling. [Copyright &y& Elsevier]

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)