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The biological factors that promote inflammation or nonalcoholic steatohepatitis (NASH) in the setting of nonalcoholic fatty liver disease remain incompletely understood. Clinical studies have demonstrated an association between obstructive sleep apnea (OSA) and both inflammation and fibrosis in NASH, but the mechanism has not been identified. In this study, we use in vitro modeling to examine the impact of intermittent hypoxia on the liver. Hepatocyte, stellate cell, and macrophage cell lines were exposed to intermittent or sustained hypoxia. Candidate genes associated with inflammation, fibrosis, and lipogenesis were analyzed. Circulating cytokines were assessed in human serum of patients with nonalcoholic fatty liver disease. Intermittent hypoxia results in significant induction of interleukin (IL)-6 expression in both hepatocytes and macrophages. The increase in IL-6 expression was independent of hypoxia inducible factor 1 induction but appeared to be in part related to antioxidant response element and nuclear factor kappa B activation. Mature microRNA 365 (miR-365) has been demonstrated to regulate IL-6 expression, and we found that miR-365 expression was decreased in the setting of intermittent hypoxia. Furthermore, macrophage cell lines showed polarization to an M1 but not M2 phenotype. Finally, we found a trend toward higher circulating levels of IL-6 in patients with OSA and NASH. Conclusion: Intermittent hypoxia acts as a potent proinflammatory stimulus, resulting in IL-6 induction and M1 macrophage polarization. Increased IL-6 expression may be due to both induction of antioxidant response element and nuclear factor kappa B as well as inhibition of miR-365 expression. Higher levels of IL-6 were observed in human samples of patients with OSA and NASH. These findings provide biological insight into mechanisms by which obstructive sleep apnea potentiates inflammation and fibrosis in patients with fatty liver disease. (Hepatology Communications 2017;1:326-337).

Background & Aims Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response. Methods We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection. Results We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties. Conclusions Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation. Lay summary In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver.

HIV/HCV co-infection accelerates progressive liver fibrosis, however the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers TGFβ1 (mediated by reactive oxygen species (ROS)) and NFκB in hepatocytes and hepatic stellate cells (HSC) in monoculture, however, they do not account for cellular cross-talk that naturally occurs. We created an in vitro co-culture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. GFP reporter cell lines driven by functional ROS (ARE), NFκB, and SMAD3 promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate co-cultures. Reporter cells lines were exposed to HIV, HCV or HIV/HCV. Activation of the 3 pathways were measured, and compared according to infection status. Extracellular matrix products (Col1A1 and TIMP1) were also measured. Both HCV and HIV independently activate TGFβ1 signaling via ROS (ARE), NFκB, and SMAD3 in both cell lines in co-culture. Activation of these profibrotic pathways was additive following HIV/HCV co-exposure. This was confirmed when examining Col1A1 and TIMP1, where mRNA and protein levels were significantly higher in LX2 cells in co-culture following HIV/HCV co-exposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the co-culture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. We conclude that HIV accentuates an HCV-driven profibrogenic program in hepatocyte and HSC lines through ROS, NFκB and TGFβ1 upregulation. Furthermore, co-culture of hepatocyte and HSC lines significantly increased expression of Col1A1 and TIMP1. Our novel co-culture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses, and may provide important insights into hepatic fibrosis. This article is protected by copyright. All rights reserved.

The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway. IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.

International audience; Poliovirus (PV)-induced apoptosis seems to play a major role in central nervous system (CNS) tissue injury, a crucial feature of the pathogenesis of poliomyelitis. We have previously shown that calcium (Ca2+) flux from the endoplasmic reticulum (ER) to the cytosol during PV infection is involved in apoptosis induction in human neuroblastoma cells. We show here that PV infection is associated with a transient upregulation of Herp (homocysteine-induced ER protein), a protein known to promote the degradation of ER-resident Ca2+ channels. Herp gene transcription is controlled by the transcription factor CREB3 (cAMP response element-binding protein 3). We found that the CREB3/Herp pathway limited the increase in cytosolic Ca2+ concentration and apoptosis early in PV infection. This may reduce the extent of PV-induced damage to the CNS during poliomyelitis.

AIM To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection. METHODS In this study, we utilize a gene editing tool, transcription activator-like effector nucleases (TALENs), to generate human hepatoma cells with a stable genetic deletion of APOB to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDA-approved antisense inhibitor of apoB100, on HCV using in vitro cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method. RESULTS We found that apoB100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALEN-mediated gene deletion of apoB (APOB KO), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apoB did not play a role in HCV entry or replication. However, the virus produced by APOB KO cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus. CONCLUSION ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1 and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2 and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

Background &/Aims The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. Methods We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. Results We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3 . However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3 , GORASP2 , ZFAND6 , and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. Conclusions The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3 , GORASP2 . SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Background & Aims Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. Methods We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. Results The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, β 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, β isoform; peroxisomal proliferator-activated receptor−γ-DBD−interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα−mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFNα; these genes represent a heretofore unknown class of non−IFN-stimulated gene IEGs. Conclusions We performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFNα against human pathogenic viruses. We found that IFNα restricts HCV via actions of general and specific IEGs.

We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca 2+ ) concentration in neuroblastoma IMR5 cells, at least partly through Ca 2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP 3 R) and ryanodine receptor (RyR) channels. This leads to Ca 2+ accumulation in mitochondria through the mitochondrial Ca 2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca 2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.

Poliovirus (PV)-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dysfunction mediated by early JNK activation in IMR5 neuroblastoma cells. We showed here that PV simultaneously activates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signaling pathway in these cells, limiting the extent of JNK activation and thereby cell death. JNK inhibition is associated with PI3K-dependent negative regulation of the apoptosis signal-regulating kinase 1, which acts upstream from JNK in PV-infected IMR5 cells. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.

The flaccid paralyses characteristic of poliomyelitis are a direct consequence of the infection of motor neurons with poliovirus (PV). In PV-infected mice, motor neurons die by apoptosis. However, the mechanisms by which PV induces cell death in neurons remain unclear. Analyses of the apoptotic pathways induced by PV infection in several cell lines have demonstrated that mitochondria play a key role in PV-induced apoptosis. Furthermore, mitochondrial dysfunction results from an imbalance between pro- and anti-apoptotic pathways. We present here an overview of the many studies of PV-induced apoptosis carried out in recent years and discuss the contribution of these studies to our understanding of poliomyelitis.