Antiviral Innate Immune Activation Research Articles

Exploration of common pathogenesis and candidate hub genes between HIV and monkeypox co-infection using bioinformatics and machine learning

This study explored the pathogenesis of human immunodeficiency virus (HIV) and monkeypox co-infection, identifying candidate hub genes and potential drugs using bioinformatics and machine learning. Datasets for HIV (GSE 37250) and monkeypox (GSE 24125) were obtained from the GEO database. Common differentially expressed genes (DEGs) in co-infection were identified by intersecting DEGs from monkeypox datasets with genes from key HIV modules screened using Weighted Gene Co-Expression Network Analysis (WGCNA). After gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and construction of protein-protein interaction (PPI) network, candidate hub genes were further screened based on machine learning algorithms. Transcriptional factors (TFs) and miRNA-candidate hub gene networks were constructed to understand regulatory mechanisms and protein-drug interactions to identify potential therapeutic drugs. Seven candidate hub genes—MX2, ADAR, POLR2H, RPL5, IFI16, IFIT2, and RPS5—were identified. TFs and miRNAs associated with these hub genes, playing a key role in regulating viral infection and inflammation due to the activation of antiviral innate immunity, were also identified through network analysis. Potential therapeutic drugs were screened based on these hub genes: AZT, a nucleotide reverse transcriptase inhibitor, suppressed viral replication in HIV and monkeypox co-infection, while mefloquine inhibited inflammation due to the activation of antiviral innate immunity. In conclusion, the study identified candidate hub genes, their transcriptional regulation, signaling pathways, and small-molecule drugs in HIV and monkeypox co-infection, contributing to understanding the pathogenesis of HIV and monkeypox co-infection and informing precise therapeutic strategies.

Abstract P3072: Effects Of Antiviral Innate Immune Activation On Cytokine Secretion And Function Of Human Ipsc-derived Cardiomyocytes

Introduction: Acute and long-term cardiovascular complications of COVID-19 have been reported, ranging from arrhythmias, ischemic heart disease, and heart failure to cerebrovascular and cardiometabolic disorders, yet the underlying molecular mechanisms are not well understood. Here, we examine the effects of stimulating the double-stranded RNA (dsRNA) pattern recognition receptor pathway, which is strongly induced to activate interferon-stimulated gene (ISG) expression in the heart during COVID-19, on excitation-contraction coupling and mitochondrial function. While providing antiviral protection, we hypothesize that, even in the absence of cardiac viral replication, this pathway might contribute to COVID-19-related cardiac dysfunction. Results: We used the viral dsRNA mimetic polyinosinic: polycytidylic (PIC) to induce a type I interferon response in iPSC-CM or A549 (lung epithelial) cells. In iPSC-CM, PIC induced robust protein expression of STAT1/pSTAT1, OAS2, OAS3, IRF7, pIRF7, IRF9 and MX1. A marked increase in cytokine protein secretion was also observed, with significant increases in CXCL-10 (~160 fold), IL6, CCL5 (~20-fold) IL8 (15-fold), and CXCL1 (~10-fold), and a 2-5-fold increase in ~30 other cytokines. Negative effects on excitation-contraction coupling were observed after 72 hours of PIC treatment, manifested as a 25% reduction in iPSC-CM Ca2+ transient amplitude. Microelectrode array analysis of iPSC-CM monolayers revealed electrophysiological dysfunction: PIC treatment increased cardiac field potential duration and spike amplitude, but reduced beat periods. A549 cells also showed increases in interferon pathway genes (STAT1, pSTAT1, IRF3, pIRF3, OAS1,2,3 and IFNb1), along with increased rates of RNA degradation after PIC treatment, consistent with activation of the OAS/RNAseL dsRNA sensor pathway. PIC-treated A549 cells also showed a large decrease in oxidative phosphorylation (maximal respiration and spare respiratory capacity), which was prevented by the mitochondrial antioxidant, mitoTEMPO. Conclusions: Activation of the viral dsRNA innate immune sensor pathway, even in the absence of active cardiac viral replication, might contribute to mitochondrial and cardiac dysfunction in COVID-19.

Cyclin-dependent kinase 5 negatively regulates antiviral immune response by disrupting myeloid differentiation primary response protein 88 self-association

ABSTRACT As a member of the pattern recognition receptors (PRRs) involving in the innate immune system, Toll-like receptors (TLRs) can sense a wide range of microbial pathogens and combat infections by producing antimicrobial products, inflammatory cytokines, and chemokines. All TLRs, with the exception of TLR3, activate a signalling cascade via the myeloid differentiation primary response gene 88 (MyD88). Therefore, the activation of MyD88-dependent signalling pathway must be finely controlled. Herein, we identified that cyclin-dependent kinase 5 (CDK5) negatively regulated TLR-MyD88 signalling pathway by targeting MyD88. Overexpression of CDK5 reduced the production of interferons (IFNs), while a deficiency in CDK5 increased the expression of IFNs in response to vesicular stomatitis virus (VSV) infection. Mechanistically, CDK5 suppressed the formation of MyD88 homodimers, resulting in the attenuated production of IFNs induced by VSV infection. Surprisingly, its kinase activity does not play a role in this process. Therefore, CDK5 can act as an internal regulator to prevent excessive production of IFNs by restricting TLR-MyD88-induced activation of antiviral innate immunity in A549 cells.

Negatively regulation of MAVS-mediated antiviral innate immune response by E3 ligase RNF5 in black carp

Mitochondrial antiviral signaling protein (MAVS) is as an adaptor in RIG-I like receptor (RLR) signaling, which plays the key role in interferon (IFN) production during host antiviral innate immune activation. MAVS is fine tuned to avoid excess IFN production, which have been extensively studied in human and mammals. However, the regulation of MAVS in teleost still remains obscure. In this manuscript, we cloned ring finger protein 5 (bcRNF5) of black carp (Mylopharyngodon piceus) and characterized this teleost E3 ubiquitin ligase as a negative regulator of MAVS. The coding region of bcRNF5 consists of 615 nucleotides which encode 205 amino acids, containing two trans-membrane domain (TM) and a ring-finger domain (RING). The transcription regulation of bcRNF5 varies in host cells in response to stimulations of LPS, poly (I:C), grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). bcRNF5 migrates around 22 KDa in immunoblot (IB) assay and distributes mainly in cytoplasm by immunofluorescent (IF) staining test. Moreover, bcRNF5 significantly inhibits bcMAVS-mediated IFN promoter transcription. In addition, both IF and co-immunoprecipitation assay showed that bcRNF5 interacts with bcMAVS. Furthermore, bcMAVS-mediated antiviral ability is distinctly impaired by bcRNF5. Taken together, these results conclude that bcRNF5, as a negative regulator of the MAVS-mediated IFN signaling, may play a key role in host protection upon virus infection in black carp.

Impact of progesterone on innate immunity and cell death after influenza A virus H1N1 2009 infection of lung and placental cells in vitro.

The influenza A virus (IAV) 2009 H1N1 pandemic was associated with an increased risk of maternal mortality, preterm birth, and stillbirth. The underlying mechanism for severe maternal lung disease and stillbirth is incompletely understood, but IAV infection is known to activate innate immunity triggering the release of cytokines. Elucidating the impact of progesterone (P4), a key hormone elevated in pregnancy, on the innate immune and inflammatory response to IAV infection is a critical step in understanding the pathogenesis of adverse maternal-fetal outcomes. IAV H1N1 pdm/09 was used to infect cell lines Calu-3 (lung adenoma) and ACH-3P (extravillous trophoblast) with or without P4 (100 nM) at multiplicity of infections (MOI) 0, 0.5, and 3. Cells were harvested at 24 and 48 hours post infection (hpi) and analyzed for cytopathic effects (CPE), replicating virus (TCID50), cytotoxicity (Lactate Dehydrogenase (LDH) assay), and NLRP3 inflammasome activation (caspase-1 activity, fluorometric assay). Activation of antiviral innate immunity was quantified (RT-qPCR, Luminex) by measuring biomarker gene and protein expression of innate immune activation (IFIT1, IFNB), inflammation (IL6), interferon signaling (MXA), chemokines (IL-8, IL-10). Both Calu-3 and ACH-3P were highly permissible to IAV infection at each timepoint as demonstrated by CPE and recovery of replicating virus. In Calu-3, progesterone treatment was associated with a significant increase in cytotoxicity, increased gene expression of IL6, and increased protein expression of IFN-β, IL-6, and IL-18. Conversely, in ACH-3P, progesterone treatment was associated with significantly suppressed cytotoxicity, decreased gene expression of IFNB, IL6 and IL1B, and increased protein expression of IFN-β and IL-6. In both cell lines, caspase-1 activity was significantly decreased after progesterone treatment, indicating NLRP3 inflammasome activation was not underlying the higher cell death in Calu-3. In summary, these data provide evidence that progesterone plays a dual role by ameliorating viral infection in the placenta but exacerbating influenza A virus-associated injury in the lung through nongenomic modulation of the innate immune response.

Rasmussen\u2019s encephalitis is characterized by relatively lower production of IFN-\u03b2 and activated cytotoxic T cell upon herpes viruses infection

BackgroundThe etiology of Rasmussen's encephalitis (RE), a rare chronic neurological disorder characterized by CD8+ T cell infiltration and unihemispheric brain atrophy, is still unknown. Various human herpes viruses (HHVs) have been detected in RE brain, but their contribution to RE pathogenesis is unclear.MethodsHHVs infection and relevant immune response were compared among brain tissues from RE, temporal lobe epilepsy (TLE) and traumatic brain injury (TBI) patients. Viral antigen or genome, CD8+ T cells, microglia and innate immunity molecules were analyzed by immunohistochemical staining, DNA dot blot assay or immunofluorescence double staining. Cytokines were measured by multiplex flow cytometry. Cell apoptosis was visualized by TUNEL staining. Viral infection, immune response and the severity of unihemispheric atrophy were subjected to correlation analysis.ResultsAntigens of various HHVs were prevalent in RE and TLE brains, and the cumulative viral score of HHVs positively correlated with the unihemispheric atrophy in RE patients. CD8+ T cells infiltration were observed in both RE and TLE brains and showed co-localization with HHV antigens, but their activation, as revealed by Granzyme B (GZMB) release and apoptosis, was found only in RE. In comparison to TLE, RE brain tissues contained higher level of inflammatory cytokines, but the interferon-β level, which was negatively correlated with cumulative viral score, was relatively lower. In line with this, the DNA sensor STING and IFI16, rather than other innate immunity signaling molecules, were insufficiently activated in RE.ConclusionsCompared with TBI, both RE and TLE had prevalently HHV infection and immune response in brain tissues. However, in comparison to TLE, RE showed insufficient activation of antiviral innate immunity but overactivation of cytotoxic T cells. Our results show the relatively lower level of antiviral innate immunity and overactivation of cytotoxic T cells in RE cases upon HHV infection, the overactivated T cells might be a compensate to the innate immunity but the causative evidence is lack in our study and need more investigation in the future.

Zebrafish prmt2 Attenuates Antiviral Innate Immunity by Targeting traf6.

TNFR-associated factor 6 (TRAF6) not only recruits TBK1/IKKε to MAVS upon virus infection but also catalyzes K63-linked polyubiquitination on substrate or itself, which is critical for NEMO-dependent and -independent TBK1/IKKε activation, leading to the production of type I IFNs. The regulation at the TRAF6 level could affect the activation of antiviral innate immunity. In this study, we demonstrate that zebrafish prmt2, a type I arginine methyltransferase, attenuates traf6-mediated antiviral response. Prmt2 binds to the C terminus of traf6 to catalyze arginine asymmetric dimethylation of traf6 at arginine 100, preventing its K63-linked autoubiquitination, which results in the suppression of traf6 activation. In addition, it seems that the N terminus of prmt2 competes with mavs for traf6 binding and prevents the recruitment of tbk1/ikkε to mavs. By zebrafish model, we show that loss of prmt2 promotes the survival ratio of zebrafish larvae after challenge with spring viremia of carp virus. Therefore, we reveal, to our knowledge, a novel function of prmt2 in the negative regulation of antiviral innate immunity by targeting traf6.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

The roles of grouper TANK in innate immune defense against iridovirus and nodavirus infections

The TRAF family member-associated nuclear factor (NF)-κB activator (TANK) was first identified as a TRAF-binding protein with both stimulatory and inhibitory properties in host innate immune activation. To elucidate the roles of TANK in teleosts, we cloned and characterized the TANK homologue of orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcTANK consists of 1026 nucleotides encoding a 342 amino acid protein with a predicted molecular mass of 38.24 kDa. EcTANK shares 89.47% and 88.89% identity with Larimichthys crocea TANK and Lates calcarifer TANK, respectively. EcTANK was distributed in all 11 examined tissues. The expression of EcTANK in the spleen increased after infection with Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV). EcTANK was mainly located in the cytoplasm of grouper spleen cells. EcTANK enhanced SGIV and RGNNV replication during viral infection in vitro. Overexpression EcTANK decreased the expression levels of interferon-associated cytokines and pro-inflammatory factors, and enhanced activation of NF-κB. Taken together, these results suggest that EcTANK may play an important role in antiviral innate immune activation in grouper.

Black carp PRMT6 inhibits TBK1-IRF3/7 signaling during the antiviral innate immune activation

Protein arginine methylation is a prevalent posttranslational modification and protein arginine methyltransferases 6 (PRMT6) has been identified as a suppressor of TBK1/IRF3 in human and mammals. To explore the role of PRMT6 in teleost fish, PRMT6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp PRMT6 (bcPRMT6) transcription in host cells varies in response to different stimuli and bcPRMT6 migrates around 43 kDa in the immunoblot assay. Like its mammalian counterpart, bcPRMT6 has been identified to distribute majorly in the nucleus through the immunofluorescent staining assay. bcPRMT6 shows little interferon (IFN) promoter-inducing activity in the reporter assay and bcPRMT6 shows no antiviral activity against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV) in plaque assay. When co-expressed with bcPRMT6, the IFN promoter-inducing abilities of black carp TBK1 (bcTBK1) and IRF3/7 (bcIRF3/7) are fiercely attenuated. Accordingly, bcTBK1-mediated antiviral activity in EPC cells is obviously dampened by bcPRMT6. The interaction between bcPRMT6 and bcIRF3/7 has been identified by co-immunoprecipitation assay; however, no direct association between bcPRMT6 and bcTBK1 has been detected. Taken together, our data elucidates for the first time in teleost fish that PRMT6 suppresses TBK1-IRF3/7 signaling during host antiviral innate immune activation.

Black carp TAB1 up-regulates TAK1/IRF7/IFN signaling during the antiviral innate immune activation

TAK1-binding protein 1 (TAB1) forms the protein complex with TAK1 and enhances its kinase activity in human and mammals. To elucidate the role of TAB1 in the innate immunity of teleost sfih, the TAB1 homologue of black carp (Mylopharyngodon piceus) (bcTAB1) has been cloned and characterized in this paper. bcTAB1 is composed of 498 amino acids and contains a typical PP2Cc domain like its mammalian counterpart. The transcription of bcTAB1 gene in vivo and ex vivo varied in response to different stimuli; and the immunofluorescence staining showed that bcTAB1 was distributed in both cytoplasm and nucleus of host cell. The reporter assay showed that neither bcTAB1-expression alone nor co-expression of bcTAB1 and bcTAK1 could activate the transcription of IFN in EPC cells. Accordingly, EPC cells expressing bcTAB1 or co-expressing bcTAB1 and bcTAK1 showed no improved antiviral activity against grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). However, EPC cells co-expressing bcTAB1, bcTAK1 and bcIRF7 showed fiercely increased IFN-inducing ability in reporter assay and obviously improved antiviral activity in plaque assay compared with EPC cells co-expressing bcTAK1 and bcIRF7. The subsequent co-immunoprecipitation assay identified that bcTAB1 associated with bcTAK1 but not interacted with bcIRF7. Based on our previous finding that bcTAK1 up-regulates bcIRF7-mediated IFN signaling during host innate immune activation, the data generated in this study support the conclusion that bcTAB1 interacts with bcTAK1 and boosts bcTAK1-activated bcIRF7/IFN signaling during host antiviral innate immune response against GCRV and SVCV.

STAT1a and STAT1b of black carp play important roles in the innate immune defense against GCRV

Signal transducer and activator of transcription 1 (STAT1) plays an important role in the Janus kinase (JAK)-STAT signaling of human and mammals; however, the mechanism of STAT1 in innate immune activation of teleost fishes remains largely unknown. In this study, two STAT1 homologues (bcSTAT1a and bcSTAT1b) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Both bcSTAT1a and bcSTAT1b transcription in host cells was obviously increased in response to the stimulation of poly (I:C), lipopolysaccharide (LPS), grass carp reovirus (GCRV) and interferon (IFN); however, the increase rate of bcSTAT1b transcription post stimulation was obviously higher than that of bcSTAT1a. bcSTAT1a and bcSTAT1b were distributed in both cytoplasm and nucleus in the immunofluorescence staining assay. Self-association of bcSTAT1a and bcSTAT1b, and the interaction between bcSTAT1a and bcSTAT1b have been detected through co-immunoprecipitation (co-IP) assay; and the data of native polyacrylamide gel electrophoresis (PAGE) implied that bcSTAT1a and bcSTAT1b might form homodimer and heterodimer in vivo like their mammalian counterparts. Both bcSTAT1a and bcSTAT1b presented IFN-inducing ability in report assay, and both bcSTAT1a and bcSTAT1b showed antiviral activities against GCRV in EPC cells. Our data support the conclusion that both bcSTAT1a and bcSTAT1b play important roles in host antiviral innate immune activation initiated by GCRV.

Molecular cloning and characterization of TANK of black carp Mylopharyngodon piceus

The TRAF family member-associated NF-κB activator (TANK) is linked to the regulation of the transcription of NF-κB in mammals; however, its role in interferon induction is unclear. To elucidate the roles of TANK in teleost, the TANK homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this paper. The open reading frame (ORF) of black carp TANK (bcTANK) comprises 1050 nucleotides and the predicted bcTANK protein contains 350 amino acids. The transcription of bcTANK in host cells increased in response to the stimulation of LPS, poly (I:C), SVCV and GCRV. bcTANK migrated around 50 KDa in immunoblot assay and was identified as a cytosolic protein by immunofluorescent staining in both EPC and HeLa cells. bcTANK could not induce the activity of IFN promoter in luciferase reporter assay in EPC cells; however, the IFN-activation ability of bcTANK was obviously enhanced when the cells were treated with LPS, poly (I:C) or virus. Both CPE ratio and virus titer in the media of EPC cells expressing bcTANK were obviously lower than those of the control cells, which were examined by violet crystal staining and plaque assay separately. Taken together, our data support the conclusion that bcTANK plays an important role in the antiviral innate immune activation of black carp.

Erratum: Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.

Nat. Immunol.; doi:10.1038/ni.3464; corrected online 9 June 2016 In the version of this article initially published online, the blot in Figure 7d was a duplicate of the blot in Figure 7c. The error has been corrected in the print, PDF and HTML versions of this article.

Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.

The DNA methyltransferase Dnmt3a has high expression in terminally differentiated macrophages; however, its role in innate immunity remains unknown. Here we report that deficiency in Dnmt3a selectively impaired the production of type I interferons triggered by pattern-recognition receptors (PRRs), but not that of the proinflammatory cytokines TNF and IL-6. Dnmt3a-deficient mice exhibited enhanced susceptibility to viral challenge. Dnmt3a did not directly regulate the transcription of genes encoding type I interferons; instead, it increased the production of type I interferons through an epigenetic mechanism by maintaining high expression of the histone deacetylase HDAC9. In turn, HDAC9 directly maintained the deacetylation status of the key PRR signaling molecule TBK1 and enhanced its kinase activity. Our data add mechanistic insight into the crosstalk between epigenetic modifications and post-translational modifications in the regulation of PRR signaling and activation of antiviral innate immune responses.

Regulation of antiviral innate immune signaling by stress-induced RNA granules.

Activation of antiviral innate immunity is triggered by cellular pattern recognition receptors. Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) detect viral non-self RNA in cytoplasm of virus-infected cells and play a critical role in the clearance of the invaded viruses through production of antiviral cytokines. Among the three known RLRs, RIG-I and melanoma differentiation-associated gene 5 recognize distinct non-self signatures of viral RNA and activate antiviral signaling. Recent reports have clearly described the molecular machinery underlying the activation of RLRs and interactions with the downstream adaptor, mitochondrial antiviral signaling protein (MAVS). RLRs and MAVS are thought to form large multimeric filaments around cytoplasmic organelles depending on the presence of Lys63-linked ubiquitin chains. Furthermore, RLRs have been shown to localize to stress-induced ribonucleoprotein aggregate known as stress granules and utilize them as a platform for recognition/activation of signaling. In this review, we will focus on the current understanding of RLR-mediated signal activation and the interactions with stress-induced RNA granules.