Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

AKT2 reduces IFNß1 production to modulate antiviral responses and systemic lupus erythematosus.

Interferon regulatory factor 3 (IRF3)-induced type I interferon (I-IFN) production plays key roles in both antiviral and autoimmune responses. IRF3 phosphorylation, dimerization, and nuclear localization are needed for its activation and function, but the precise regulatory mechanisms remain to be explored. Here, we show that the serine/threonine kinase AKT2 interacts with IRF3 and phosphorylates it on Thr207, thereby attenuating IRF3 nuclear translocation in a 14-3-3ε-dependent manner and reducing I-IFN production. We further find that AKT2 expression is downregulated in viral-infected macrophages or in monocytes and tissue samples from systemic lupus erythematosus (SLE) patients and mouse models. Akt2-deficient mice exhibit increased I-IFN induction and reduced mortality in response to viral infection, but aggravated severity of SLE. Overexpression of AKT2 kinase-inactive or IRF3-T207A mutants in zebrafish supports that AKT2 negatively regulates I-IFN production and antiviral response in a kinase-dependent manner. This negative role of AKT2 in IRF3-induced I-IFN production suggests that AKT2 may be therapeutically targeted to differentially regulate antiviral infection and SLE.

Optimizing the Semisynthesis towards glycosylated interferon-ß-polypeptide by utilizing bacterial protein expression and chemical modification.

The synthesis of a sufficient amount of homogeneous glycoprotein is of great interest because natural glycoproteins show considerable heterogeneity in oligosaccharide structures, making the studies on glycan structure-function relationship difficult. Herein, we report optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion. Peptide thioesters and peptides with Cys residues at their N-terminals are necessary intermediates to perform native chemical ligation. We successfully performed thioesterification for a peptide prepared in E. coli via Cys-cyanylation at its C-terminal followed by hydrazinolysis and acidic thiolysis. These optimized conditions could tolerate an acid labile Thz protected Cys at the N-terminal of a peptide-hydrazide and specific cyanylation of the C-terminal Cys to yield a peptide thioester. To reduce the amount of precious oligosaccharide that is required in the conventional SPPS method, an improved liquid phase glycopeptide coupling was also optimized in a good yield (46% over four steps). Lastly, chemoselective protection of the internal cysteines and activation of the N-terminal cysteine were optimized toward a long peptide prepared in E. coli. By using these strategies, a full-length interferon-ß glycosyl polypeptide as a model was successfully obtained.

Administration of an adeno-associated viral vector expressing interferon-ß in patients with inflammatory hand arthritis, results of a phase I/II study.

OBJECTIVE: Inflammatory hand arthritis (IHA) results in impaired function. Local gene therapy with ART-I02, a recombinant adeno-associated virus (AAV) serotype 5 vector expressing interferon (IFN)-ß, under the transcriptional control of nuclear factor κ-B responsive promoter, was preclinically shown to have favorable effects. This study aimed to investigate the safety and tolerability of local gene therapy with ART-I02 in patients with IHA. METHODS: In this first-in-human, dose-escalating, cohort study, 12 IHA patients were to receive a single intra-articular (IA) injection of ART-I02 ranging 0.3 × 1012-1.2 × 1013 genome copies in an affected hand joint. Adverse events (AEs), routine safety laboratory and the clinical course of disease were periodically evaluated. Baseline- and follow-up contrast enhanced magnetic resonance images (MRIs), shedding of viral vectors in bodily fluids, and AAV5 and IFN-ß immune responses were evaluated. A data review committee provided safety recommendations. RESULTS: Four patients were enrolled. Long-lasting local AEs were observed in 3 patients upon IA injection of ART-I02. The AEs were moderate in severity and could be treated conservative. Given the duration of the AEs and their possible or probable relation to ART-I02, no additional patients were enrolled. No systemic treatment emergent AEs were observed. The MRIs reflected the AEs by (peri)arthritis. No T-cell response against AAV5 or IFN-ß, nor IFN-ß antibodies could be detected. Neutralizing antibody titers against AAV5 raised post-dose. CONCLUSION: Single IA doses of 0.6 × 1012 or 1.2 × 1012 ART-I02 vector genomes were administered without systemic side effects or serious AEs. However, local tolerability was insufficient for continuation. TRIAL REGISTRATION: NCT02727764.

Myeloid Protease-Activated Receptor-2 Contributes to Influenza A Virus Pathology in Mice.

Background: Innate immune responses to influenza A virus (IAV) infection are initiated in part by toll-like receptor 3 (TLR3). TLR3-dependent signaling induces an antiviral immune response and an NFκB-dependent inflammatory response. Protease-activated receptor 2 (PAR2) inhibits the antiviral response and enhances the inflammatory response. PAR2 deficiency protected mice during IAV infection. However, the PAR2 expressing cell-types contributing to IAV pathology in mice and the mechanism by which PAR2 contributes to IAV infection is unknown. Methods: IAV infection was analyzed in global (Par2-/- ), myeloid (Par2fl/fl;LysMCre+) and lung epithelial cell (EpC) Par2 deficient (Par2fl/fl ;SPCCre+) mice and their respective controls (Par2+/+ and Par2fl/fl). In addition, the effect of PAR2 activation on polyinosinic-polycytidylic acid (poly I:C) activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM). Lastly, we determined the effect of PAR2 inhibition in wild-type (WT) mice. Results: After IAV infection, Par2-/- and mice with myeloid Par2 deficiency exhibited increased survival compared to infected controls. The improved survival was associated with reduced proinflammatory mediators and reduced cellular infiltration in bronchoalveolar lavage fluid (BALF) of Par2-/- and Par2fl/fl;LysMCre+ 3 days post infection (dpi) compared to infected control mice. Interestingly, Par2fl/fl;SPCCre+ mice showed no survival benefit compared to Par2fl/fl . In vitro studies showed that Par2-/- BMDM produced less IL6 and IL12p40 than Par2+/+ BMDM after poly I:C stimulation. In addition, activation of PAR2 on Par2+/+ BMDM increased poly I:C induction of IL6 and IL12p40 compared to poly I:C stimulation alone. Importantly, PAR2 inhibition prior to IAV infection protect WT mice. Conclusion: Global Par2 or myeloid cell but not lung EpC Par2 deficiency was associated with reduced BALF inflammatory markers and reduced IAV-induced mortality. Our study suggests that PAR2 may be a therapeutic target to reduce IAV pathology.

The Kinase MAP4K1 Inhibits Cytosolic RNA-Induced Antiviral Signaling by Promoting Proteasomal Degradation of TBK1/IKKε.

TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines in response to acute viral infection. However, excessive or prolonged production of IFN-I is harmful and even fatal to the host by causing autoimmune disorders. In this study, we identified mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) as a negative regulator in the RIG-I-like receptor (RLR) signaling pathway. MAP4K1, a member of Ste20-like serine/threonine kinases, was previously known as a prominent regulator in adaptive immunity by downregulating T-cell receptor (TCR) signaling and B-cell receptor (BCR) signaling. However, its role in regulating antiviral innate immune signaling is still unclear. This study reports an undiscovered role of MAP4K1, which inhibits RLR signaling by targeting TBK1/IKKε for proteasomal degradation via the ubiquitin ligase DTX4. We initially identify MAP4K1 as an interacting partner of TBK1 by yeast two-hybrid screens and subsequently investigate its function in RLR-mediated antiviral signaling pathways. Overexpression of MAP4K1 significantly inhibits RNA virus-triggered activation of IFN-ß and the production of proinflammatory cytokines. Consistently, knockdown or knockout experiments show opposite effects. Furthermore, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via DTX4. Knockdown of DTX4 abrogated the ubiquitination and degradation of TBK1/IKKε. Collectively, our results identify that MAP4K1 acts as a negative regulator in antiviral innate immunity by targeting TBK1/IKKε, discover a novel TBK1 inhibitor, and extend a novel functional role of MAP4K1 in immunity. IMPORTANCE TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines to restrict the spread of invading viruses. However, excessive or prolonged production of IFN-I is harmful to the host by causing autoimmune disorders. In this study, we identified that mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) is a negative regulator in the RLR signaling pathway. Notably, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via the ubiquitin ligase DTX4, leading to the negative regulation of the IFN signaling pathway. Previous studies showed that MAP4K1 has a pivotal function in adaptive immune responses. This study identifies that MAP4K1 also plays a vital role in innate immunity and outlines a novel mechanism by which the IFN signaling pathway is tightly controlled to avoid excessive inflammation. Our study documents a novel TBK1 inhibitor, which serves as a potential therapeutic target for autoimmune diseases, and elucidated a significant function for MAP4K1 linked to innate immunity in addition to subsequent adaptive immunity.

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation.

The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.

Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo.

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

SARS-CoV-2 3C-like protease antagonizes interferon-beta production by facilitating the degradation of IRF3.

The prevalence of SARS-CoV-2 is a great threat to global public health. However, the relationship between the viral pathogen SARS-CoV-2 and host innate immunity has not yet been well studied. The genome of SARS-CoV-2 encodes a viral protease called 3C-like protease. This protease is responsible for cleaving viral polyproteins during replication. In this investigation, 293T cells were transfected with SARS-CoV-2 3CL and then infected with Sendai virus (SeV) to induce the RIG-I like receptor (RLR)-based immune pathway. q-PCR, luciferase reporter assays, and western blotting were used for experimental analyses. We found that SARS-CoV-2 3CL significantly downregulated IFN-ß mRNA levels. Upon SeV infection, SARS-CoV-2 3CL inhibited the nuclear translocation of IRF3 and p65 and promoted the degradation of IRF3. This effect of SARS-CoV-2 3CL on type I IFN in the RLR immune pathway opens up novel ideas for future research on SARS-CoV-2.

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection.

Murine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and demonstrated its role as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host. Using reverse genetics, we generated MNV-3 viruses carrying a single or a triple termination codon inserted in the VF1 ORF. VF1-deleted MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. C57BL/6 mice infected with VF1-deleted MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a 3 month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo. Our results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonize the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilize multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

DeSUMOylation of Apoptosis Inhibitor 5 by Avibirnavirus VP3 Supports Virus Replication.

SUMOylation is a reversible posttranslational modification involved in the regulation of diverse biological processes. Growing evidence suggests that virus infection can interfere with the SUMOylation system. In the present study, we discovered that apoptosis inhibitor 5 (API5) is a SUMOylated protein. Amino acid substitution further identified that Lys404 of API5 was the critical residue for SUMO3 conjugation. Moreover, we found that Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. In addition, our results further revealed that viral protein VP3 inhibited the SUMOylation of API5 by targeting API5 and promoting UBC9 proteasome-dependent degradation through binding to the ubiquitin E3 ligase TRAF3. Furthermore, we revealed that wild-type but not K404R mutant API5 inhibited IBDV replication by enhancing MDA5-dependent IFN-ß production. Taken together, our data demonstrate that API5 is a UBC9-dependent SUMOylated protein and deSUMOylation of API5 by viral protein VP3 aids in viral replication. IMPORTANCE Apoptosis inhibitor 5 (API5) is a nuclear protein initially identified for its antiapoptotic function. However, so far, posttranslational modification of API5 is unclear. In this study, we first identified that API5 K404 can be conjugated by SUMO3, and Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. Mechanically, viral protein VP3 directly interacts with API5 and inhibits SUMOylation of API5. Additionally, the cellular E3 ligase TNF receptor-associated factor 3 (TRAF3) is employed by VP3 to facilitate UBC9 proteasome-dependent degradation, leading to the reduction of API5 SUMOylation. Moreover, our data reveal that SUMOylation of API5 K404 promotes MDA5-dependent beta interferon (IFN-ß) induction, and its deSUMOylation contributes to IBDV replication. This work highlights a critical role of conversion between SUMOylation and deSUMOylation of API5 in regulating viral replication.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-ß production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-ß promoter activity, rather than IFN-ß transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-ß production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host-influenza NS1 protein interactions.

COPD Is Associated with Elevated IFN-ß Production by Bronchial Epithelial Cells Infected with RSV or hMPV.

IFN treatment may be a viable option for treating COPD exacerbations based on evidence of IFN deficiency in COPD. However, in vitro studies have used primarily influenza and rhinoviruses to investigate IFN responses. This study aims to investigate the susceptibility to infection and IFN response of primary bronchial epithelial cells (BECs) from COPD donors to infection with RSV and hMPV. BECs from five COPD and five healthy donors were used to establish both submerged monolayer and well-differentiated (WD) cultures. Two isolates of both RSV and hMPV were used to infect cells. COPD was not associated with elevated susceptibility to infection and there was no evidence of an intrinsic defect in IFN production in either cell model to either virus. Conversely, COPD was associated with significantly elevated IFN-ß production in response to both viruses in both cell models. Only in WD-BECs infected with RSV was elevated IFN-ß associated with reduced viral shedding. The role of elevated epithelial cell IFN-ß production in the pathogenesis of COPD is not clear and warrants further investigation. Viruses vary in the responses that they induce in BECs, and so conclusions regarding antiviral responses associated with disease cannot be made based on single viral infections.

SARS-CoV-2 NSP12 Protein Is Not an Interferon-ß Antagonist.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

IKKε isoform switching governs the immune response against EV71 infection.

The reciprocal interactions between pathogens and hosts are complicated and profound. A comprehensive understanding of these interactions is essential for developing effective therapies against infectious diseases. Interferon responses induced upon virus infection are critical for establishing host antiviral innate immunity. Here, we provide a molecular mechanism wherein isoform switching of the host IKKε gene, an interferon-associated molecule, leads to alterations in IFN production during EV71 infection. We found that IKKε isoform 2 (IKKε v2) is upregulated while IKKε v1 is downregulated in EV71 infection. IKKε v2 interacts with IRF7 and promotes IRF7 activation through phosphorylation and translocation of IRF7 in the presence of ubiquitin, by which the expression of IFNß and ISGs is elicited and virus propagation is attenuated. We also identified that IKKε v2 is activated via K63-linked ubiquitination. Our results suggest that host cells induce IKKε isoform switching and result in IFN production against EV71 infection. This finding highlights a gene regulatory mechanism in pathogen-host interactions and provides a potential strategy for establishing host first-line defense against pathogens.

Poly (I: C) inhibits reticuloendothelial virus replication in chicken macrophage-like cells through the activation of toll-like receptor-3 signaling.

Reticuloendothelial virus (REV) is widely found in many domestic poultry areas and results in severe immunosuppression of infected chickens. This increases the susceptibility to other pathogens, which causes economic losses to the poultry industry. The aim of our study was to determine whether polyinosinic-polycytidylic acid [Poly (I: C)] treatment could inhibit REV replication in chicken macrophage-like cell line, HD11. We found that Poly (I: C) treatment could markedly inhibit REV replication in HD11 from 24 to 48 h post infection (hpi). Additionally, Poly (I: C) treatment could switch HD11 from an inactive type into M1-like polarization from 24 to 48 hpi. Furthermore, Poly (I: C) treatment promoted interferon-ß secretion from HD11 post REV infection. Moreover, Toll-like receptor-3 (TLR-3) mRNA and protein levels in HD11 treated with Poly (I: C) were markedly increased compared to those of HD11 not treated with Poly (I: C). The above results suggested that Poly (I: C) treatment switches HD11 into M1-like polarization to secret more interferon-ß and activate TLR-3 signaling, which contributes to block REV replication. Our findings provide a theoretical reference for further studying the underlying pathogenic mechanism of REV and Poly (I: C) as a potential therapeutic intervention against REV infection.

Dengue virus induces interferon-ß by activating RNA sensing pathways in megakaryocytes.

Activation of innate receptors in megakaryocytes (MKs) may affect the ability to produce functional platelets. Low platelet count is one of the clinical manifestations of dengue virus (DENV) infection. In MKs, the effect of innate receptors during DENV-infection is not well studied. Here we used MEG-01 cells to investigate DENV serotype 2 induced innate receptors in these cells. DENV RNA was estimated by qRT-PCR in the culture supernatant. The expression of innate receptors was determined by western blot and qPCR. DENV infection led to increased expression of RIG-I at 24 hrs post-infection (hpi) and MDA-5 at 48 and 72 hpi (p<0.05). However, no change in the expression of TLR3 at protein level was observed. Activation of MDA-5 resulted in increased expression of IFN-ß and ISG-15 in DENV infected MEG-01 cells, which was further confirmed by MDA-5 siRNA treatment. Apart from inducing innate receptors, DENV significantly decreases the expression of CD61, an activation marker of megakaryocyteson MEG-01 cells as observed by flow cytometry analysis (p<0.01). Results from this study confirm that DENV infection activates the type-I interferon in megakaryocytes and may play a significant role in maturation and development.

MiR-139 Induces an Interferon-ß Response in Prostate Cancer Cells by Binding to RIG-1.

BACKGROUND: We previously identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Expression of one of the down-regulated miRNAs, miR-139-5p, was significantly associated with a lower incidence of biochemical recurrence and metastasis. Transcriptome profiling of miR-139-expressing prostate cancer cells revealed up-regulation of genes involved in interferon (IFN) stimulation. The association between miR-139 and IFN-ß was further explored in this study. MATERIALS AND METHODS: We examined miR-139 transfected PC3, Du145 and LNCaP cells and the associated IFN response by transcriptome sequencing, immunoblotting and pulldown assays. RESULTS: Treatment of prostate cancer cells by miR-139 resulted in the up-regulation of IFN-related genes. Specifically, miR-139 induced expression of the IFN-ß protein. The ability of miR-139 to induce IFN-ß was due to its binding to RIG-1 and the induction of IFN-related genes was found to be dependent on RIG-1 expression. CONCLUSION: miR-139 acts as an immune agonist of RIG-1 to enhance IFN-ß response in prostate cancer cells.

Swine Acute Diarrhea Syndrome Coronavirus Nucleocapsid Protein Antagonizes Interferon-ß Production via Blocking the Interaction Between TRAF3 and TBK1.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-ß promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-ß production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.