Hepatitis C Virus Infected Cells Research Articles

Hepatitis B virus and hepatitis C virus affect mitochondrial function through different metabolic pathways, explaining virus-specific clinical features of chronic hepatitis.

Hepatitis C virus (HCV) and hepatitis B virus (HBV) cause chronic hepatitis with important clinical differences. HCV causes hepatic steatosis and insulin resistance, while HBV confers increased risk of liver cancer. We hypothesised these differences may be due to virus-specific effects on mitochondrial function. Seahorse technology was utilised to investigate effects of virus infection on mitochondrial function. Cell based assays were used to measure mitochondrial membrane potential and quantify pyruvate and lactate. Mass spectrometry was performed on mitochondria isolated from HBV expressing, HCV infected and control cells cultured with isotope-labelled amino acids, to identify proteins with different abundance. Altered expression of key mitochondrial proteins was confirmed by real time PCR and western blot. Reduced mitochondrial function and ATP production were observed with HCV infection and HBV expression. HCV impairs glycolysis and reduces expression of genes regulating fatty acid oxidation, promoting lipid accumulation. HBV causes lactate accumulation by increasing expression of lactate dehydrogenase A, which converts pyruvate to lactate. In HBV expressing cells there was marked enrichment of pyruvate dehydrogenase kinase, inhibiting conversion of pyruvate to acetyl-CoA and thereby reducing its availability for mitochondrial oxidative phosphorylation. HCV and HBV impair mitochondrial function and reduce ATP production. HCV reduces acetyl-CoA availability for energy production by impairing fatty acid oxidation, causing lipid accumulation and hepatic steatosis. HBV has no effect on fatty oxidation but reduces acetyl-CoA availability by disrupting pyruvate metabolism. This promotes lactic acidosis and oxidative stress, increasing the risk of disease progression and liver cancer.

Targeting ER stress/PKA/GSK-3β/β-catenin pathway as a potential novel strategy for hepatitis C virus-infected patients

BackgroundChronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). The HCC risk, while decreased compared with active HCV infection, persists in HCV-cured patients by direct-acting antiviral agents (DAA). We previously demonstrated that Wnt/β-catenin signaling remained activated after DAA-mediated HCV eradication. Developing therapeutic strategies to both eradicate HCV and reverse Wnt/β-catenin signaling is needed.MethodsCell-based HCV long term infection was established. Chronically HCV infected cells were treated with DAA, protein kinase A (PKA) inhibitor H89 and endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA). Western blotting analysis and fluorescence microscopy were performed to determine HCV levels and component levels involved in ER stress/PKA/glycogen synthase kinase-3β (GSK-3β)/β-catenin pathway. Meanwhile, the effects of H89 and TUDCA were determined on HCV infection.ResultsBoth chronic HCV infection and replicon-induced Wnt/β-catenin signaling remained activated after HCV and replicon eradication by DAA. HCV infection activated PKA activity and PKA/GSK-3β-mediated Wnt/β-catenin signaling. Inhibition of PKA with H89 both repressed HCV and replicon replication and reversed PKA/GSK-3β-mediated Wnt/β-catenin signaling in both chronic HCV infection and replicon. Both chronic HCV infection and replicon induced ER stress. Inhibition of ER stress with TUDCA both repressed HCV and replicon replication and reversed ER stress/PKA/GSK-3β-dependent Wnt/β-catenin signaling. Inhibition of either PKA or ER stress both inhibited extracellular HCV infection.ConclusionTargeting ER stress/PKA/GSK-3β-dependent Wnt/β-catenin signaling with PKA inhibitor could be a novel therapeutic strategy for HCV-infected patients to overcomes the issue of remaining activated Wnt/β-catenin signaling by DAA treatment.3KsUMj7wB7yx5QgLH83TLMVideo

Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes.

BackgroundCell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive.AimTo decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection.MethodsWe used Huh7 cells–JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques.ResultsHCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes.ConclusionRTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.

Nonstructural NS5A Protein Regulates LIM and SH3 Domain Protein 1 to Promote Hepatitis C Virus Propagation.

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP-1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.

STAT3 signaling pathway plays importantly genetic and functional roles in HCV infection.

BackgroundHepatitis C virus (HCV) infection is an extensive health problem, which leads to serious liver diseases. Host genetic polymorphisms were associated with HCV infection, progression, and treatment effect of patients. Signal transducers and activators of transcription 3 (STAT3) signaling pathway was important to HCV infection, but no genetic association was studied between STAT3 signaling pathway and HCV infection.MethodsTo investigate the genetic and functional roles of the STAT3 signaling pathway, we collected 394 HCV patients and 395 normal controls to genotype 25 signal nucleotide polymorphisms (SNPs) of six genes (Interleukin 6 [IL6, OMIM 147620], Interleukin 6 receptor [IL6R, OMIM 147880], Hepatocyte nuclear factor 1 alpha [HNF1A, OMIM 142410], Hepatocyte nuclear factor 4 alpha [HNF4A, OMIM 600281], STAT3 [OMIM 102582], and ATP binding cassette subfamily C member 2 [ABCC2, OMIM 601107]). Then expression level of these genes were analyzed in HCV infected cells with or without IL6 transfection.ResultsOur results identified that the SNPs in STAT3 signaling pathway were associated with HCV infection or biochemical features of Yunnan HCV patients. Genotype AA of rs4075015 (IL6R) and GG of rs3212172 (HNF4A) increased the risk of HCV infection (p = 0.024 and 0.029), but the genotype AA of rs7553796 (IL6R) played a protective role in HCV infection (p = 0.0008). High‐density lipoprotein cholesterol (HDL‐C) and Glutamyl transpeptidase (GGT) level were associated with genotypes of rs4845617 (IL6R, p = 0.045) and rs1053023 (STAT3, p = 0.034), respectively. Cell assays suggested that IL6 transfection could suppress HCV proliferation. RNA and protein levels of the IL6R, HNF1A, STAT3, and ABCC2 genes significantly increased after HCV infection.ConclusionWe identified STAT3 signaling pathway influenced HCV infection and biochemical characteristics of HCV patients through genetic and functional aspects.

Interaction of miR-125b-5p with Human antigen R mRNA: Mechanism of controlling HCV replication

Cellular miRNAs influence Hepatitis C virus (HCV) infection in multiple ways. In this study, we demonstrate that miR-125b-5p is upregulated in HCV infected patient serum samples as well as in HCV infected liver carcinoma cells and is involved in translational regulation of one of its predicted targets, Human antigen R (HuR). We used miRNA mimics and antagomiRs to confirm that HuR is a bonafide miR-125b target. Previously, we have shown that HuR is a positive regulator of HCV replication, whereas we noticed that miR-125b is a negative regulator of HCV infection. As a connecting link between these two observations, we showed that knockdown of miR-125b-5p increased HuR protein levels and rescued HCV replication when the availability of HuR in the cytoplasm was compromised using siRNAs against HuR or an inhibitor of HuR export to the cytoplasm. Overall, the study sheds light on the ability of host cell to use a miRNA as a tool to control virus propagation.

Visualisation and analysis of hepatitis C virus non-structural proteins using super-resolution microscopy

Hepatitis C virus (HCV) RNA replication occurs in the cytosol of infected cells within a specialised membranous compartment. How the viral non-structural (NS) proteins are associated and organised within these structures remains poorly defined. We employed a super-resolution microscopy approach to visualise NS3 and NS5A in HCV infected cells. Using single molecule localisation microscopy, both NS proteins were resolved as clusters of localisations smaller than the diffraction-limited volume observed by wide-field. Analysis of the protein clusters identified a significant difference in size between the NS proteins. We also observed a reduction in NS5A cluster size following inhibition of RNA replication using daclatasvir, a phenotype which was maintained in the presence of the Y93H resistance associated substitution and not observed for NS3 clusters. These results provide insight into the NS protein organisation within hepatitis C virus RNA replication complexes and the mode of action of NS5A inhibitors.

Neuregulin 1 discovered as a cleavage target for the HCV NS3/4A protease by a microfluidic membrane protein array

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is essential for HCV maturation. The NS3/4A protease is a target for several HCV treatments and is a well-known target for HCV drug discovery. The protein is membrane associated and thus probably interacts with other membrane proteins. However, the vast majority of known NS3 host partners are soluble proteins rather than membrane proteins, most likely due to lack of appropriate platforms for their discovery. Utilization of an integrated microfluidics platform enables analysis of membrane proteins in their native form. We screened over 2800 membrane proteins for interaction with NS3 and 90 previously unknown interactions were identified. Of these, several proteins were selected for validation by co-immunoprecipitation and for NS3 proteolytic activity. Bearing in mind the considerable number of interactions formed, together with the popularity of NS3/4A protease as a drug target, it was striking to note its lack of proteolytic activity. Only a single protein, Neuregulin1, was observed to be cleaved, adding to the 3 known NS3/4A cleavage targets. Neuregulin1 participates in neural proliferation. Recent studies have shown its involvement in HCV infection and hepatocellular carcinoma. We showed that NS3/4A triggers an increase in neuregulin1 mRNA levels in HCV infected cells. Despite this increase, its protein concentration is decreased due to proteolytic cleavage. Additionally, its EGF-like domain levels were increased, possibly explaining the ErbB2 and EGFR upregulation in HCV infected cells. The newly discovered protein interactions may provide insights into HCV infection mechanisms and potentially provide new therapeutic targets against HCV.

HCV Reporter System (Viral Infection-Activated Split-Intein-Mediated Reporter System) for Testing Virus Cell-to-cell Transmission ex-vivo.

Hepatitis C virus (HCV) spread involves two distinct entry pathways: cell-free transmission and cell-to-cell transmission. Cell-to-cell transmission is not only an efficient way for viruses to spread but also an effective method for escaping neutralizing antibodies. We adapted the viral infection-activated split-intein-mediated reporter system (VISI) and developed a straightforward model for Live-cell monitoring of HCV cell-to-cell transmission ex-vivo: co-culture of HCV infected donor cells (red signal) with uninfected recipient cells (green signal) and elimination of the cell-free transmission by adding potent neutralizing antibody AR3A in the supernatant. With this model, the efficiency of cell-to-cell transmission can be evaluated by counting the number of foci designated by the green signal of recipient cells.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells.

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes.

IntroductionTelomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines.ResultsPrimary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors.ConclusionsHCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner.

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release.

Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein.

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread.

ID: 231: IFN-λ4 positive pDC stimulated with HCV infected cells mount reduced IFN-α responses

Host genetic variability, as manifested in single nucleotide (SNP) variations found within distinct alleles of genes that are relevant for the immune system, may have dramatic consequences on infection susceptibility and disease outcome. A recently identified SNP (rs12979860, minor TT allele) within the promoter region of IFN- λ 3 (IL28B) turned out to be associated with reduced spontaneous clearance of hepatitis C virus (HCV) as well as impaired sustained viral response in chronically HCV infected patients. rs12979860 shows high linkage disequilibrium with the ss469415590 ( Δ G/ Δ G, minor allele) that results in frameshift and reconstitution of the IFN- λ 4 gene. Although IFN- λ 4 positive individuals cleared HCV less efficiently than IFN- λ 4 negative ones, recombinant IFN- λ 4 exhibits anti-HCV activity in vitro. To test whether the minor allele is associated with compromised immune cell functions, we stimulated human peripheral blood mononuclear cells (PBMC) carrying either the minor or the major allele with synthetic TLR ligands. Interestingly, under such conditions all PBMC mounted similar IFN- α response. Furthermore, primary human plasmacytoid dendritic cells (pDC) stimulated with the HCV variant Jc1 responded similarly, irrespective of whether they carried the major or the minor allele. On the contrary, pDC carrying the minor allele mounted reduced IFN- α responses upon stimulation with Jc1 infected hepatoma cells. In conclusion, this observation correlated with impaired responses to IFN- α 2/ribavirin therapy and reduced spontaneous virus clearance of chronically HCV infected Δ G/ Δ G patients, highlighting the option to now in depth analyse the functional role of IFN- λ 4.

The interaction of the Hepatitis C Virus with intra-cellular signaling converts EGF into a potent inducer of the production of CXCR2-ligands by the infected host cell

Introduction: To establish persistent infection the hepatitis C virus (HCV) must have evolved strategies to escape antiviral and innate immunity and to influence the inflammatory response. Migration of the different cell types of adaptive and innate immunity is largely controlled by chemokines. Hence chemokines are decisive for the local composition of the different types of immune- or inflammatory cells and therefore play a major role for the formation of the local inflammatory- or immune-milieu. It is therefore not surprising that HCV has evolved strategies to gain influence on the expression of chemokines. The molecular mechanisms, which are employed by HCV to modify the expression of these factors, are incompletely understood. Likewise the influence of HCV on the regulation of chemokine expression in response to different stimuli is not clear. The present study investigates the impact of HCV on the production of the major chemo-attractants of neutrophils, the CXCR2 ligands CXCL1, 2, 3 and 8, in its host cell upon stimulation with the growth factor EGF. Methods: Huh-7 cells either infected with the HCV cc strain JC1 or harbouring the HCV subgenomic replicon were used and the impact of HCV on cellular signalling and gene expression was analysed employing targeted gene knockdown by siRNA, inhibitor-studies, rtPCR and Immunoblot-analysis. Results: Expression of the CXCR2 ligands in the hepatoma cell lines Huh-7 or Huh-7.5 is not or only weakly influenced upon stimulation with EGF. Therefore, the observation was surprising, that in the presence of either the sub-genomic or the full-length HCV replicon or upon infection of Huh-7.5 cells with the HCVcc strain JC1 EGF is converted in to a potent inducer of the different CXCR2 ligands. The further analysis of the molecular mechanism involved in this phenomenon revealed that with respect to the regulation of CXCL8 the NS3/4A mediated down-regulation of the endogenous feedback inhibitor of the EGF receptor (EGFR), the ubiquitously expressed T cell protein-tyrosine phosphatase (TC-PTP) and subsequent sensitization of the EGFR is important. Furthermore inhibitor studies and studies using targeted gene knockdown by specific siRNA suggest that downstream of the EGFR activation of the mitogen activated protein kinase (MAPK) activating protein kinase (MEK)1 and of its down-stream effector kinases the extracellular signal stimulated kinase (Erk)1 and 2 play a role for EGF inducible expression of CXCL8 in the presence of HCV. Interestingly this mechanism is not responsible for the EGFR-dependent up-regulation of the other CXCR2 ligands CXCL-1, 2 and 3 in HCV infected cells upon stimulation with EGF. Conclusion: The study provide evidence that HCV utilizes EGFR and EGFR-dependent signalling not only as cofactors for viral entry and replication but also interferes with EGF-dependent signalling thereby modifying the inter-cellular communication of the infected host cell.

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis.

To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus (HCV) nonstructural protein 5A (NS5A). The human hepatoma cell lines, Huh7 and Huh7.5, were infected with HCV or transiently-transfected with a vector expressing HCV NS5A. The effect of HCV NS5A on the status of the critical players involved in insulin signaling was analyzed using real-time quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0. To investigate the effect of insulin treatment on the players involved in insulin signaling pathway, we analyzed the status of insulin receptor substrate-1 (IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1 (Ser(307)) in HCV infected or NS5A transfected Huh7.5 cells compared to their respective controls. Furthermore, an increased phosphorylation of Akt (Ser(473)) was observed in HCV infected and NS5A transfected cells compared to their mock infected cells. In contrast, we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue (Ser(307)) followed by decreased phosphorylation of Akt Thr(308), Fox01 Ser(256) and GSK3β Ser(9), the downstream players of the insulin signaling pathway. Furthermore, increased expression of PECK and glucose-6-phosphatase, the molecules involved in gluconeogenesis, in HCV NS5A transfected cells was observed. Taken together, our results suggest the role of HCV NS5A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.

Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Screening the anti infectivity potentials of native N- and C-lobes derived from the camel lactoferrin against hepatitis C virus

BackgroundHepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain.MethodsLactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry.ResultsN- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively.ConclusionGenerated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.

Visualizing hepatitis C virus infection in humanized mice

Hepatitis C virus (HCV) establishes frequently persistent infections. Chronic carriers can develop severe liver disease. HCV has been intensely studied in a variety of cell culture systems. However, commonly used cell lines and primary hepatocyte cultures do not or only in part recapitulate the intricate host environment HCV faces in the liver. HCV infects readily only humans and chimpanzees, which poses challenges in studying HCV infection in vivo. Consequently, tractable small animal models are needed that are not only suitable for analyzing HCV infection but also for testing novel therapeutics. Here, we will focus our discussion on humanized mice, i.e. mice engrafted with human tissues or expressing human genes, which support HCV infection. We will further highlight novel methods that can be used to unambiguously detect HCV infected cells in situ, thereby facilitating a spatio-temporal dissection of HCV infection in the three dimensional context of the liver.

GP73 Is Upregulated by Hepatitis C Virus (HCV) Infection and Enhances HCV Secretion

Hepatitis C virus (HCV) is a major cause of chronic liver disease. However, little is known about the details of its assembly and secretion. Golgi-related proteins have been recently proven to have a key function in HCV secretion. Golgi protein 73 (GP73), a resident Golgi membrane protein, is a potential serum biomarker for the diagnosis of liver diseases and hepatocellular carcinoma. Previous studies have demonstrated the upregulation of GP73 in the liver samples and sera of HCV-infected patients. However, the function and regulatory mechanism of GP73 in HCV infection at the cellular level remain unknown. In this study, we examined the expression level of GP73 in HCV infected cells and its effect on HCV life cycle in cell culture systems. Both the protein expression and mRNA levels of GP73 significantly increased in HCV subgenomic replicon-harboring cells and HCV-infected cells, which imply that GP73 was upregulated by HCV infection. HCV production was significantly enhanced when GP73 was overexpressed, but dramatically inhibited when GP73 was silenced. However, the overexpression and knockdown of GP73 showed no evident effect on the entry, protein translation, RNA replication, and assembly of HCV, which indicates that GP73 enhanced the secretion process. Moreover, the coiled-coil domain of GP73 was required to increase HCV secretion. GP73 increased and interacted with apolipoprotein E, an identified host factor that assists in HCV secretion. These results demonstrate the critical function of GP73 in HCV secretion and provide new insights into the therapeutic design of antiviral strategies.