Abstract: Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases 1-4 . Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade 5-7 . Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.

Abstract: In mammals, 2'-O-methylation of RNA is a molecular signature by which the cellular innate immune system distinguishes endogenous from exogenous messenger RNA1-3. However, the molecular functions of RNA 2'-O-methylation are not well understood. Here we have purified TAR RNA-binding protein (TRBP) and its interacting partners and identified a DICER-independent TRBP complex containing FTSJ3, a putative 2'-O-methyltransferase (2'O-MTase). In vitro and ex vivo experiments show that FTSJ3 is a 2'O-MTase that is recruited to HIV RNA through TRBP. Using RiboMethSeq analysis4, we identified predominantly FTSJ3-dependent 2'-O-methylations at specific residues on the viral genome. HIV-1 viruses produced in FTSJ3 knockdown cells show reduced 2'-O-methylation and trigger expression of type 1 interferons (IFNs) in human dendritic cells through the RNA sensor MDA5. This induction of IFN-α and IFN-β leads to a reduction in HIV expression. We have identified an unexpected mechanism used by HIV-1 to evade innate immune recognition: the recruitment of the TRBP-FTSJ3 complex to viral RNA and its 2'-O-methylation.

Abstract: Specialized receptors that recognize molecular patterns such as double stranded RNA duplexes-indicative of viral replication-are potent triggers of the innate immune system Although their activation is beneficial during viral infection, RNA transcribed from endogenous mobile genetic elements may also act as ligands potentially causing autoimmunity Recent advances indicate that the adenosine deaminase ADAR1 through RNA editing is involved in dampening the canonical antiviral RIG-I-like receptor-, PKR-, and OAS-RNAse L pathways to prevent autoimmunity However, this inhibitory effect must be overcome during viral infections In this review we discuss ADAR1's critical role in balancing immune activation and self-tolerance

Abstract: RIG-I-Like Receptors (RLRs) RIG-I, MDA5, and LGP2, are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) infections continue to grow in the US. Here, we use a systems biology approach to define the contributions of each RLR in the innate immune response to WNV. Genome-wide RNAseq and bioinformatics analyses of macrophages from mice lacking either RLR reveal that the RLRs drive distinct immune gene activation and response polarization to mediate an M1/inflammatory signature while suppressing the M2/wound healing phenotype. While LGP2 functions to modulate inflammatory signaling, RIG-I and MDA5 together are essential for M1 macrophage polarization in vivo and the control of WNV infection through potential downstream control of ATF4 and SMAD4 to regulate target gene expression for cell polarization. These analyses reveal the RLR-driven signature of macrophage polarization, innate immune protection, and immune programming against WNV infection.

Abstract: The retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I– and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I–dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.

Abstract: RNA helicases play an important role in the response to microbial infection. Retinoic acid inducible gene-I (RIG-I) and members of the RIG-I-like receptor (RLR) family of helicases function as cytoplasmic pattern recognition receptors (PRRs) whose actions are essential for recognition of RNA viruses. RIG-I association with pathogen-associated molecular patterns (PAMPs) within viral RNA leads to its activation and signaling via the mitochondrial antiviral signaling (MAVS) adapter protein. This interaction mediates downstream signaling events that drive the innate immune response to virus infection. Here we identify the DEAH-box RNA helicase DHX15 as a RLR binding partner and signaling cofactor. In human cells, DHX15 is required for virus-induced RLR signaling of innate immune gene expression. Knockdown of DHX15 increased susceptibility to infection by RNA viruses of diverse genera, including Paramyxoviridae, Rhabdoviridae, and Picornaviridae. DHX15 associates with RIG-I caspase activation and recruitment domains (CARDs) through its amino terminus, in which the complex is recruited to MAVS on virus infection. Importantly, although DHX15 cannot substitute for RIG-I in innate immune signaling, DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation in response to viral RNA. Our results define DHX15 as a coreceptor required for RLR innate immune responses to control RNA virus infection.

Abstract: Zika virus (ZIKV) is a mosquito-transmitted, emerging Flavivirus that causes Guillain-Barre syndrome and microcephaly in adults and fetuses, respectively. Since ZIKV was first isolated in 1947, severe outbreaks have occurred at various places worldwide, including Yap Island in 2007, French Polynesia in 2013, and Brazil in 2015. Although incidences of ZIKV infection and dissemination have drastically increased, the mechanisms underlying the pathogenesis of ZIKV have not been sufficiently studied. In addition, despite extensive research, the exact roles of individual ZIKV genes in the viral evasion of the host innate immune responses remain elusive. Besides, it is still possible that more than one ZIKV-encoded protein may negatively affect type I interferon (IFN) induction. Hence, in this study, we aimed to determine the modulations of the IFN promoter activity, induced by the MDA5/RIG-I signaling pathway, by over-expressing individual ZIKV genes. Our results show that two nonstructural proteins, NS2A and NS4A, significantly down-regulated the promoter activity of IFN-β by inhibiting multiple signaling molecules involved in the activation of IFN-β. Interestingly, while NS2A suppressed both full-length and constitutively active RIG-I, NS4A had inhibitory activity only on full-length RIG-I. In addition, while NS2A inhibited all forms of IRF3 (full-length, regulatory domain-deficient, and constitutively active), NS4A could not inhibit constitutively active IRF3-5D. Taken together, our results showed that NS2A and NS4A play major roles as antagonists of MDA5/RIG-I-mediated IFN-β induction and more importantly, these two viral proteins seem to inhibit induction of the type I IFN responses in differential mechanisms. We believe this study expands our understanding regarding the mechanisms via which ZIKV controls the innate immune responses in cells and may pave the way to development of ZIKV-specific therapeutics.

Abstract: Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-β production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-β production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-β production and transcription factor IRF3 activation, but did not block IFN-β production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.

Abstract: Infection by enteroviruses can cause severe neurological complications in humans. The interactions between the enteroviral and host proteins may facilitate the virus replication and be involved in the pathogenicity of infected individuals. It has been shown that human enteroviruses possess various mechanisms to suppress host innate immune responses in infected cells. Previous studies showed that infection by enterovirus 71 (EV71) causes the degradation of MDA5, which is a critical cytoplasmic pathogen sensor in the recognition of picornaviruses for initiating transcription of type I interferons. In the present study, we demonstrated that the RNA-dependent RNA polymerase (RdRP; also denoted 3Dpol) encoded by EV71 interacts with the caspase activation and recruitment domains (CARDs) of MDA5 and plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation and mRNA expression. In addition, we found that the 3Dpol protein encoded by coxsackievirus B3 also interacted with MDA5 and downregulated the antiviral signaling initiated by MDA5. These findings indicate that enteroviral RdRP may function as an antagonist against the host antiviral innate immune response.IMPORTANCE Infection by enteroviruses causes severe neurological complications in humans. Human enteroviruses possess various mechanisms to suppress the host type I interferon (IFN) response in infected cells to establish viral replication. In the present study, we found that the enteroviral 3Dpol protein (or RdRP), which is a viral RNA-dependent RNA polymerase for replicating viral RNA, plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation. We further demonstrated that enteroviral 3Dpol protein interacts with the caspase activation and recruitment domains (CARDs) of MDA5. These findings indicate that enteroviral RdRP functions as an antagonist against the host antiviral response.

Abstract: Hemoglobin is an important oxygen-carrying protein and plays crucial roles in establishing host resistance against pathogens and in regulating innate immune responses. The hemoglobin subunit beta (HB) is an essential component of hemoglobin, and we have previously demonstrated that the antiviral role of the porcine HB (pHB) is mediated by promoting type I interferon pathways. Thus, considering the high homology between human HB (hHB) and pHB, we hypothesized that hHB also plays an important role in the antiviral innate immunity. In this study, we characterized hHB as a regulatory factor for the replication of RNA viruses by differentially regulating the RIG-I- and MDA5-mediated antiviral signaling pathways. Furthermore, we showed that hHB directly inhibited MDA5-mediated signaling by reducing the MDA5-double-stranded RNA (dsRNA) interaction. Additionally, hHB required hHB-induced reactive oxygen species (ROS) to promote RIG-I-mediated signaling through enhancement of K63-linked RIG-I ubiquitination. Taken together, our findings suggest that hHB is a pleiotropic regulator of RIG-I/MDA5-mediated antiviral responses and further highlight the importance of the intercellular microenvironment, including the redox state, in regulating antiviral innate immune responses.IMPORTANCE Hemoglobin, the most important oxygen-carrying protein, is involved in the regulation of innate immune responses. We have previously reported that the porcine hemoglobin subunit beta (HB) exerts antiviral activity through regulation of type I interferon production. However, the antiviral activities and the underlying mechanisms of HBs originating from other animals have been poorly understood. Here, we identified human HB (hHB) as a pleiotropic regulator of the replication of RNA viruses through regulation of RIG-I/MDA5-mediated signaling pathways. hHB enhances RIG-I-mediated antiviral responses by promoting RIG-I ubiquitination depending on the hHB-induced reactive oxygen species (ROS), while it blocks MDA5-mediated antiviral signaling by suppressing the MDA5-dsRNA interaction. Our results contribute to an understanding of the crucial roles of hHB in the regulation of the RIG-I/MDA5-mediated signaling pathways. We also provide novel insight into the correlation of the intercellular redox state with the regulation of antiviral innate immunity.

Abstract: The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that the addition of Riplet to these cells reduced HCV Y16F replication, whereas the addition of Riplet lacking the RING domain restored HCV Y16F replication. In addition, TBK1 inhibition or IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a noncanonical Riplet-TBK1-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection.IMPORTANCE The HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. Since Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.

Abstract: Teleost fish, as with other vertebrates, rely on their innate immune system as a first line of defense against invading pathogens. A very important characteristic of the innate immune response is its ability to recognize conserved molecular structures, such as viral dsRNA and ssRNA. Mda5 is one of the three pattern recognition receptors (PRRs) that recognize cytoplasmic viral ligands. Teleost Mda5 is widely conserved among several fish species and possesses the same structural domains as those seen in their mammalian counterparts. Fish Mda5 has been shown to be capable of initiating an inflammatory response both in vitro (in different fish cell lines) and in vivo using synthetic viral analogs or virus. The interferon (IFN) pathway is triggered as a result of Mda5 activation, leading to the expression of type I IFNs, IFN- stimulated genes and pro-inflammatory cytokines. Although it is known that Mda5 acts as a receptor for virally-produced ligands, it has been shown more recently that it can also initiate an immune response against bacterial challenges. This review discusses recent advances in the characterization of teleost Mda5 and its potential role in antiviral and antibacterial immunity in teleost fish.

Abstract: Chikungunya virus (CHIKV) is a single-stranded positive-sense RNA virus, belonging to the genus Alphavirus of the Togaviridae family. It causes multiple symptoms, including headache, fever, severe joint and muscle pain, and arthralgia. Since CHIKV was first isolated in Tanzania in 1952, there have been multiple outbreaks of chikungunya fever. However, its pathogenesis and mechanisms of viral immune evasion have been poorly understood. In addition, the exact roles of individual CHIKV genes on the host innate immune response remain largely unknown. To investigate if CHIKV-encoded genes modulate the type I interferon (IFN) response, each and every CHIKV gene was screened for its effects on the induction of the IFN-β promoter. Here we report that CHIKV nsP2, E2 and E1 strongly suppressed activation of the IFN-β promoter induced by the MDA5/RIG-I receptor signaling pathway, suggesting that nsP2, E2, and E1 are the major antagonists against induction of IFN-β. Delineation of underlying mechanisms of CHIKV-mediated inhibition of the IFN-β pathway may help develop virus-specific therapeutics and vaccines.

Abstract: Flaviviruses are a diverse group of arthropod-borne viruses responsible for numerous significant public health threats; therefore, understanding the interactions between these viruses and the human immune response remains vital. West Nile virus (WNV) and Zika virus (ZIKV) infect human dendritic cells (DCs) and can block antiviral immune responses in DCs. Previously, we used mRNA sequencing and weighted gene coexpression network analysis (WGCNA) to define molecular signatures of antiviral DC responses following activation of innate immune signaling (RIG-I, MDA5, or type I interferon [IFN] signaling) or infection with WNV. Using this approach, we found that several genes involved in T cell cosignaling and antigen processing were not enriched in DCs during WNV infection. Using cis-regulatory sequence analysis, STAT5 was identified as a regulator of DC activation and immune responses downstream of innate immune signaling that was not activated during either WNV or ZIKV infection. Mechanistically, WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN-β, and interleukin-4 (IL-4), but not granulocyte-macrophage colony-stimulating factor (GM-CSF), signaling. Unexpectedly, dengue virus serotypes 1 to 4 (DENV1 to DENV4) and the yellow fever 17D vaccine strain (YFV-17D) did not antagonize STAT5 phosphorylation. In contrast to WNV, ZIKV inhibited JAK1 and TYK2 phosphorylation following type I IFN treatment, suggesting divergent mechanisms used by these viruses to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert the immune response in infected DCs.IMPORTANCE Flaviviruses are a diverse group of insect-borne viruses responsible for numerous significant public health threats. Previously, we used a computational biology approach to define molecular signatures of antiviral DC responses following activation of innate immune signaling or infection with West Nile virus (WNV). In this work, we identify STAT5 as a regulator of DC activation and antiviral immune responses downstream of innate immune signaling that was not activated during either WNV or Zika virus (ZIKV) infection. WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN-β, and IL-4, but not GM-CSF, signaling. However, other related flaviviruses, dengue virus serotypes 1 to 4 and the yellow fever 17D vaccine strain, did not antagonize STAT5 phosphorylation. Mechanistically, WNV and ZIKV showed differential inhibition of Jak kinases upstream of STAT5, suggesting divergent countermeasures to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert antiviral immune responses in human DCs.

Abstract: Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the beta coronavirus subfamily and causes severe morbidity and mortality in humans especially when infected patients have underlying diseases such chronic obstructive pulmonary disease (COPD). Previously, we demonstrated that MERS-CoV-encoded ORF8b strongly inhibits MDA5- and RIG-I-mediated induction of the interferon beta (IFN-β) promoter activities. Here, we report that ORF8b seem to regulate MDA5 or RIG-I differentially as protein levels of MDA5 were significantly down-regulated while those of RIG-I were largely unperturbed. In addition, ORF8b seemed to efficiently suppress phosphorylation of IRF3 at the residues of 386 and 396 in cells transfected with RIG-I while total endogenous levels of IRF3 remained largely unchanged. Furthermore, ORF8b was able to inhibit all forms of RIG-I; full-length, RIG-I-1-734, and RIG-I-1-228, last of which contains only the CARD domains. Taken together, it is tempting to postulate that ORF8b may interfere with the CARD-CARD interactions between RIG-I and MAVS. Further detailed analysis is required to delineate the mechanisms of how ORF8b inhibits the MDA5/RIG-I receptor signaling pathway.

Abstract: The airway epithelial barrier is critical for preventing pathogen invasion and translocation of inhaled particles into the lung. Epithelial cells also serve an important sentinel role after infection and release various pro-inflammatory mediators that recruit and activate immune cells. Airway epithelial barrier disruption has been implicated in a growing number of respiratory diseases including viral infections. It is thought that when a pathogen breaks the barrier and gains access to the host tissue, pro-inflammatory mediators increase, which further disrupts the barrier and initiates a vicious cycle of leak. However, it is difficult to study airway barrier integrity in vivo, and little is known about relationship between epithelial barrier function and airway inflammation. Current assays of pulmonary barrier integrity quantify the leak of macromolecules from the vasculature into the airspaces (or "inside/out" leak). However, it is also important to measure the ease with which inhaled particles, allergens, or pathogens can enter the subepithelial tissues (or "outside/in" leak). We challenged mice with inhaled double stranded RNA (dsRNA) and explored the relationship between inside/out and outside/in barrier function and airway inflammation. Using wild-type and gene-targeted mice, we studied the roles of the dsRNA sensors Toll Like Receptor 3 (TLR3) and Melanoma Differentiation-Associated protein 5 (MDA5). Here we report that after acute challenge with inhaled dsRNA, airway barrier dysfunction occurs in a TLR3-dependent manner, whereas leukocyte accumulation is largely MDA5-dependent. We conclude that airway barrier dysfunction and inflammation are regulated by different mechanisms at early time points after exposure to inhaled dsRNA.

Abstract: Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon β (IFN-β) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-β expression would provide basis of the cell culture adaptation of HEV.

Abstract: Hypoxia is a common phenomenon in solid tumours and is considered a hallmark of cancer. Increasing evidence shows that hypoxia promotes local immune suppression. Type I IFN is involved in supporting cytotoxic T lymphocytes by stimulating the maturation of dendritic cells (DCs) and enhancing their capacity to process and present antigens. However, there is little information about the relationship between hypoxia and the type I interferon (IFN) pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA), followed by IFNα/β secretion and transcription activation of IFN-stimulated genes (ISGs). The aims of this study were to determine both the effect and mechanisms of hypoxia on the I IFN pathway in breast cancer. There was a downregulation of the type I IFN pathway expression at mRNA and protein level in cancer cell lines under hypoxia in vitro and in vivo in xenografts. This pathway was suppressed at each level of signalling, from the dsRNA sensors (RIG-I, MDA5), the adaptor (MAVS), transcription factors (IRF3, IRF7, STAT1) and several ISGs (RIG-I, IRF7, STAT1, ADAR-p150). There was also lower IFN secretion under hypoxic conditions. HIF1 and HIF2 regulation of gene expression did not explain most of the effects. However, ATAC-Seq data revealed that in hypoxia peaks with STAT1 and IRF3 motifs had decreased accessibility. Thus hypoxia leads to an overall 50% downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in a HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumours.

Abstract: The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that addition of Riplet to these cells reduced HCV Y16F replication. In addition, IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a non-canonical Riplet-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection. Importance The HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet, but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. As Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.

Abstract: The molecular mechanisms of innate immunity are closely associated with the development of non-alcoholic fatty liver disease (NAFLD). TNF-α is a key cytokine involved in the pathogenesis of metabolic inflammation like NAFLD. Melanoma differentiation-associated gene 5 (MDA5) is a member of the intracellular RNA helicase family proteins that play a pivotal role in an antiviral immune response. Previous studies have demonstrated that TNF-α induces the expression of MDA5 in some types of cells. However, the correlation between TNF-α and the expression of MDA5 in hepatocytes remains unknown. In the present study, we used two human hepatocellular carcinoma cell lines, HuH-7 and HLE, and examined the expression of MDA5 in these cells upon stimulation with TNF-α. The expression of MDA5 induced by TNF-α was analyzed by quantitative real-time RT-PCR and western blotting. Next, RNA interference against MDA5 was performed and the expressions of CXCL10 and STAT1 were examined. We found that the expression of MDA5 had increased upon stimulation with TNF-α in a concentration-dependent manner. Gene silencing against MDA5 suppressed the expression of TNF-α-induced CXCL10 in both cells. In HLE cells, gene silencing of MDA5 impaired STAT1 phosphorylation 24 h after stimulation with TNF-α. On the other hand, TNF-α-induced STAT1 phosphorylation was not detected in HuH-7 cells. These results indicated that MDA5 positively modulated the TNF-α-induced expression of CXCL10 in both STAT1-dependent and -independent manner and may be associated with metabolic inflammation in the liver.