Expressing Hepatitis C Virus Core Research Articles

Association between breast cancer and hepatitis C: A joint study of hospitalized patients and nationwide cohorts

Whether hepatitis C virus (HCV) infection is associated with breast cancer risk remains elusive, and we aimed to elucidate it. A nationwide population-based cohort study of the Taiwan National Health Insurance Research Database (TNHIRD) was conducted. Additionally, breast cancer risk factors, and HCV core expression were surveyed in breast cancer patients of a tertiary care center. Three TNHIRD cohorts (1:4:4, propensity score-matched, 2003-2012), including HCV-treated (3646 HCV-infected females with interferon-based therapy ≥6 months), HCV-untreated (n=14,584) and HCV-uninfected (n=14,584) cohorts, were enrolled. The HCV-untreated cohort had the highest 9-year breast cancer cumulative incidence (2.017%; 95% confidence interval [CI]: 1.382%-2.846%), while the HCV-treated (1.073%; 0.414%-2.356%), and HCV-uninfected (1.453%; 0.785%-2.486%) cohorts showed no difference. Untreated HCV infection (hazard ratio [HR]: 1.701; 95% CI: 1.205%-2.400), urban residency (1.658, 1.183-2.323), and baseline cardiovascular events (1.920; 1.005-3.668) were associated with incident breast cancers. The interaction analysis showed that particularly among patients <49 years, HCV infection was associated with breast cancer development (2.193; 1.097-4.384). Of 12,170 hospitalized breast cancer patients, 4.90% were HCV Ab-positive. HCV Ab-positive patients were older (60.92+/-10.82 vs 53.91+/-11.38 years, P < 0.0001) and had a higher body mass index (25.39+/-5.1 vs 24.5+/-4.3 kg/m2, P = 0.007), rates of diabetes (30.60 vs 19.98%, P < 0.0001), hypertension (46.9 vs 30.39%, P < 0.0001), dyslipidemia (25.52 vs 20.28%, P = 0.031), and hyperuricemia (11.38 vs 5.52%, P < 0.0001) than their counterparts. No HCV core-positive cells were demonstrated in breast cancer tissues. Conclusions: Untreated HCV infection, urbanization, and cardiovascular events were potential risk factors for breast cancer. The HCV-associated risk was most prominent among patients <49 years, might not be associated with in situ HCV core-related oncogenesis but with metabolic alterations, and was reversed by anti-HCV therapy.

Marmoset Viral Hepatic Inflammation Induced by Hepatitis C Virus Core Protein via IL-32

Common marmosets infected with GB virus-B (GBV-B) chimeras containing hepatitis C virus (HCV) core and envelope proteins (CE1E2p7) developed more severe hepatitis than those infected with HCV envelope proteins (E1E2p7), suggesting that HCV core protein might be involved in the pathogenesis of viral hepatitis. The potential role of HCV core in hepatic inflammation was investigated. Six individual cDNA libraries of liver tissues from HCV CE1E2p7 or E1E2p7 chimera-infected marmosets (three animals per group) were constructed and sequenced. By differential expression gene analysis, 30 of 632 mRNA transcripts were correlated with the immune system process, which might be associated with hepatitis. A protein–protein interaction network was constituted by STRING database based on these 30 differentially expressed genes (DEGs), showing that IL-32 might play a central regulatory role in HCV core-related hepatitis. To investigate the effect of HCV core protein on IL-32 production, HCV core expressing and mock constructs were transfected into Huh7 cells. IL-32 mRNA and secretion protein were detected at significantly higher levels in cells expressing HCV core protein than in those without HCV core expression (P < 0.01 and P < 0.001, respectively). By KEGG enrichment analysis and using the specific signaling pathway inhibitor LY294002 for inhibition of PI3K, IL-32 expression was significantly reduced (P < 0.001). In conclusion, HCV core protein induces an increase of IL-32 expression via the PI3K pathway in hepatic cells, which played a major role in development of HCV-related severe hepatitis.

The Synergistic Effect of Fluvastatin and IFN-λ on Peripheral Blood Mononuclear Cells of Chronic Hepatitis C Virus (HCV) Patients with IL-28B rs12979860 CC Genotype.

There is a relationship between the life cycle of the hepatitis C virus (HCV) and the synthesis and hemostasis of lipids as well as lipid metabolism and interferon (IFN) regulatory system. This study was aimed to examine the effect of fluvastatin and IFN-ƛ in the expression of mediators involved in lipid metabolism and HCV proliferation in patients with rs12979860 CC polymorphism. Thirteen patients with HCV and five controls with rs1297986CC polymorphism were included in this study. Peripheral blood mononuclear cells (PBMCs) of patients and controls were treated by fluvastatin, IFN-λ or fluvastatin+IFN-λ. Assessment of IL-28B polymorphism, RNA extraction, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed. The mRNA expression of sterol regulatory element-binding protein 1 c (SREBP1c), ATP-binding cassette transporter A1 (ABCA1), diacylglycerol acyltransferase 1 (DGAT1), and HCV core as well as measurement of ABCA1 protein level were evaluated before and after treatment. The results indicated that IFN-λ +fluvastatin acted as an inhibitor in mRNA expression of SREBP1c; while acting as an inducer in the expression of ABCA-1. The results of ABCA1 assay showed a significant increase of this protein after treatment with fluvastatin and IFN-λ compared with untreated cells (p=0.02). Moreover, the mRNA expression of HCV core was suppressed in all experimental groups treated with fluvastatin, IFN-λ or their combination which was more significant after treatment with fluvastatin+IFN-λ (p<0.001). The results of this study demonstrated the significant effect of treatment with fluvastatin+IFN-λ in PBMCs of HCV patients with rs12979860 CC polymorphism. According to the drug resistance of viruses and prevention of virus-induced steatosis in patients with HCV, using regulatory agents of lipid mediators in parallel with current medications could be considered as an effective therapeutic strategy.

Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress

ABSTRACTObjectives: The occurrence of oxidative stress and endoplasmic reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated and play an important role in liver injury. During viral infection, hepatocytes must handle not only the replication of the virus, but also inflammatory signals generating oxidative stress and damage. Although several mechanisms exist to overcome cellular stress, little attention has been given to the adaptive response of hepatocytes during exposure to multiple noxious triggers.Methods: In the present study, Huh-7 cells and hepatocytes expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER stress, were additionally challenged with the superoxide anion generator menadione to mimic external oxidative stress. The production of reactive oxygen species (ROS) as well as the response to oxidative stress and ER stress were investigated.Results: We demonstrate that hepatocytes diminish oxidative stress through a reduction in ROS production, ER-stress markers (HSPA5 [GRP78], sXBP1) and apoptosis (caspase-3 activity) despite external oxidative stress. Interestingly, the level of the autophagy substrate protein p62 was downregulated together with HCV Core degradation, suggesting that hepatocytes can overcome excess oxidative stress through autophagic degradation of one of the stressors, thereby increasing cell survival.Duscussion: In conclusion, hepatocytes exposed to direct and indirect oxidative stress inducers are able to cope with cellular stress associated with viral hepatitis and thus promote cell survival.

Hepatitis C virus core protein triggers abnormal porphyrin metabolism in human hepatocellular carcinoma cells.

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.

Target delivery of small interfering RNAs with vitamin E- loaded lipid nanoparticles in inhibition of hepatitis C virus core protein expression in mouse model

Objective: To study the efficacy of vitamin E-loaded lipid nanoparticles (VE-DC) in the mouse model to target small interfering RNA (siRNA) for inhibition of hepatitis C virus(HCV) core protein expression. Methods: A high-pressure hydrodynamic method was adopted to construct an animal model of liver-specific expression to inject the plasmid containing HCV core protein into mice tail vein. Western blotting and immunofluorescence techniques were used to evaluote the liver targeting property of VE - DC/siRNA nanoparticles and the effectiveness to repress HCV Core expression. Dual luciferase reporter gene assays and in vivo imaging in mice further confirmed the inhibiting effect of VE-DC/siRNA on gene expression mediated by HCV 5' untranslated region. The adverse reactions of VE-DC/siRNA were reported by detecting serum creatinine, white blood cells and interferon. Student's t - test and one -way analysis of variance were used to compare the difference between the two groups, and P < 0.05 was considered statically significant. Results: The dual luciferase reporter gene analysis showed that the luciferase activity of the VE-DC/siRNA treated group was 39.67 ± 15.53, which was significantly lower than 77.33±11.06 of the DC/siRNA group and 91.67 ± 13.65 of the siRNA treated group, P < 0.05. The difference was statistically significant, and there was no obvious organ toxicity and obvious immune response to VE-DC/siRNA. Nanoparticle VE-DC has a good liver targeting ability, which can transport siRNA to the liver and effectively inhibit the expression of HCV Core, with an average inhibition rate of 83.01%. Conclusion: VE-DC could target the delivery of siRNA to the liver and inhibit the expression of HCV- related genes in a mouse model, showing high effectiveness and low toxicity.

Hepatitis C virus core protein induces dysfunction of liver sinusoidal endothelial cell by down-regulation of silent information regulator 1.

Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-β1 (TGF-β1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-β1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes. HCV core protein may down-regulate the activity and the expression of SIRT1 of LSEC, then decreasing synthesis of adiponectin and the expression of AdipoR2, thus inducing contraction of LSEC and hepatic sinusoidal capillarization and increasing oxidative stress, ultimately cause hepatic stellate cell (HSC) activation. Treatment with SIRT1 activator restored the function of LSEC and inhibited the activation of HSC.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

HepatitisC virus core protein increases Snail expression and induces epithelial-mesenchymal transition through the signal transducer and activator of transcription3 pathway in hepatoma cells.

Aberrant expression of Snail, a mediator of epithelial-mesenchymal transition (EMT), is crucial for cancer invasiveness and metastasis. Although hepatitisC virus (HCV) core protein has been implicated in hepatocarcinogenesis, the relationship between HCV core and Snail expression has not been clarified. HepG2 and Huh7 stable cell lines were established by transfection with pcDNA-HCVc. HepG2-HCVc and Huh7-HCVc cells were co-administered with AG490. Cell migration and invasiveness were tested. STAT3 and Snail expression was analyzed by Real-time PCR and Western blot. We found that HCV core is capable of increasing Snail expression and inducing EMT in hepatoma cells. HCV core-induced Snail expression was accompanied by activation of signal transducer and activator of transcription3 (STAT3), inhibition of STAT3 abrogated HCV core-induced Snail expression and EMT. Furthermore, chromatin immunoprecipitation showed that phosphorylated STAT3 directly binds to the Snail promoter. Collectively, these results suggest that HCV core would play a role in hepatocellular carcinoma invasiveness and metastasis by activating the STAT3 pathway, increasing Snail expression and subsequently triggering EMT. These findings would advance the understanding of HCV-mediated invasiveness and metastasis, and might provide a new potential therapeutic target for HCV-related hepatocellular carcinoma.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis.

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin–proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells.

Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4

Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.

The structural HCV genes delivered by MPG cell penetrating peptide are directed to enhance immune responses in mice model

One of the significant problems in vaccination projects is the lack of an effective vaccine against hepatitis C virus (HCV). The goal of the current study is to evaluate and compare two DNA constructs encoding HCV core and coreE1E2 genes alone or complexed with MPG peptide as a delivery system for stimulation of antibody responses and IFN-γ secretion in Balb/c mice model. Indeed, MPG cell penetrating peptide was used to improve DNA immunization in mice. Our results demonstrated that MPG forms stable non-covalent nanoparticles with pcDNA-core and pcDNA-coreE1E2 at an N/P ratio of 10:1. The in vitro transfection efficiency of core or coreE1E2 DNA using MPG and TurboFect delivery systems was confirmed by western blot analysis. The results indicated the expression of the full-length core (∼21 kDa), and coreE1E2 (∼83 kDa) proteins using an anti-His monoclonal antibody. In addition, the expression of HCV core and coreE1E2 proteins was performed in bacteria and the purified recombinant proteins were injected to mice with Montanide 720 adjuvant. Our data showed that the immunized mice with HCV core and coreE1E2 proteins generated the mixture of sera IgG1 and IgG2a isotypes considerably higher than other groups. Furthermore, DNA constructs encoding core and coreE1E2 complexed with MPG could significantly induce IFN-γ secretion in lower concentrations than the naked core and coreE1E2 DNAs. Taken together, the DNA formulations as well as protein regimens used in this study triggered high-level IFN-γ production in mice, an important feature for the development of Th1 immune responses.

Hepatitis C virus core protein interacts with Snail and histone deacetylases to promote the metastasis of hepatocellular carcinoma.

Downregulation of E-cadherin by the transcriptional repressor Snail is associated with acquisition of metastatic potential. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, it is unclear whether Snail is involved in HCV core-induced dysregulation of E-cadherin. Herein, we investigated the mechanism by which HCV core induces E-cadherin repression and the role of Snail in HCV core-mediated invasiveness and metastasis. We found that HCV infection, especially HCV core expression, effectively induced the epithelial-mesenchymal transition (EMT) in hepatoma cells by repressing E-cadherin. HCV core interacted with Snail and enhanced its binding to the E-box in the promoter region of E-cadherin, leading to decreased E-cadherin promoter activity. We found that HCV core, Snail, and the histone deacetylases HDAC1/HDAC2 formed a co-repressor complex at the E-cadherin promoter. Moreover, HCV core was shown to stabilize Snail through activation of the PI3K/Akt/GSK3β pathway. Silencing Snail expression restored E-cadherin expression and inhibited HCV core-promoted tumor growth and distant lung metastasis in vivo. Collectively, these results demonstrated that HCV core induced EMT by interacting with the transcriptional repressor complex Snail/HDACs at the E-cadherin promoter, which led to E-cadherin repression and increased invasiveness of hepatoma cells. These findings increase understanding of factors regulating metastasis in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma.

Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains.

Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.

Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection.

Multispecific, broad, and potent T cell responses have been correlated with viral clearance in hepatitis C virus (HCV) infection. However, the majority of infected patients develop chronic infection, suggesting that natural infection mostly leads to development of inefficient T cell immunity. Multiple mechanisms of immune modulation and evasion have been shown in HCV infection through various investigations. This study examined the generation and modulation of T cell responses against core and frameshift (F) proteins of HCV. A single immunization of mice with replication incompetent recombinant adenovirus vectors encoding for F or core antigens induces poor T cell responses and leads to generation of CD4+ and CD8+ T cells with low granzyme B (GrB) expression. These T cells have impaired GrB enzyme activity and are unable to kill peptide loaded target cells. The low intracellular expression of GrB is not due to degranulation of cytotoxic granules containing cytotoxic T cells. Addition of exogenous IL-2 in in vitro cultures leads to partial recovery of GrB production, whereas immunization with the Toll-like receptor (TLR) agonist poly I:C leads to complete restoration of GrB expression in both CD4+ and CD8+ T cells. Thus, a possible new strategy of T cell modulation is recognized wherein effector T cells are caused to be dysfunctional by HCV-derived antigens F or core, and strategies are also delineated to overcome this dysfunction. These studies are important in the investigation of prophylactic vaccine and immunotherapy strategies for HCV infection.

Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture.

BackgroundHepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.AimTo investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.MethodHCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.ResultsFFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).ConclusionOur study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.

Inhibition of expression of hepatitis C virus 1b genotype core and NS4B genes in HepG2 cells using artificial microRNAs.

The present study aimed to evaluate the silencing effect of artificial microRNAs (amiRNAs) against the hepatitis C virus (HCV) 1b (HCV1b) genotype core (C) and non-structural protein 4B (NS4B) genes. pDsRed-monomer-Core and pDsRed-monomer-NS4B plasmids, containing the target genes were constructed. A total of eight artificial micro RNA (amiRNA)-expressing plasmids, namely, pmiRE-C-mi1 to -mi4 and pmiRE-NS4B-mi1 to -mi4, were designed and constructed to interfere with various sites of the core and NS4B genes, and the amiRNA interfering plasmid and the corresponding target gene-expressing plasmid were co-transfected into HepG2 cells. At 48 h after transfection, HCV core and NS4B gene expression levels were detected using fluorescence microscopy, flow cytometry, reverse transcription quantitative polymerase chain reaction and western blot analysis. Fluorescence microscopy revealed that the target gene-transfected cells expressed red fluorescent protein, whereas the interfering plasmid-transfected cells exhibited expression of green fluorescent protein. The percentage of red fluorescent proteins and mean fluorescence intensity, as well as protein expression levels of the core and NS4B genes within the cells, which were co-transfected by the amiRNA interfering plasmid and the target gene, were significantly decreased. The results of the present study confirmed that amiRNAs may effectively and specifically inhibit the expression of HCV1b core and NS4B genes in HepG2 cells, potentially providing a novel therapeutic strategy for the treatment of HCV.

Enhanced Immune Responses of a Hepatitis C Virus core DNA Vaccine by co-Inoculating Interleukin-12 Expressing Vector in Mice

Hepatitis C virus (HCV) infection is a worldwide problem without an effective vaccine with more than 170 million chronically infected people worldwide. DNA vaccines expressing antigenic portions of the virus with adjuvants have recently been developed as a novel vaccination technology. In the present study, a DNA vaccine expressing HCV core protein was developed with IL12 as a genetic adjuvant and different aspects of cell immune responses due to this vaccine were evaluated in an animal model of the infection. Material and Methods: HCV core gene was inserted into pcDNA3.1(-) eukaryotic expression vector and the recombinant plasmid was transformed into DH5α competent cells and a large scale DNA vaccine was prepared. The expression of the vector was verified in CHO cell line. Female C57BL/6 mice were immunized 3 times with 90 ng doses of the DNA vaccine on days 0, 14 and 28. Two weeks after the last immunization, the immune responses against HCV core antigen were assessed by lymphocyte proliferation, CTL cytotoxicity and cytokines secretion assays. Results: The results showed that the co-administration of IL12, as a genetic adjuvant, increased the ability of HCV core DNA vaccine to enhance cytolytic T lymphocyte activity, lymphocyte proliferation and shifting of the immune response toward a T helper type 1 pattern and altogether improved the protective immunity. Conclusion: This study demonstrated that intramuscular injection of HCV core DNA vaccine with a genetic adjuvant induced significant cellular immune responses in C57BL/6 mice.

Abstract 17: Hepatitis C virus (HCV) core protein potentiates proangiogenic activity of hepatocellular carcinoma (HCC) cells

Abstract Background: Recent clinical trials found that sorafenib may have better efficacy for HCV-related HCC than HCC with other etiologies. HCV core protein has been found to influence the mitogen-activated protein kinase pathway, which is one of the targets of sorafenib. We thus planned this study to examine the effect of HCV core protein overexpression on HCC cells. Methods: We stably expressed HCV core protein in Huh7, a HCC cell line, using a lentivirus based vector S2. Two stable clones were selected for experiments. One clone (Huh7-core-high) expressed a higher amount of HCV core protein than the other (Huh7-core-low). Huh7 expressing the empty vector (Huh7-S2) served as a control. We compared the cytotoxicity of sorafenib in these cells using the MTT assay. Tube formation analysis and Matrigel plug assay were performed to evaluate the angiogenic activity. Results: Huh7-core-high and Huh7-core-low cells did not show increased cell proliferation compared to Huh7-S2 cells. Sorafenib showed similar in vitro cytotoxicity for Huh7-core-high, Huh7-core-low, and Huh7-S2 cells. Overexpression of HCV core protein potentiates proangiogenic activity of Huh7 cells dose-dependently. In tube formation analysis, Huh7-core-high induced more potent angiogenesis than Huh7-S2 cells with more total tube lengths (p = 0.02), mean tube lengths (p = 0.04), mean tube areas (p = 0.04), branch points (p = 0.03), and nodes (p &amp;lt; 0.01). Huh7-core-low cells induced stronger angiogenesis than Huh7-S2 cells with more total tube lengths (p = 0.05) and branch points (p = 0.01). By Matrigel plug assay, Huh7-core-high and Huh7-core-low cells dose-dependently induced stronger angiogenesis in mice compared to Huh7-S2 cells with significantly higher hemoglobin levels in the Matrigel plugs. Conclusions: HCV core protein expression potentiates the proangiogenic activity of HCC, both in vitro and in vivo. (This work was supported by grants NSC98-3112-B-002-038, NSC101-2314-B-002-141, 100CAP1020-2 &amp; 102-2314-B-002-120.) Citation Format: Yu-Yun Shao, Chung-Yi Huang, Jun-Wei Chen, Chih-Hung Hsu, Ann-Lii Cheng. Hepatitis C virus (HCV) core protein potentiates proangiogenic activity of hepatocellular carcinoma (HCC) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2014-17

Effects of alcohol on HCV replication and its mechanism

Objective To investigate the effects of alcohol on hepatitis C virus（ HCV） replication and type I interferon signaling pathway in human hepatocytes .Methods Primary hepatocytes were treated with different concentrations of alcohol , and then infected with HCV .The infected cells were collected to measure the level of HCV RNA .The alcohol-treated hepatocytes were also collected to detect the expression of HCV Core, IFN-α, IFN-β, IRF-7, suppressor of cytokine signaling SOCS-2 and SOCS-3 at mRNA and protein levels by real-time quantitative PCR and ELISA or Western blot , respectively .Results Alcohol treatment enhanced HCV infection and replication in primary hepatocytes at concentrations higher than 10 mmol/L in a dose-dependent manner （P<0.05）.Treatment with 40 mmol/L of alcohol significantly reduced the expression of IFN-α, IFN-βand IRF-7 at mRNA and protein levels , and increased the expression of SOCS-2 and SOCS-3 at mRNA and protein levels .Conclusion Alcohol treatment could damage the host in-nate immunity in human hepatocytes and promote HCV replication by reducing the expression of type Ⅰinter-feron （ IFN-αand IFN-β） and IRF-7 and increasing the expression of negative regulators including SOCS-2 and SOCS-3.These results demonstrated that the impairment of innate immunity in liver of alcohol abusers might contribute to the enhancement of HCV infection and result in poor therapeutic effect of IFN -α. Key words: Hepatitis C virus; Alcohol; Type I interferon; Innate immunity