Interferon Antiviral Response Research Articles

Sulforaphane suppresses dengue virus replication by inhibition of dengue protease and enhancement of antiviral interferon response through Nrf2-mediated heme oxygenase-1 induction

Dengue virus (DENV) infection is a life-threatening disease that causes severe dengue hemorrhagic fever and dengue shock syndrome. There is currently no effective therapeutic agent or widely used vaccine for treating DENV infections. In this study, sulforaphane (SFN), an antioxidant phytocompound, exhibited significant antiviral activity, with a half-inhibitory concentration value of 5.8 ± 0.7 μM to suppress viral RNA replication. Mechanistic studies demonstrated that SFN-induced heme oxygenase-1 (HO-1) expression, which inhibited viral protease activity, leading to the restoration of antiviral interferon (IFN) responses against DENV replication. Its antiviral activity can be attenuated by the knockdown of HO-1 expression by short hairpin RNA and blockage of activity with a specific inhibitor, supporting the contention that HO-1 induction contributes to the anti-DENV activity of SFN. SFN also stimulated HO-1 induction through an Nrf2-dependent pathway. SFN effectively prolonged the survival rate and reduced the virus titer in DENV-infected ICR suckling mice with activated expression of interferon-stimulated genes. The anti-DENV activity of SFN was also observed in the IFN-deficient AG129 mouse model. Collectively, SFN represents significant anti-DENV activity in vitro and in vivo and is a promising drug candidate or dietary supplement against DENV.

Respiratory and systemic monocytes, dendritic cells, and myeloid-derived suppressor cells in COVID-19: Implications for disease severity.

Since the beginning of the SARS‐CoV‐2 pandemic in 2020, researchers worldwide have made efforts to understand the mechanisms behind the varying range of COVID‐19 disease severity. Since the respiratory tract is the site of infection, and immune cells differ depending on their anatomical location, studying blood is not sufficient to understand the full immunopathogenesis in patients with COVID‐19. It is becoming increasingly clear that monocytes, dendritic cells (DCs), and monocytic myeloid‐derived suppressor cells (M‐MDSCs) are involved in the immunopathology of COVID‐19 and may play important roles in determining disease severity. Patients with mild COVID‐19 display an early antiviral (interferon) response in the nasopharynx, expansion of activated intermediate monocytes, and low levels of M‐MDSCs in blood. In contrast, patients with severe COVID‐19 seem to lack an early efficient induction of interferons, and skew towards a more suppressive response in blood. This is characterized by downregulation of activation markers and decreased functional capacity of blood monocytes and DCs, reduced circulating DCs, and increased levels of HLA‐DRloCD14+ M‐MDSCs. These suppressive characteristics could potentially contribute to delayed T‐cell responses in severe COVID‐19 cases. In contrast, airways of patients with severe COVID‐19 display hyperinflammation with elevated levels of inflammatory monocytes and monocyte‐derived macrophages, and reduced levels of tissue‐resident alveolar macrophages. These monocyte‐derived cells contribute to excess inflammation by producing cytokines and chemokines. Here, we review the current knowledge on the role of monocytes, DCs, and M‐MDSCs in COVID‐19 and how alterations and the anatomical distribution of these cell populations may relate to disease severity.

Rubella Virus Triggers Type I Interferon Antiviral Response in Cultured Human Neural Cells: Involvement in the Control of Viral Gene Expression and Infectious Progeny Production

The type I interferon (IFN) response is one of the primary defense systems against various pathogens. Although rubella virus (RuV) infection is known to cause dysfunction of various organs and systems, including the central nervous system, little is known about how human neural cells evoke protective immunity against RuV infection, leading to controlling RuV replication. Using cultured human neural cells experimentally infected with RuV RA27/3 strain, we characterized the type I IFN immune response against the virus. RuV infected cultured human neural cell lines and induced IFN-β production, leading to the activation of signal transducer and activator of transcription 1 (STAT1) and the increased expression of IFN-stimulated genes (ISGs). Melanoma-differentiation-associated gene 5 (MDA5), one of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, is required for the RuV-triggered IFN-β mRNA induction in U373MG cells. We also showed that upregulation of RuV-triggered ISGs was attenuated by blocking IFN-α/β receptor subunit 2 (IFNAR2) using an IFNAR2-specific neutralizing antibody or by repressing mitochondrial antiviral signaling protein (MAVS) expression using MAVS-targeting short hairpin RNA (shRNA). Furthermore, treating RuV-infected cells with BX-795, a TANK-binding kinase 1 (TBK1)/I kappa B kinase ε (IKKε) inhibitor, robustly reduced STAT1 phosphorylation and expression of ISGs, enhancing viral gene expression and infectious virion production. Overall, our findings suggest that the RuV-triggered type I IFN-mediated antiviral response is essential in controlling RuV gene expression and viral replication in human neural cells.

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases

ABSTRACT Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.

Anti-Alphaviral Alkaloids: Focus on Some Isoquinolines, Indoles and Quinolizidines.

The discovery and the development of safe and efficient therapeutics against arthritogenic alphaviruses (e.g., chikungunya virus) remain a continuous challenge. Alkaloids are structurally diverse and naturally occurring compounds in plants, with a wide range of biological activities including beneficial effects against prominent pathogenic viruses and inflammation. In this short review, we discuss the effects of some alkaloids of three biologically relevant structural classes (isoquinolines, indoles and quinolizidines). Based on various experimental models (viral infections and chronic diseases), we highlight the immunomodulatory effects of these alkaloids. The data established the capacity of these alkaloids to interfere in host antiviral and inflammatory responses through key components (antiviral interferon response, ROS production, inflammatory signaling pathways and pro- and anti-inflammatory cytokines production) also involved in alphavirus infection and resulting inflammation. Thus, these data may provide a convincing perspective of research for the use of alkaloids as immunomodulators against arthritogenic alphavirus infection and induced inflammation.

Transcriptional response to VZV infection is modulated by RNA polymerase III in lung epithelial cell lines.

Ancestral RNA polymerase III (Pol III) is a multi-subunit polymerase responsible for transcription of short non-coding RNA, such as double-stranded short interspersed nuclear elements (SINEs). Although SINE ncRNAs are generally transcriptionally repressed, they can be induced in response to viral infections and can stimulate immune signaling pathways. Indeed, mutations in RNA Pol III have been associated with poor antiviral interferon response following infection with varicella zoster virus (VZV). In this study, we probed the role of Pol III transcripts in the detection and initial immune response to VZV by characterizing the transcriptional response following VZV infection of wild type A549 lung epithelial cells as well as A549 cells lacking specific RNA sensors MAVS and TLR3, or interferon-stimulated genes RNase L and PKR in presence or absence of functional RNA Pol III. Multiple components of the antiviral sensing and interferon signaling pathways were involved in restricting VZV replication in lung epithelial cells thus suggesting an innate defense system with built-in redundancy. In addition, RNA Pol III silencing altered the antiviral transcriptional program indicating that it plays an essential role in the sensing of VZV infection.

SARS-CoV-2 Omicron Induces Enhanced Mucosal Interferon Response Compared to other Variants of Concern, Associated with Restricted Replication in Human Lung Tissues.

SARS-CoV-2 Omicron variant has been characterized by decreased clinical severity, raising the question of whether early variant-specific interactions within the mucosal surfaces of the respiratory tract could mediate its attenuated pathogenicity. Here, we employed ex vivo infection of native human nasal and lung tissues to investigate the local-mucosal susceptibility and innate immune response to Omicron compared to Delta and earlier SARS-CoV-2 variants of concern (VOC). We show that the replication of Omicron in lung tissues is highly restricted compared to other VOC, whereas it remains relatively unchanged in nasal tissues. Mechanistically, Omicron induced a much stronger antiviral interferon response in infected tissues compared to Delta and earlier VOC-a difference, which was most striking in the lung tissues, where the innate immune response to all other SARS-CoV-2 VOC was blunted. Notably, blocking the innate immune signaling restored Omicron replication in the lung tissues. Our data provide new insights to the reduced lung involvement and clinical severity of Omicron.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1–69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

Variability in Susceptibility to Type I Interferon Response and Subgenomic RNA Accumulation Between Clinical Isolates of Dengue and Zika Virus From Oaxaca Mexico Correlate With Replication Efficiency in Human Cells and Disease Severity.

Dengue and Zika viruses cocirculate annually in endemic areas of Mexico, causing outbreaks of different magnitude and severity every year, suggesting a continuous selection of Flavivirus variants with variable phenotypes of transmissibility and virulence. To evaluate if Flavivirus variants with different phenotypes cocirculate during outbreaks, we isolated dengue and Zika viruses from blood samples of febrile patients from Oaxaca City during the 2016 and 2019 epidemic years. We compared their replication kinetics in human cells, susceptibility to type I interferon antiviral response, and the accumulation of subgenomic RNA on infected cells. We observed correlations between type I interferon susceptibility and subgenomic RNA accumulation, with high hematocrit percentage and thrombocytopenia. Our results suggest that Flaviviruses that cocirculate in Oaxaca, Mexico, have variable sensitivity to the antiviral activity of type I interferons, and this phenotypic trait correlates with the severity of the disease.

Fish female-biased gene cyp19a1a leads to female antiviral response attenuation between sexes by autophagic degradation of MITA.

From insects to mammals, both innate and adaptive immune response are usually higher in females than in males, with the sex chromosome and hormonal differences considered the main reasons. Here, we report that zebrafish cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a), an autosomal gene with female-biased expression, causes female fish to exhibit a lower antiviral response. First, we successfully constructed an infection model by intraperitoneal injection of spring viremia of carp virus (SVCV) into zebrafish (Danio rerio) and Carassius auratus herpesvirus (CaHV) in gibel carp (Carassius gibelio). Specifically, female fish were more vulnerable to viral infection than males, accompanied by a significantly weaker interferon (IFN) expression. After screening several candidates, cyp19a1a, which was highly expressed in female fish tissues, was selected for further analysis. The IFN expression and antiviral response were significantly higher in cyp19a1a-/- than in cyp19a1a+/+. Further investigation of the molecular mechanism revealed that Cyp19a1a targets mediator of IRF3 activation (MITA) for autophagic degradation. Interestingly, in the absence of MITA, Cyp19a1a alone could not elicit an autophagic response. Furthermore, the autophagy factor ATG14 (autophagy-related 14) was found interacted with Cyp19a1a to either promote or attenuate Cyp19a1a-mediated MITA degradation by either being overexpressed or knocked down, respectively. At the cellular level, both the normal and MITA-enhanced cellular antiviral responses were diminished by Cyp19a1a. These findings demonstrated a sex difference in the antiviral response based on a regulation mechanism controlled by a female-biased gene besides sex chromosome and hormonal differences, supplying the current understanding of sex differences in fish.

SARS-CoV-2: A Master of Immune Evasion.

Viruses and their hosts have coevolved for a long time. This coevolution places both the pathogen and the human immune system under selective pressure; on the one hand, the immune system has evolved to combat viruses and virally infected cells, while viruses have developed sophisticated mechanisms to escape recognition and destruction by the immune system. SARS-CoV-2, the pathogen that is causing the current COVID-19 pandemic, has shown a remarkable ability to escape antibody neutralization, putting vaccine efficacy at risk. One of the virus’s immune evasion strategies is mitochondrial sabotage: by causing reactive oxygen species (ROS) production, mitochondrial physiology is impaired, and the interferon antiviral response is suppressed. Seminal studies have identified an intra-cytoplasmatic pathway for viral infection, which occurs through the construction of tunneling nanotubes (TNTs), hence enhancing infection and avoiding immune surveillance. Another method of evading immune monitoring is the disruption of the antigen presentation. In this scenario, SARS-CoV-2 infection reduces MHC-I molecule expression: SARS-CoV-2’s open reading frames (ORF 6 and ORF 8) produce viral proteins that specifically downregulate MHC-I molecules. All of these strategies are also exploited by other viruses to elude immune detection and should be studied in depth to improve the effectiveness of future antiviral treatments. Compared to the Wuhan strain or the Delta variant, Omicron has developed mutations that have impaired its ability to generate syncytia, thus reducing its pathogenicity. Conversely, other mutations have allowed it to escape antibody neutralization and preventing cellular immune recognition, making it the most contagious and evasive variant to date.

Omicron variant of SARS-CoV-2 exhibits an increased resilience to the antiviral type I interferon response.

The new variant of concern (VOC) of SARS-CoV-2, Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype (wt) strain (B.1) with respect to their interactions with the antiviral interferon (IFN-alpha/beta) response in infected cells. Our data indicate that IFN induction by Omicron is low and comparable to the wt, whereas Delta showed an increased IFN induction. However, Omicron exceeded both the wt and the Delta strain with respect to the ability to withstand the antiviral state imposed by IFN-alpha.

A STING agonist preconditions against ischaemic stroke via an adaptive antiviral Type 1 interferon response.

Converging lines of inquiry have highlighted the importance of the Type I antiviral response not only in defending against viruses but also in preconditioning the brain against ischaemic stroke. Despite this understanding, treatments that foster brain resilience by driving antiviral interferon responses have yet to be developed for human use. Studies from our laboratory showed that tilorone, the first human antiviral immunomodulatory agent to be developed, robustly preconditioned against stroke in mice and rats. Tilorone is a DNA intercalator; therefore, we hypothesized that it stabilizes cytosolic DNA (released from the mitochondria or the nucleus), thereby activating cyclic GMP–AMP synthase, a homeostatic DNA sensor, and its downstream pathway. This pathway involves stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1), and interferon regulatory protein-3 and culminates in a protective Type I interferon response. We tested this hypothesis by examining the ability of structurally diverse small-molecule agonists of STING to protect against oxygen/glucose deprivation in vitro in mouse cortical cultures and in vivo against transient ischaemia in mice. The STING agonists significantly reduced cell death both in vitro and in vivo but failed to do so in STING knockout mice. As expected, STING agonist-induced protection was associated with the induction of interferon related genes and the effects could be abrogated in vitro by a TBK1 inhibitor. Taken together, these findings in mice identify STING as a therapeutic target for preconditioning the brain against ischaemic stroke in vitro and in vivo. Moreover, they suggest that clinically approved STING agonists such as Ganciclovir or α-Mangostin are candidate drugs that could be tested in humans as a prophylactic treatment to alleviate brain injury associated with ischaemic stroke.

Interferon‐alpha or ‐beta Facilitates SARS‐CoV‐2 Pulmonary Vascular Infection by Inducing ACE2

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is characterized by a hyperinflammatory state typified by elevated circulating pro‐inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID‐19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS‐CoV‐2 binding receptor ACE2. Herein we describe SARS‐CoV‐2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID‐19, demonstrating both heterogeneous ACE2 expression and endothelial damage (Figure). In primary endothelial cell cultures, we show that SARS‐CoV‐2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon‐alpha (IFNα) or ‐beta(β) ‐ two of the main anti‐viral cytokines induced in severe SARS‐CoV‐2 infection ‐ but not significantly by other cytokines (including interleukin 6 and interferon g /λ). Our findings suggest that the stereotypical anti‐viral interferon response may paradoxically facilitate the propagation of COVID‐19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID‐19.

The preterm infant microbiome impairs lung immune responses to respiratory syncytial virus infection

Abstract The gut microbiome plays a critical role in neonatal immune development; early-life perturbations to the microbiome have been linked to later susceptibility to respiratory conditions, including asthma and chronic obstructive pulmonary disease. Preterm infants are at higher risk for adverse outcomes to respiratory infections such as respiratory syncytial virus (RSV). The microbiome in premature infants is significantly altered, but it remains unclear how this affects lung immune cell development and susceptibility to RSV infection independent of lung immaturity. In this study, we have generated a biorepository of stool specimens from preterm and term infants during the first weeks of life. Metagenomic sequencing studies indicate significant alterations in the gut microbiome between these two groups, including a lack of fiber-fermenting Bacteroides species in preterm infants. Using these stool specimens, we find that the altered gut microbiome in premature infants induces IL-17/neutrophil-biased immune responses but weaker anti-viral interferon responses in the lung, both at steady state and following RSV infection. Mice colonized with preterm infant stool bacteria produced lower levels of neutralizing antibodies against RSV than mice colonized with term infant stool bacteria. Taken together, our findings suggest that the altered gut microbiome in premature infants dysregulates immune cell development in the lung and impairs the infant’s ability to develop long-term immunity against respiratory infections. These findings might be potentially leveraged in the development of gut bacteria-based interventions to improve the development of protective immunity against respiratory viral pathogens in preterm infants. Supported by grants from NIH/NCATS (TL1-TR-002386) and the Biocodex Microbiota Foundation

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Infection in a Bat Gastrointestinal Organoid Model

Abstract The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is thought to have originated in bats, since multiple viruses with a high level of genetic similarity have been identified in bats. In addition to respiratory symptoms, COVID-19 frequently involves gastrointestinal symptoms such as diarrhea and vomiting, indicating the SARS-CoV-2 can target the gastrointestinal (GI) tract. However, there is no robust in vitro model for assessing the SARS-CoV-2 infection in the bat GI tract. Here, we established gastrointestinal organoid cultures from Jamaican fruit bats (JFB),( Artibeus jamaicensis), which replicated the characteristic morphology of the gastrointestinal epithelium and showed tissue specific gene expression patterns and cell differentiation. To analyze whether JFB intestinal epithelial cells are susceptible to SARS-CoV-2, we performed in vitro infection experiments. While increased SARS-CoV-2 RNA was detected in both cell lysates and supernatants from the infected organoids after 48 h, there was no evidence of active viral replication, and no infectious virus was produced. However, the JFB distal organoids significantly upregulated anti-viral and pro-inflammatory genes in response to SARS-CoV-2. Unexpectedly, SARS-CoV-2 infected JFB organoids had a decreased incidence of apoptotic cell death. Collectively, our data suggest that primary intestinal epithelial cells from JFBs are resistant to SARS-CoV-2 infection and cell damage, likely because they are able to mount a strong antiviral interferon response.

Eosinophil-mediated suppression and anti–IL-5 enhancement of plasmacytoid dendritic cell interferon responses in asthma

Virus-induced IFN-α secretion by plasmacytoid dendritic cells (pDCs) is negatively impacted by IgE and has been linked to asthma exacerbations. Eosinophils, another contributor to type 2 inflammation, are also associated with asthma severity. We sought to investigate the impact of eosinophils on pDC antiviral interferon responses and determine whether anti-IL-5/5Rα therapy enhances pDC antiviral function. Blood pDCs purified from anonymous donors were stimulated invitro with rhinovirus (RV)-16 in the presence or absence of eosinophils/eosinophil supernatants. IFN-α was measured in supernatants and RNA collected for bulk RNA-sequencing. Next, purified pDCs from 8 individuals with moderate to severe asthma, treated or not treated with anti-IL-5/5Rα therapy, were cultured exvivo with or without RV; IFN-α secretion and differential gene expression analysis were compared between groups. Exposure to either eosinophils or eosinophil supernatants inhibited RV-induced pDC IFN-α secretion in a dose-dependent manner and did not impact pDC viability. Eosinophil-derived neurotoxin and TGF-β partially recapitulated pDC IFN-α inhibition. Transcriptome analysis revealed global repression of pDC interferon response patterns by eosinophils, most notably in basal expression of interferon-stimulated genes. Increased RV-induced IFN-α secretion and transcription as well as increased basal interferon-stimulated gene expression was detected in pDCs from participants treated with anti-IL-5/5Rα therapy. Our findings highlight a novel mechanism through which type 2 inflammation regulates pDC IFN-α responses relevant to RV respiratory infections in the context of eosinophilic airway disease, suggesting a potential mechanism through which eosinophil-depleting therapies may reduce severity of RV illnesses.

Promoter Binding and Nuclear Retention Features of Zebrafish IRF Family Members in IFN Response.

Interferon regulatory factors (IRFs) constitute a family of transcription factors that synchronize interferon (IFN) antiviral response through translocating to nucleus and binding to the promoters of IFN and IFN-stimulated genes (ISGs). Fish contain 11 IRF members; however, whether or how fish IRF family genes function in IFN response remains limited. Herein, we determine the regulatory roles of 11 zebrafish IRF family members in IFN response relevant to their subcellular localization and promoter binding. Zebrafish IRF family members display three patterns of constitutive localization, only in nucleus (IRF1/2/9/11), only in cytoplasm (IRF3/5/7), and largely in nucleus with small amounts in cytoplasm (IRF4b/6/8/10). DNA pull-down assays confirm that all zebrafish IRF proteins are capable to bind fish IFN promoters, albeit to various degrees, thus regulating IFN gene transcription as activators (IRF1/3/5/6/7/8/9/11) or repressors (IRF2/4b/10). Further characterization of distinct IFN gene activation reveals that IRF1/3/5/6/7/8/9/11 efficiently stimulate zebrafish IFNφ1 expression, and IRF1/7/11 are responsible for zebrafish IFNφ3 expression. Two conserved basic residues within the helix α3 of DNA binding domains (DBDs) contribute to constitutive or inducible nuclear import for all zebrafish IRF family members and DNA binding for most members, thereby enabling them to function as transcription factors. Our results reveal a conserved and general mechanism that specifies zebrafish IRF family proteins to nuclear import and DNA binding, thereby regulating fish IFN response.

Conserved function of crucian carp cGAS in the MITA-mediated interferon signaling

Mammalian cyclic GMP-AMP synthase (cGAS) is pivotal for cytosolic DNA-triggered interferon (IFN) response. However, the function of cGAS in fish IFN response remains unclear. Our recent study has reported that cGAS from crucian and grass carps downregulates the IFN response by attenuating the K63-linked ubiquitination of retinoic acid-inducible gene-I (RIG-I) and its interaction with mitochondrial antiviral signaling protein (MAVS). Here, the function of crucian carp cGAS was further investigated. We found that crucian carp cGAS directly binds to poly deoxyadenylic-deoxythymidylic acid (poly (dA:dT)) and exhibits mediator of IFN regulatory factor 3 (IRF3) activation (MITA)-dependent activation of the IFN response, indicating a conserved function of crucian carp cGAS in the MITA-mediated IFN signaling. However, crucian carp cGAS could suppress the IFN activation stimulated by polyinosinic: polycytidylic acid (poly (I:C)) in time- and dose-dependent manners. These data collectively suggest complicated functions of crucian carp cGAS in the IFN antiviral response.

Reduced IFN-ß inhibitory activity of Lagos bat virus phosphoproteins in human compared to Eidolon helvum bat cells.

Eidolon helvum bats are reservoir hosts for highly pathogenic lyssaviruses often showing limited disease upon natural infection. An enhanced antiviral interferon (IFN) response combined with reduced inflammation might be linked to the apparent virus tolerance in bats. Lyssavirus phosphoproteins inhibit the IFN response with virus strain-specific efficiency. To date, little is known regarding the lyssavirus P-dependent anti-IFN countermeasures in bats, mainly due to a lack of in vitro tools. By using E. helvum bat cell cultures in a newly established bat-specific IFN-promoter activation assay, we analyzed the IFN-ß inhibitory activity of multiple lyssavirus P in E. helvum compared to human cells. Initial virus infection studies with a recently isolated E. helvum-borne Lagos bat virus street strain from Ghana showed enhanced LBV propagation in an E. helvum lung cell line compared to human A549 lung cells at later time points suggesting effective viral countermeasures against cellular defense mechanisms. A direct comparison of the IFN-ß inhibitory activity of the LBV-GH P protein with other lyssavirus P proteins showed that LBV-GH P and RVP both strongly inhibited the bat IFN-β promotor activation (range 75-90%) in EidLu/20.2 and an E. helvum kidney cell line. Conversely, LBV-GH P blocked the activation of the human IFN-β promoter less efficiently compared to a prototypic Rabies virus P protein (range LBV P 52-68% vs RVP 71-95%) in two different human cell lines (HEK-293T, A549). The same pattern was seen for two prototypic LBV P variants suggesting an overall reduced LBV P IFN-ß inhibitory activity in human cells as compared to E. helvum bat cells. Increased IFN-ß inhibition by lyssavirus P in reservoir host cells might be a result of host-specific adaptation processes towards an enhanced IFN response in bat cells.