Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular JAK-STAT pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 h of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV infected cells, the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. In conclusion, we describe the kinetics of gene induction as being fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes suggesting distinct roles of these cytokines within the immune response. Furthermore, we demonstrate that the observed differences are substantially altered by infection with the hepatitis C virus.

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.

We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.

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.Poliovirus (PV), from the Picornaviridae family, causes paralytic poliomyelitis, a disease in which the motor neurons are destroyed in association with PV replication. PV consists of a single-stranded positive-sense RNA genome surrounded by a nonenveloped icosahedral protein capsid. The human PV receptor CD155 and its simian counterparts belong to the immunoglobulin superfamily (24,25,31) and are related to the nectin family of adhesion molecules (28,38).PV is transmitted mostly via the fecal-oral route. It first infects the oropharynx and the digestive tract and then spreads to the central nervous system (CNS), in which it targets mostly motor neurons. Studies with mouse models have shown that PV-infected motor neurons in the spinal cord die by apoptosis (10, 19). PV-induced apoptosis therefore seems to play a major role in the tissue injury occurring in the CNS.PV triggers apoptosis in vitro in tissue cultures of human colon carcinoma (Caco-2) cells (4), promonocytic cells (U937) (29), dendritic cells (41), murine L cells expressing CD155 (21, 36), HeLa cells (8,39), and cultures of mixed mouse primary nerve cells (12) from the cerebral cortexes of mice transgenic for CD155. Analyses of the apoptotic pathways induced following PV infection in several cell lines have demonstrated that mitochondria are key actors of PV-induced apoptosis. In particular, mitochondrial outer membrane permeabilization (MOMP) following PV infection leads to a loss of mitochondrial transmembrane potential and the release of proapoptotic molecules, including cytochrome c, from the mitochondria to the cytosol (8, 21). We recently demonstrated that MOMP in PV-infected neuronal IMR5 cells was dependent on Bax, a proapoptotic member of the Bcl-2 family. Bax activation was mediated by c-Jun NH2-terminal kinase (JNK) phosphorylation after PV infection (6). JNK activation occurred early after PV infection, whereas apoptotic features were observed later in PV-infected cells. These events may involve a balance between pro-and antiapoptotic signals following PV infection. Pro-and antiapoptotic events potentially acting in synergy or competing with each other during the reproduction cycle of PV have been described by Agol's group (1, 39). However, the mechanisms involved in maintaining this delicate balance remain uncle...

eWe 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.

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 HCVinfected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. H epatitis C virus (HCV) poses a threat to public health by infecting approximately 170 million people worldwide and causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). Fifty to ninety percent of acute infections become chronic due to failure to mount a productive immune response to clear the virus. The innate immune system is the first line of defense responsible for recognizing viral pathogens and is therefore one of the crucial elements in determining the outcome of HCV infection.It is known that HCV RNA is recognized by the combined actions of protein kinase R (PKR), retinoic acid-inducible gene 1 (RIG-I), and Toll-like receptor 3 (TLR3) after viral entry into host cells (3-5). Pathogen recognition triggers downstream signaling to activate transcription factors, such as interferon (IFN) regulatory factors (IRFs), to induce type I interferon secretion and stimulation of the JAK-STAT signaling pathway, leading to the expression of IFN-stimulated genes (ISGs), which presumptively mediate control of viral replica-

Background & Aims Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon (IFN)-α is an important component of anti-HCV therapy; it upregulates transcription of IFN-stimulated genes (ISGs)—many of which have been investigated for their anti-viral effects. However, all the genes required for the anti-viral function of IFN-α (IFN effector genes, IEGs) are not known. IEGs include not only ISGs, but other non-transcriptionally induced genes that are required for the anti-viral effect of IFN-α. In contrast to candidate approaches based on analyses mRNA expression, identification of IEGs requires a broad functional approach. Methods We performed an unbiased genome-wide small-interfering (si)RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with siRNAs, incubated with IFN-α, 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 9 (ALG10, BCHE, DPP4, GCKR, GUCY1B3, MYST1, PPP3CB, PDIP1, SLC27A2) and demonstrated that they enabled IFN-α–mediated suppression of HCV at multiple steps of its lifecycle. Expression of these genes had more potent effects against flaviviridae, because a subset were required for IFN-α to suppress dengue virus but not influenza A virus. Furthermore, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFN-α; these genes represent a heretofore unknown class of non-ISG IEGs. Conclusion We performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFN-α against human pathogenic viruses. We found that IFN-α restricts HCV via actions of general and specific IEGs.

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.