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CREB-binding protein (CBP) participates in numerous transcription events. However, cell-intrinsic inhibitors of CBP are poorly defined. Here, we found that cellular USP12 interacts with the HAT domain of CBP and inhibits CBP’s acetyltransferase activity. Interestingly, USP12 positively regulates interferon (IFN) antiviral signaling independently of its deubiquitinase activity. Furthermore, we found that in IFN signaling USP12 translocates from the cytoplasm to the nucleus. The decrease in cytoplasmic USP12 facilitates CBP-induced acetylation and activation of IFN signaling proteins in the cytoplasm. Moreover, USP12 accumulation in the nucleus blocks CBP-induced acetylation of phosphorylated STAT1 (p-STAT1) and therefore inhibits the dephosphorylation effects of TCPTP on p-STAT1, which finally maintains nuclear p-STAT1 levels and IFN antiviral efficacy. USP12 nuclear translocation extends our understanding of the regulation of the strength of IFN antiviral signaling. Our study uncovers a cell-intrinsic regulation of CBP acetyltransferase activity and may provide potential strategies for IFN-based antiviral therapy., Author summary Activated p-STAT1 is a determinant for the strength of IFN antiviral signaling. We and other groups have demonstrated that activated p-STAT1 is regulated by multiple protein post-translational modifications, including phosphorylation, acetylation and ubiquitination. In this study, we revealed that CBP-mediated acetylation regulation of p-STAT1 is modulated by the deubiquitinase USP12 in a deubiquitinase activity-independent manner. USP12 translocates into the nucleus in IFN signaling, which critically regulates nuclear p-STAT1 levels and IFN antiviral activity by inhibiting CBP’s acetyltransferase activity. Importantly, we demonstrated that USP12 is a cell-intrinsic inhibitor of the acetyltransferase CBP. These findings promote the understanding of delicate regulation of both CBP-mediated acetylation and IFN antiviral signaling.

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1’s antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons., Author summary After decades of research on the innate immune system, we still struggle to understand exactly how this first line of defense protects cells against viral infections. Our gap in knowledge stems, on one hand, from the sheer number of effector genes, few of which have been characterized in mechanistic detail. On the other hand, our understanding of innate gene transcription is constantly evolving. We know that different regulatory mechanisms greatly influence the quality, magnitude, and timing of gene expression, all of which may contribute to the antiviral power of the innate response. Deciphering these regulatory mechanisms is indispensable for harnessing the power of innate immunity in novel antiviral therapies. Here, we report a novel transcriptional program as part of the cell-intrinsic immune system, raised by E74-like ETS transcription factor 1 (ELF1). ELF1 potently restricts multi-cycle propagation of all viruses tested in our study. Reduced levels of ELF1 significantly diminish host defenses against influenza A virus in vitro and in vivo, suggesting a critical but previously overlooked role of this ETS transcription factor. The ELF1 program is complex and comprises over 300 potentially antiviral genes, which are almost entirely distinct from those known to be induced by interferon. Taken together, our data provide evidence for a program of antiviral protection that expands the previously known arsenal of the innate immune response.

Mediator of IRF3 activation (MITA, also named as STING/ERIS/MPYS/TMEM173), is essential to DNA virus- or cytosolic DNA-triggered innate immune responses. In this study, we demonstrated the negative regulatory role of RING-finger protein (RNF) 90 in innate immune responses targeting MITA. RNF90 promoted K48-linked ubiquitination of MITA and its proteasome-dependent degradation. Overexpression of RNF90 inhibited HSV-1- or cytosolic DNA-induced immune responses whereas RNF90 knockdown had the opposite effects. Moreover, RNF90-deficient bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMMs) and mouse embryonic fibroblasts (MEFs) exhibited increased DNA virus- or cytosolic DNA-triggered signaling and RNF90 deficiency protected mice from DNA virus infection. Taken together, our findings suggested a novel function of RNF90 in innate immunity., Author summary After the invasion of viruses, the genetic materials, RNA and DNA generated by the viruses, could be recognized by the cytosolic RNA or DNA sensors and then these sensors will initiate the innate immune signaling pathway and protect the host from viral invasion. However, excessive immune responses may cause tissue damage and do harm to the host. Therefore, the innate immune responses should be tightly controlled and turned off timely. MITA is a key adaptor in DNA-sensor signaling pathway. Here, we find that RNF90, a known E3 ubiquitin-protein ligase, interacted with MITA, enhanced the ubiquitination of MITA and promoted the degradation of MITA. RNF90-defient mice showed increased innate immune responses upon DNA virus HSV-1 infection. Our research revealed a new negative regulatory mechanism of the host to manipulate the antiviral innate immune responses.

The stimulator of interferon genes (STING) protein has been shown to play a pivotal role in response to both cytosolic RNA and dsDNA to elicit interferon (IFN) production in mammals. However, the role of duck STING (DuSTING) in antiviral innate immunity, especially in anti-RNA virus infection, has yet to be elucidated. In this study, the function of DuSTING in IFN induction and its role in anti-RNA virus infections were studied. DuSTING was amplified via reverse transcription-polymerase chain reaction (RT-PCR) from Pekin duck, showing that its cDNA sequence contains an open reading frame (ORF) of 1,149 bp and encodes 382 amino acids (aa). Sequence alignment showed that DuSTING protein shares 71.1, 43.4, and 33.3% identity with chickens, humans, and zebra fish, respectively. Overexpression of DuSTING in duck embryo fibroblasts (DEFs) strongly activated IFN-β promotor activity. Deletion mutant analysis revealed that the first 42 aa containing the first transmembrane (TM) domains and the last 32 aa containing a part of the C-terminal tail (CTT) are essential for its IFN-β activation. In vitro experiments showed that the mRNA levels of DuSTING and IFNs were all upregulated when the DEFs were infected with H9N2 avian influenza virus (AIV) SH010, while overexpression of DuSTING inhibited the replication of this virus. In vivo studies showed that DuSTING mRNA was widely expressed in different tissues, and was up-regulated in the spleen and lung of ducks challenged with SH010. In conclusion, our results indicate that DuSTING is an essential IFN mediator and plays a role in anti-RNA virus innate immunity.

Herpes simplex virus type 1 (HSV-1), one of the human pathogens widely epidemic and transmitted among various groups of people in the world, often causes symptoms known as oral herpes or lifelong asymptomatic infection. HSV-1 employs many sophisticated strategies to escape host antiviral immune response based on its multiple coding proteins. However, the functions involved in the immune evasion of miRNAs encoded by HSV-1 during lytic (productive) infection remain poorly studied. Dual-luciferase reporter gene assay and bioinformatics revealed that Asp-Glu-Ala-Asp (DEAD)-box helicase 41 (DDX41), a cytosolic DNA sensor of the DNA-sensing pathway, was a putative direct target gene of HSV-1-encoded miR-H2-3p. The transfection of miR-H2-3p mimics inhibited the expression of DDX41 at the level of mRNA and protein, as well as the expression of interferon beta (IFN-&beta, ) and myxoma resistance protein I (MxI) induced by HSV-1 infection in THP-1 cells, and promoted the viral replication and its gene transcription. However, the transfection of miR-H2-3p inhibitor showed opposite effects. This finding indicated that HSV-1-encoded miR-H2-3p attenuated cytosolic DNA&ndash, stimulated antiviral immune response by manipulating host DNA sensor molecular DDX41 to enhance virus replication in cultured cells.

Many studies have demonstrated that immunobiotics with immunoregulatory functions improve the outcomes of several bacterial and viral infections by modulating the mucosal immune system. However, the precise mechanisms underlying the immunoregulatory and antiviral activities of immunobiotics have not yet been elucidated in detail. The present study was conducted to determine whether selected lactic acid bacteria (LAB) modulate toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (PolyI:C) induced viral response in human intestinal epithelial cells (IECs). PolyI:C increased the expression of interferon-β (IFN-β), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein (MCP-1), and interleukin-1β (IL-1β) in HCT116 cells, and these up-regulations were significantly altered when cells were pre-stimulated with LAB isolated from Korean fermented foods. LAB strains were capable to up-regulate IFN-β but down-regulated IL-6, IL-8, MCP-1, and IL-1β mRNA levels as compared with PolyI: C. HCT-116 cell treatment with LABs beneficially modulated the mRNA levels of C-X-C motif chemokine 10 (CXCL-10), 2-5A oligoadenylate synthetase 1 (OSA1), myxovirus resistance protein (MxA), TLR3, and retinoic acid inducible gene-I (RIG-I), and TLR negative regulators. In addition, LABs increased IFN-β, IFN-α, and interleukin-10 (IL-10) and decreased tumor necrosis factor-α (TNF-α) and IL-1β protein/mRNA levels in THP-1 cells. LABs also protected the cells by maintaining tight-junction proteins (zonula occludens-1 and occludin). The beneficial effects of these LABs were mediated via modulation of the interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) pathways. Overall, the results of this study indicate that immunobiotics have potent antiviral and anti-inflammatory activities that may use as antiviral substitutes for human and animal applications.

Helicase DDX41 is a cytosolic sensor capable of detecting double-stranded DNA in mammals. However, the function of DDX41 remains poorly understood in invertebrates. In a previous study, we identified the first DDX41 sensor in the black tiger shrimp Penaeus monodon (PmDDX41) and showed that it played a role in anti-viral response. In the present study, we demonstrated that PmDDX41 was localized in the cytoplasm of shrimp hemocytes. However, PmDDX41 was localized in both the cytoplasm and nucleus of hemocytes in the presence of white spot syndrome virus (WSSV) infection or when stimulated by the nucleic acid mimics, poly(dA:dT) and poly(I:C). Similar results were observed when PmDDX41 was transfected into human embryonic kidney 293T (HEK293T) cells. Immunoprecipitation further demonstrated that PmDDX41 bound to biotin-labeled poly(dA:dT) but not poly(I:C). The overexpression of shrimp PmDDX41 and mouse stimulator of interferon gene (MmSTING) in HEK293T cells synergistically promoted IFN-β and NF-κB promoter activity via the DEADc domain. Co-immunoprecipitation (Co-IP) also confirmed that there was an interaction between PmDDX41 and STING after stimulation with poly(dA:dT) but not poly(I:C). Our study is the first to demonstrate that PmDDX41 acts as a cytosolic DNA sensor that interacts with STING via its DEADc domain to trigger the IFN-β and NF-κB signaling pathways, thus activating antiviral innate immune responses.

Cyclic GMP-AMP synthase (cGAS) senses viral DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the ER-localized adaptor protein Mediator of IRF3 Activator (MITA) to initiate innate antiviral response. Human cytomegalovirus (HCMV) proteins can antagonize host immune responses to promote latent infection. Here, we identified HCMV UL42 as a negative regulator of cGAS/MITA-dependent antiviral response. UL42-deficiency enhances HCMV-induced production of type I interferons (IFNs) and downstream antiviral genes. Consistently, wild-type HCMV replicates more efficiently than UL42-deficient HCMV. UL42 interacts with both cGAS and MITA. UL42 inhibits DNA binding, oligomerization and enzymatic activity of cGAS. UL42 also impairs translocation of MITA from the ER to perinuclear punctate structures, which is required for MITA activation, by facilitating p62/LC3B-mediated degradation of translocon-associated protein β (TRAPβ). These results suggest that UL42 can antagonize innate immune response to HCMV by targeting the core components of viral DNA-triggered signaling pathways., Author summary Recognition of viral DNA by the cytosolic DNA sensor cGAS and subsequent induction of type I IFNs via the cGAS-MITA signaling axis are important for host antiviral innate immunity. The human cytomegalovirus (HCMV) causes complications in immunodeficient populations and is a major cause of birth defects. It is known that HCMV suppresses innate immunity, which is pivotal for establishing immune evasion and latent infection. In this study, we found that HCMV protein UL42 inhibits innate antiviral responses thus promotes HCMV replication. UL42 functions by targeting cGAS and MITA through distinct mechanisms. UL42 inhibits cGAS activation by interrupting its DNA binding and oligomerization, while it targets MITA by interfering trafficking of MITA from the ER to perinuclear punctate structures, a process required for MITA activation. These findings defined an important mechanism for HCMV immune evasion, which may provide a therapeutic target for the treatment of HCMV infection.

Next generation sequencing has revealed the widespread occurrence of persistent virus infections in insects but little is known regarding to what extent persistent infections can affect cellular physiology and how they might contribute to the development of disease. In contrast to the pathogenic infections occurring in Drosophila S2 cells, it was observed that Cricket Paralysis virus (CrPV; Dicistroviridae) causes persistent infections in 9 lepidopteran and 2 coleopteran cell lines. The status of the persistent infection was subsequently investigated in more detail using silkworm-derived Bm5 cells, where the infection eventually becomes pathogenic after 3-4 weeks. The short-term persistence period in Bm5 cells is characterized by low levels of viral replication and virion production as well as by the production of viral siRNAs. However, during this period cellular physiology also becomes altered since the cells become susceptible to infection by the nodavirus Flock House virus (FHV). Pathogenicity and widespread mortality at 4 weeks is preceded by a large increase in virion production and the transcriptional activation of immune-related genes encoding RNAi factors and transcription factors in the Toll, Imd and Jak-STAT pathways. During the infection of Bm5 cells, the infective properties of CrPV are not altered, indicating changes in the physiology of the host cells during the transition from short-term persistence to pathogenicity. The in vitro system of Bm5 cells persistently infected with CrPV can therefore be presented as an easily accessible model to study the nature of persistent virus infections and the processes that trigger the transition to pathogenicity, for instance through the application of different "omics" approaches (transcriptomics, proteomics, metabolomics). The different factors that can cause the transition from persistence to pathogenicity in the Bm5-CrPV infection model are discussed. ispartof: Journal of Insect Physiology vol:115 pages:1-11 ispartof: location:England status: published

Objectives Coronavirus disease 2019 (COVID-19) remains a global challenge. Corticosteroids constitute a group of anti-inflammatory and immunosuppressive drugs that are widely used in the treatment of COVID-19. Comprehensive reviews investigating the comparative proportion and efficacy of corticosteroid use are scarce. Therefore, we conducted a systematic review and meta-analysis of clinical trials to evaluate the proportion and efficacy of corticosteroid use for the treatment of COVID-19. Methods We conducted a comprehensive literature review and meta-analysis of research articles, including observational studies and clinical trials, by searching the PubMed, EMBASE, Cochrane Controlled Trials Registry, and China Academic Journal Network Publishing databases. Patients treated between December 1, 2019, and January 1, 2021, were included. The outcome measures were the proportion of patients treated with corticosteroids, viral clearance and mortality. The effect size with the associated 95% confidence interval is reported as the weighted mean difference for continuous outcomes and the odds ratio for dichotomous outcomes. Results Fifty-two trials involving 15710 patients were included. The meta-analysis demonstrated that the proportion of COVID-19 patients who received corticosteroids was significantly lower than that of patients who did not receive corticosteroids (35.19% vs. 64.49%). In addition, our meta-analysis demonstrated no significant difference in the proportions of severe and nonsevere cases treated with corticosteroids (27.91% vs. 20.91%). We also performed subgroup analyses stratified by whether patients stayed in the intensive care unit (ICU) and found that the proportion of patients who received corticosteroids was significantly higher among those who stayed in the ICU than among those who did not. The results of our meta-analysis indicate that corticosteroid treatment significantly delayed the viral clearance time. Finally, our meta-analysis demonstrated no significant difference in the use of corticosteroids for COVID-19 between patients who died and those who survived. This result indicates that mortality is not correlated with corticosteroid therapy. Conclusion The proportion of COVID-19 patients who received corticosteroids was significantly lower than that of patients who did not receive corticosteroids. Corticosteroid use in subjects with severe acute respiratory syndrome coronavirus 2 infections delayed viral clearance and did not convincingly improve survival; therefore, corticosteroids should be used with extreme caution in the treatment of COVID-19.

Increasing evidence suggests important roles for long noncoding RNAs (lncRNAs) as new gene modulators involved in various biological processes. However, the function roles of lncRNAs in lower vertebrates are still unknown. Here, we firstly identify a lncRNA, named MAVS antiviral-related lncRNA (MARL), as a key regulator for antiviral immunity in teleost fish. The results indicate that fish MAVS play essential roles in host antiviral responses and inhibition of Siniperca chuatsi rhabdovirus (SCRV) replication. miR-122 reduces MAVS expression and suppress MAVS-mediated antiviral responses, which may help viruses evade host antiviral responses. Further, MARL functions as a competing endogenous RNA (ceRNA) for miR-122 to control protein abundance of MAVS, thereby inhibiting SCRV replication and promoting antiviral responses. Our data not only shed new light on understanding the function role of lncRNA in biological processes in lower vertebrates, but confirmed the hypothesis that ceRNA regulatory networks exist widely in vertebrates., Author summary Increasing evidence indicates that lncRNAs participate in the regulation of various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and host antiviral responses remains largely unknown, particularly in lower vertebrates. Our results provided the first direct evidence that a lncRNA, termed MAVS antiviral-related lncRNA (MARL), acts as a key regulator for antiviral immunity in lower vertebrates. lncRNAs have been identified to function as competing endogenous RNAs (ceRNAs) and cross-talk with mRNAs by competing shared for miRNAs. Such ceRNAs regulate the distribution of miRNA molecules on their targets and thereby apply an additional level of post-transcriptional regulation. In our study, MARL functions as a ceRNA for miR-122 to control protein abundance of fish MAVS, thereby inhibiting virus replication and promoting antiviral responses. This is the first study to demonstrate ceRNA regulatory networks existing in lower vertebrates, which can provide new insights into understanding the effects of lncRNAs on host-virus interactions.

Background MicroRNAs (miRs) control gene expression and the development of the immune system and antiviral responses. MiR-155 is an evolutionarily-conserved molecule consistently induced during viral infections in different cell systems. Notably, there is still an unresolved paradox for the role of miR-155 during viral respiratory infections. Despite being essential for host antiviral TH1 immunity, miR-155 may also contribute to respiratory disease by enhancing allergic TH2 responses and NFkB-mediated inflammation. The central goal of this study was to define how airway miR-155 production is related to TH1, TH2, and pro-inflammatory cytokine responses during naturally occurring viral respiratory infections in young children. Methods Normalized nasal airway levels of miR-155 and nasal protein levels of IFN-γ, TNF-α, IL-1β, IL-13, IL-4 were quantified in young children (≤2 years) hospitalized with viral respiratory infections and uninfected controls. These data were linked to individual characteristics and respiratory disease parameters. Results A total of 151 subjects were included. Increased miR-155 levels were observed in nasal samples from patients with rhinovirus, RSV and all respiratory viruses analyzed. High miR-155 levels were strongly associated with high IFN-γ production, increased airway TH1 cytokine polarization (IFN-γ/IL-4 ratios) and increased pro-inflammatory responses. High airway miR-155 levels were linked to decreased respiratory disease severity in individuals with high airway TH1 antiviral responses. Conclusions The airway secretion of miR-155 during viral respiratory infections in young children is associated with enhanced antiviral immunity (TH1 polarization). Further studies are needed to define additional physiological roles of miR-155 in the respiratory tract of human infants and young children during health and disease.

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression., Author summary Infection with viruses and other pathogens requires appropriate cellular countermeasures, which involve swift and accurate adaptation of gene expression profiles. mRNAs encoding for immune-regulatory and effector proteins need to be transported into the cytoplasm in order to generate proteins necessary to fight the pathogen. Here we show that this process requires proper functionality of the Nuclear cap protein 3 (NCBP3), a protein recently identified to contribute to an alternative mRNA cap-binding complex. An Ncbp3-deficient mouse model allowed higher virus growth in vitro and showed high susceptibility to influenza A virus challenge in vivo. While NCBP3-deficient cells were able to transcriptionally upregulate cytokine mRNAs, generation of cytokines was significantly reduced in the absence of NCBP3. Our data shows a non-redundant function of NCBP3 and the alternative cap-binding complex in antiviral responses. More broadly, this work demonstrates a yet unappreciated aspect of post-transcriptional gene regulation.

FAS-associated factor-1 (FAF1) is a component of the death-inducing signaling complex involved in Fas-mediated apoptosis. It regulates NF-κB activity, ubiquitination, and proteasomal degradation. Here, we found that FAF1 positively regulates the type I interferon pathway. FAF1gt/gt mice, which deficient in FAF1, and FAF1 knockdown immune cells were highly susceptible to RNA virus infection and showed low levels of inflammatory cytokines and type I interferon (IFN) production. FAF1 was bound competitively to NLRX1 and positively regulated type I IFN signaling by interfering with the interaction between NLRX1 and MAVS, thereby freeing MAVS to bind RIG-I, which switched on the MAVS-RIG-I-mediated antiviral signaling cascade. These results highlight a critical role of FAF1 in antiviral responses against RNA virus infection., Author summary Type I interferon-mediated antiviral response is critical for controlling virus infections. However, interferon-mediated immune responses need to be tightly regulated to maintain host immune homeostasis. Recently, molecules involved in regulating interferon-mediated innate immune response are the subject of much research. Among these, the first protein to be identified as a negative regulator of MAVS was the nucleotide-binding domain and leucine-rich repeat containing family member, NLRX1. NLRX1 associates with MAVS to inhibit antiviral signaling by interrupting virus-induced RLR-MAVS interactions. Interestingly, we found that FAF1 interacts with NLRX1 in response to RNA virus infection and this interaction inhibits binding of MAVS to NLRX1, which in turn switches on RIG-I mediated antiviral immune responses. As results, we showed that FAF1gt/gt mice, which deficient in FAF1, and FAF1 knockdown immune cells were highly susceptible to RNA virus infection and showed low levels of inflammatory cytokines and type I interferon (IFN) production. Our findings suggest that FAF1 is a crucial regulator that induces the antiviral innate immune responses against RNA virus infection.

The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion., Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (MS) and P2013/ABI-2906 (co-financed by Autonomous Community of Madrid and EC FEDER funds) (FS)

During RNA virus infection, the adaptor protein MAVS recruits TRAF3 and TRAF6 to form a signalosome, which is critical to induce the production of type I interferons (IFNs) and proinflammatory cytokines. While activation of the MAVS/TRAF3/TRAF6 signalosome is well studied, the negative regulation of the signalosome remains largely unknown. Here we report that RNA viruses specifically promote the deubiquitinase OTUD1 expression by NF-κB-dependent mechanisms at the early stage of viral infection. Furthermore, OTUD1 upregulates protein levels of intracellular Smurf1 by removing Smurf1 ubiquitination. Importantly, RNA virus infection promotes the binding of Smurf1 to MAVS, TRAF3 and TRAF6, which leads to ubiquitination-dependent degradation of every component of the MAVS/TRAF3/TRAF6 signalosome and subsequent potent inhibition of IFNs production. Consistently, OTUD1-deficient mice produce more antiviral cytokines and are more resistant to RNA virus infection. Our findings reveal a novel immune evasion mechanism exploited by RNA viruses, and elucidate a negative feedback loop of MAVS/TRAF3/TRAF6 signaling mediated by the OTUD1-Smurf1 axis during RNA virus infection., Author summary Extensive studies have demonstrated redundant roles of multiple antiviral signaling pathways in host defense against viral infection. However, the strategies viruses have evolved to efficiently antagonize the complementary effect of alternative IFNs-inducing signaling remain largely unknown. Here we report that RNA viruses specifically promote the expression of the deubiquitinase OTUD1. RNA viruses-induced OTUD1 upregulates intracellular Smurf1 protein levels by deubiquitination modification, and promotes the binding of Smurf1 to MAVS, TRAF3 and TRAF6, which leads to degradation of every component of the MAVS/TRAF3/TRAF6 signalosome and inhibition of IFNs production. Our finding identifies a potent negative regulation of innate antiviral response, which could be crucial for RNA viruses to establish efficient infection at the early stage.

The innate immune system plays a critical role in the initial antiviral response. However, the timing and duration of these responses must be tightly regulated during infection to ensure appropriate immune cell activation and anti-viral defenses. Here we demonstrate that during antiviral response, a negative regulator miR-221 was also induced in an ELF4-dependent manner. We further show that ELF4 promotes miR-221 expression through direct binding to its promoter. Overexpression and knockdown assay show that miR-221 can negatively regulate IFNβ production in time of virus infection. RNA-seq analysis of miR-221 overexpressed cells revealed multiple candidate targets. Taken together, our study identified a novel negative microRNA regulator of innate antiviral response, which is dependent on ELF4.

Background: Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viralstimulus to mimic in vivo and in vitro infection induced by some viruses. Objective: To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture models of peripheral blood mononuclear cells (PBMC) and A549 cells. Methods: T and NK cell activation was evaluated by flow cytometry and levels of TNF-α, IFN-γ, IL-10, IL-29, and IL-17 by ELISA. Cells in direct contact with A549 (PBMC-A549) and cells with no contact with it (PBMC//A549) were used for this purpose. PBMCs alone and both co-culture systems were stimulated for 24 h with the following stimuli: LPS (10 µg/ml), LcM (106 UFC/ml), Poly I:C (2 µg/ml), Poly I:C+LcM, and LcM (3 h)+Poly I:C. Moreover, unstimulated cells were used as a control. Results: Poly I:C and LcM (3 h)+Poly I:C in PBMC-A549 showed a significant increase in the percentage of CD8+ expression (p

Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P

Apstrakt: Hepatitis S virusna infekcija (HCV), jedan od najčešćih uzroka bolesti jetre, česta je komplikacija kod pacijenata sa terminalnom bubrežnom insuficijencijom (eng. end-stage renal disease, ESRD). ESRD se definiše kao smanjena glomerularna filtracija, a takođe je praćena i poremećenom funkcijom imunskog sistema. Galektin-3 je lektin koji povezuje β-galaktozide, uključen u različite biološke procese, uključujući patogenezu hronične bubrežne bolesti. Cilj naše studije je bio da se ispita težina bolesti kod ESRD+ HCV+ pacijenata i analiziraju serumske koncentracije citokina; Anti- HCV antitela; i galektina-3. Takođe, pokušali smo da odredimo potencijalnu korelaciju između nivoa galektina-3 i parametara težine bolesti ALT i AST. Naši rezultati pokazuju smanjene nivoe ALT i AST (p = 0.00), što ukazuje na manje oštećenje jetre kod ESRD+ HCV+ pacijenata u odnosu na HCV+ pacijenate. Povećana koncentracija IL-6 (p = 0.03) implicira hepatoprotektivnu ulogu IL -6 kod ovih pacijenata. Takođe, nivo galektina-3 (r = 0.00) u serumu ESRD+ HCV+ pacijenata veći je nego kod HCV+ pacijenata. Ovu promenu „prati― negativna korelacija između galektina-3 i AST-a i ALT-a (p = 0.029; p = 0.033). Prisustvo povećanih sistemskih vrednosti IL-6 i Gal-3 kod ESRD+ HCV+ pacijenata može biti kompenzatorni mehanizam da se izbegnu ili ograniče tekući proinflamacijski procesi i da se suprimira hronična inflamacija, što ukazuje na nove aspekte HCV infekcije kod pacijenata sa ESRD. Abstract: Hepatitis C virus infection (HCV), one of the greatest causes of liver disease, is a frequent complication in patients with end-stage renal disease (ESRD) on dialysis. ESRD is defined as decreased glomerular filtration and also accompanied by impaired function of the immune system. Galectin-3 is a β-galactoside-binding lectin, involved in various biological processes including pathogenesis of chronic renal disease. The aim of our study was to estimate disease severity in ESRD HCV+ patients and analyze the serum concentrations of cytokines; anti-HCV antibodies; and galectin-3. Also, we attempted to determine potential correlation between galectin-3 level and parameters of disease severity ALT and AST. Our results showed decreased levels of ALT and AST (p = 0 00), demonstrating less liver destruction in ESRD HCV+ patients in comparison to HCV+ patients. Increased levels of IL-6 (p = 0 03) implicate a hepatoprotective role of IL-6 in these patients. Also, level of galectin-3 (p = 0 00) in the serum of ESRD HCV+ patients was higher than that of HCV+ patients. This alteration was accompanied with negative correlation between galectin-3 and AST and ALT, respectively (p = 0 029; p = 0 033). The presence of increased systemic levels of IL-6 and Gal-3 in ESRD HCV+ patients may be an attempt to counteract or limit ongoing proinflammatory processes and to downregulate chronic inflammation, suggesting the new aspects of HCV infection in ESRD patients.

RIG-I plays important roles in pathogen sensing and activation of antiviral innate immune responses in response to RNA viruses. RIG-I-mediated signaling must be precisely controlled to maintain innate immune signaling homeostasis. Previous studies demonstrated that lysine 63 (K63)-linked polyubiquitination of RIG-I is vital for its activation, but the mechanisms through which RIG-I is deubiquitinated to control innate immune responses are not well understood. Here we identified USP27X as a negative regulator of antiviral signaling in response to RNA viruses through siRNA library screening. Further functional studies indicated that USP27X negatively modulated RIG-I-mediated antiviral signaling in a deubiquitinase-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I interferon signaling. Collectively, these studies uncover a novel negative regulatory role of USP27X in targeting RIG-I to balance innate immune responses., Author summary Innate antiviral immunity provides the first line of the host defense system that rapidly detects invading virus through pattern recognition receptors, and subsequently inducing type I interferon production. Protein ubiquitination/deubiquitination play important roles in antiviral signaling, however how the dynamic regulation of ubiquitin modification controls innate immune signaling against RNA virus infection remains unclear. In this study, we screened E3 ligases and deubiquitinases (DUBs) using a siRNA library, and identified USP27X, a DUB, as a negative regulator of antiviral signaling in response to RNA virus infection. Moreover, we employed knockdown and knockout of USP27X various cell lines to perform functional analyses, and found that USP27X suppressed RIG-I-mediated antiviral signaling in a DUB activity-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I IFN signaling. Collectively, our study reveals that in addition to removing K48-linked polyubiquitin chains, USP27X can target RIG-I for removing the K63-linked polyubiquitin chains, thereby balancing antiviral signaling to avoid deleterious effects on host cells.

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In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ag) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase (AID), we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in anti-viral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses, and found that mutations producing Arg codons in anti-viral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with antigen (Ag). In many cases, mutations producing codons for these alternative amino acids in anti-viral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which 2/3rds of random mutations generate codons for these key residues. Finally, by directly analyzing x-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via somatic hypermutation occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

The cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I), which is also known as an IFN-stimulated gene (ISG), senses viral RNA to activate antiviral signaling. It is therefore thought that RIG-I is regulated in a STAT1-dependent manner. Although RIG-I-mediated antiviral signaling is indispensable for the induction of an appropriate adaptive immune response, the mechanism underlying the regulation of RIG-I expression remains elusive. Here, we examined the direct regulation of RIG-I expression by interferon regulatory factor 3 (IRF-3), which is an essential molecule for antiviral innate immunity. We initially found that RIG-I can be induced by dsRNA in both IFN-independent and IRF-3-dependent manners. A sequence analysis revealed that the RIG-I gene has putative IRF-3-binding sites in its promoter region. Using a combination of cellular, molecular biological, and mutational approaches, we first showed that IRF-3 can directly regulate the expression of RIG-I via a single IRF-element (IRF-E) site in the proximal promoter region of the RIG-I gene in response to dsRNA. IRF-3 is considered a master regulator in antiviral signaling for the generation of type I interferons (IFNs). Thus, our findings demonstrate that RIG-I expression induced by the IRF-3-mediated pathway may serve as a crucial antiviral factor for reinforcing a surveillance system against viral invasion through the regulation of the cytoplasmic viral sensor RIG-I.

Hepatitis B virus (HBV) suppresses innate immune signaling to establish persistent infection. Although HBV is a DNA virus, its pre-genomic RNA (pgRNA) can be sensed by RIG-I and activates MAVS to mediate interferon (IFN) λ synthesis. Despite of the activation of RIG-I-MAVS axis by pgRNA, the underlying mechanism explaining how HBV infection fails to induce interferon-αβ (IFN) synthesis remained uncharacterized. We demonstrate that HBV induced parkin is able to recruit the linear ubiquitin assembly complex (LUBAC) to mitochondria and abrogates IFN β synthesis. Parkin interacts with MAVS, accumulates unanchored linear polyubiquitin chains on MAVS via LUBAC, to disrupt MAVS signalosome and attenuate IRF3 activation. This study highlights the novel role of parkin in antiviral signaling which involves LUBAC being recruited to the mitochondria. These results provide avenues of investigations on the role of mitochondrial dynamics in innate immunity., Author Summary Hepatitis B virus (HBV) chronic infection is one of the major causes of hepatocellular carcinoma. HBV infection is associated with mitochondrial dysfunction. We previously showed that persistent infection of HBV requires rapid clearance of impaired mitochondria by mitophagy, a cellular quality control process that insures survival of HBV infected cells. During the process, Parkin, an RBR E3 ligase, is recruited to mitochondria to induce mitophagy. In this study, we show that the Parkin, plays a critical role in the modulation of innate immune signaling. Using HBV expressing cells, we show that the Parkin recruits linear ubiquitin assembly complex (LUBAC) to the mitochondria and subsequently inhibits downstream signaling of mitochondrial antiviral signaling protein (MAVS). Mitochondrial LUBAC then catalyzes linear ubiquitin chains on MAVS, which abrogates its downstream events such as MAVS-TRAFs interaction and abolishes IRF3 phosphorylation. The results of this study highlight the molecular details explaining how HBV can suppress interferon synthesis implicating a mitophagy-independent role of Parkin. HBV-induced mitochondrial damage serves as the platform for recruitment of Parkin and LUBAC, which together modify MAVS by ubiquitination and cripples its downstream signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-β and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-β production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities. Fil: Ishizuka, Takamasa. Tohoku University; Japón Fil: Kanmani, Paulraj. Tohoku University; Japón Fil: Kobayashi, Hisakazu. Tohoku University; Japón Fil: Miyazaki, Ayako. National Institute of Animal Health. Viral Diseases and Epidemiology Research Division; Japón Fil: Soma, Junichi. National Federation of Agricultural Cooperative Associations. Institute of Animal Health. Research and Development Section; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Nochi, Tomonori. Tohoku University; Japón Fil: Iwabuchi, Noriyuki. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón Fil: Xiao, Jin-zhong. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón Fil: Saito, Tadao. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Haruki Kitazawa. Tohoku University; Japón

Summary: The Ebola virus glycoprotein (EBOV-GP) forms GP-containing microvesicles, so-called virosomes, which are secreted from GP-expressing cells. However, determinants of GP-virosome release and their functionality are poorly understood. We characterized GP-mediated virosome formation and delineated the role of the antiviral factor tetherin (BST2, CD317) in this process. Residues in the EBOV-GP receptor-binding domain (RBD) promote GP-virosome secretion, while tetherin suppresses GP-virosomes by interactions involving the GP-transmembrane domain. Tetherin from multiple species interfered with GP-virosome release, and tetherin from the natural fruit bat reservoir showed the highest inhibitory activity. Moreover, analyses of GP from various ebolavirus strains, including the EBOV responsible for the West African epidemic, revealed the most efficient GP-virosome formation by highly pathogenic ebolaviruses. Finally, EBOV-GP-virosomes were immunomodulatory and acted as decoys for EBOV-neutralizing antibodies. Our results indicate that GP-virosome formation might be a determinant of EBOV immune evasion and pathogenicity, which is suppressed by tetherin. : Nehls et al. demonstrate that the glycoprotein of the highly pathogenic Ebola virus is incorporated into secretory vesicles, called GP-virosomes, to dampen the immune response and capture neutralizing antibodies. The lack of replication competence and the incorporation of antigenically intact GP might qualify GP-virosomes as safe vaccine candidates. Keywords: Ebola virus, glycoprotein, microvesicles, virosome, exosome, tetherin, immune modulation, immune evasion, antiviral immune response, neutralizing antibody