Hepatitis C Virus Core Protein Expression Research Articles

HCV core protein promotes heparin binding EGF‐like growth factor expression and activates Akt

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction and is closely associated with the development of human hepatocellular carcinoma (HCC). Among HCV components, core protein is implicated in cell growth regulation, and we previously demonstrated that HCV core protein interacted with 14-3-3 protein and activated the kinase Raf-1 and mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. In the present study, we investigated the expression levels and function of downstream molecules in the MAPK/ERK signaling pathway in cells expressing HCV core protein. Heparin-binding EGF-like growth factor (HB-EGF) mRNA, in HepG2 cells stably expressing HCV core protein, was detected by RT-PCR. The soluble HB-EGF in culture media was measured by heparin agarose chromatography/Western blot analysis. Immunodetection of Akt and IKK and IB, in HeLa cells and HepG2 cells expressing HCV core protein, were performed with neutralizing antibody for HB-EGF, phospatidylinositol-3-kinase [PI(3)K] inhibitor and dominant-negative mutant of Ras (DN-Ras). HB-EGF expression was significantly elevated in cells expressing HCV core protein. HCV core protein activated Akt through the Ras/PI(3)K pathway by autocrine secretion of HB-EGF. Also, HCV core protein activated IKK through Ras/PI(3)K/Akt pathway by autocrine secretion of HB-EGF. As the Ras/PI(3)K/Akt pathway is critical in anti-apoptotic HB-EGF signaling, we examined the possible role of this pathway in cells expressing HCV core protein. In addition, we investigated the relationship between IB kinases (IKK) and Akt in cells expressing HCV core protein, since IKKs are known to be activated by HCV core protein and by Akt in the presence of potent mitogen. We showed that HCV core protein promoted autocrine secretion of HB-EGF and activated Akt through the Ras/PI(3)K pathway. This model indicates a new approach to mechanism of proliferation and anti-apoptosis in HCC. HCV core protein is a potent activator of mitogenic and anti-apoptotic signaling involved in hepatocarcinogenesis.

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.

HCV synergizes with body weight in the promotion of insulin resistance

Hepatitis C virus (HCV) infection appears to contribute to the development of insulin resistance (IR). Among the multiple determinants of IR, body mass index (BMI) is the most important. We investigated the contribution of HCV to BMI-associated IR using a transgenic mouse model expressing HCV core protein. Eight transgenic and five nontransgenic littermate controls were evaluated. Glucose and insulin tolerance tests (ITT) were performed on two separate occasions. Multivariate linear mixed modelling was used to evaluate and compare the effect of weight on IR between HCV core transgenic and nontransgenic controls. There were no statistically significant differences in glucose or ITT (P = 0.58 and P = 0.59, respectively) between the two groups, and no difference in median weights between transgenic and control mice (P = 0.11). However, there was greater variance in the distributions of Tg when compared to nontransgenic mice for both glucose and insulin tolerance. When evaluating this closely, a differential contribution of weight to IR curves between these groups was noted (P = 0.05). Among nontransgenic mice, IR curves for mice of different weights were comparable, however, for transgenic mice, higher weights resulted in larger levels of IR curves with slower decay. In all animals, steatosis was absent or minimal. We conclude that weight has a greater effect on IR in HCV core expressing transgenic mice than littermate controls. HCV therefore synergizes with weight in the promotion of IR. Steatosis was not a prerequisite for the development of IR, implying that HCV's effects on IR may be independent of steatosis.

Synonymous Mutations in the Core Gene Are Linked to Unusual Serological Profile in Hepatitis C Virus Infection

The biological role of the protein encoded by the alternative open reading frame (core+1/ARF) of the Hepatitis C virus (HCV) genome remains elusive, as does the significance of the production of corresponding antibodies in HCV infection. We investigated the prevalence of anti-core and anti-core+1/ARFP antibodies in HCV-positive blood donors from Cambodia, using peptide and recombinant protein-based ELISAs. We detected unusual serological profiles in 3 out of 58 HCV positive plasma of genotype 1a. These patients were negative for anti-core antibodies by commercial and peptide-based assays using C-terminal fragments of core but reacted by Western Blot with full-length core protein. All three patients had high levels of anti-core+1/ARFP antibodies. Cloning of the cDNA that corresponds to the core-coding region from these sera resulted in the expression of both core and core+1/ARFP in mammalian cells. The core protein exhibited high amino-acid homology with a consensus HCV1a sequence. However, 10 identical synonymous mutations were found, and 7 were located in the aa(99–124) region of core. All mutations concerned the third base of a codon, and 5/10 represented a T>C mutation. Prediction analyses of the RNA secondary structure revealed conformational changes within the stem-loop region that contains the core+1/ARFP internal AUG initiator at position 85/87. Using the luciferase tagging approach, we showed that core+1/ARFP expression is more efficient from such a sequence than from the prototype HCV1a RNA. We provide additional evidence of the existence of core+1/ARFP in vivo and new data concerning expression of HCV core protein. We show that HCV patients who do not produce normal anti-core antibodies have unusually high levels of antit-core+1/ARFP and harbour several identical synonymous mutations in the core and core+1/ARFP coding region that result in major changes in predicted RNA structure. Such HCV variants may favour core+1/ARFP production during HCV infection.

617 Translational opportunities from genomic and chemical biology of cancer

Hepatitis C virus (HCV) Core protein is implicated in the development of hepatocellular carcinoma (HCC). We utilized a HepG2 human hepatocyte cell line with inducible expression of HCV Core protein (HCV-1b) to investigate the early effects of Core protein on hepatocyte gene expression and to identify molecular processes modulated by the Core protein. A significant change was observed in the expression of 407 genes, which included genes regulating apoptosis, immune response, and cell cycle. Some of these genes were previously known to be tumor markers. The decreased expression of chemo-attractants such as TNFSF10, CCL20, and osteopontin was observed, which suggested that HCV Core expression could lead to suppression of inflammatory response as well as trafficking of macrophages and neutrophils to the site of HCV infection. An increased expression of anti-apoptosis factors including PAK2, API5, BH1, Tax1BP1, DAXX, and TNFAIP3/A20 was observed. Some of these genes were also linked to the regulation of NFKB activation and that the alteration of their expression levels, by HCV Core, might lead to the suppression NFKB activation of inflammatory responses. Our data suggested that Core expression may contribute to the viral persistence by protecting infected hepatocytes from cell death by the suppressing apoptosis and inflammatory reaction to HCV viral infection.

Abstract 5723: A role of retinoic acid in preventive effect of hepatitis C virus- and oxidative stress-related hepatocarcinogenesis

Abstract Aims; It has been reported that hepatitis C virus (HCV) core expression causes hepatocellular carcinoma (HCC) in transgenic mice by reactive oxygen species (ROS). On the other hand, we have reported that loss of signals of retinoic acid (RA), a metabolite of vitamin A, leads to steatohepatitis, eventually to HCC in the receptor for RA (RARα)-knocked-down mice. We have demonstrated that the mechanism by which loss of RA signal causes HCC is mediated by increased oxidative stress via impaired metabolisms of fatty acids and iron. In turn, the effect of oxidative stress on RA signals remains to be clarified. The present study aims to explore the effect of oxidative stress and HCV core expression on RA signals in the context of hepatocarcinogenesis. Methods; The first parts of experiments were done using hydrogen peroxide as the source of ROS Human HCC cell lines HuH7, HLF, HepG2, Hep3B, Li7, Alexander and HuH6 were used. The expression level of RARα and activation of MAPKs such as ERK, JNK and p38, and RA signal by DR5-reporter assay were examined with 0.1∼1μM hydrogen peroxide. Anisomycin, an activator of JNK signal, SP600125, an inhibitor of JNK, and constitutive active MKK7-JNK1 was explored on level of RARα and activation of MAPKs. Phosphorylation and ubiquitination of mutant RARα, of which three phosphorylation sites by JNK are changed from serines to alanines, were examined. The second parts of experiments were performed using tetracycline-inducible HCV core system (Tet-On system), which is constructed in HuH7 cells. The intensity of RA signal after induction of HCV core protein was measured by DR5-reporter assay. The expression Sp101b, which is induced by RA and an adaptive protein of HCV core, was examined on expression and cellular translocation. The level of SRC-3, a coactivator of RAR/RXR, and p38 MAPK, an upstream signal of SRC-3 and a downstream signal of oxidative stress, were examined. The cellular antioxidative ability and hydroxyl radical formation were measured during this process. Results; In the first parts experiment using hydrogen peroxide, oxidative stress suppressed RA signal through degradation of RARα. The degradation of RARα was mediated by its phosphorylation by JNK and followed by ubiquitination. In the second parts of experiments, expression of HCV core protein reached its maximum at 72 h at 5 μg/ml of Tet. RA signal increased at 36 h, however then decreased at 48 h and 72 h. The decreased RA signal was reverted by N-acetyl cystein (NAC), an antioxidative protein. The cellular antioxidative ability was decreased at 24h, and hydroxyl radical formation was increased at 48 h. Activation of p38 and SRC-3 was observed after induction of HCV core expression, however it was inhibited by NAC. Conclusions; The data of the present study suggest that RA plays an important role in preventive effect of hepatitis C virus- and oxidative stress-related hepatocarcinogenesis Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5723.

Involvement of PA28γ in the propagation of hepatitis C virus

We have reported previously that the proteasome activator PA28gamma participates not only in degradation of hepatitis C virus (HCV) core protein in the nucleus but also in the pathogenesis in transgenic mice expressing HCV core protein. However, the biological significance of PA28gamma in the propagation of HCV has not been clarified. PA28gamma is an activator of proteasome responsible for ubiquitin-independent degradation of substrates in the nucleus. In the present study, knockdown of PA28gamma in cells preinfection or postinfection with the JFH-1 strain of HCV impaired viral particle production but exhibited no effect on viral RNA replication. The particle production of HCV in PA28gamma knockdown cells was restored by the expression of an small interfering RNA (siRNA)-resistant PA28gamma. Although viral proteins were detected in the cytoplasm of cells infected with HCV, suppression of PA28gamma expression induced accumulation of HCV core protein in the nucleus. HCV core protein was also degraded in the cytoplasm after ubiquitination by an E3 ubiquitin ligase, E6AP. Knockdown of PA28gamma enhanced ubiquitination of core protein and impaired virus production, whereas that of E6AP reduced ubiquitination of core protein and enhanced virus production. Furthermore, virus production in the PA28gamma knockdown cells was restored through knockdown of E6AP or expression of the siRNA-resistant wild-type but not mutant PA28gamma incapable of activating proteasome activity. Our results suggest that PA28gamma participates not only in the pathogenesis but also in the propagation of HCV by regulating the degradation of the core protein in both a ubiquitin-dependent and ubiquitin-independent manner.

The hepatitis C virus core protein indirectly induces alpha-smooth muscle actin expression in hepatic stellate cells via interleukin-8

Progressive deposition of liver fibrosis is a common feature of chronic hepatitis associated with hepatitis C virus (HCV) infection, and it may eventually lead to cirrhosis and liver failure. Although this fibrogenic process appears to be linked to HCV protein expression and replication via indirect mechanisms, i.e., to be mediated by virally-driven inflammation, a direct role of HCV in inducing fibrosis deposition has never been entirely excluded. We established an in vitro system in which the human hepatic stellate cell line LX-2 was cultured in the presence of conditioned medium from human hepatoma Huh-7 cells transduced with a lentiviral vector expressing HCV core proteins of different genotypes. Treatment of LX-2 cells, with conditioned medium from Huh-7 cells expressing HCV core protein, led to the activation of alpha-smooth muscle actin expression. Among the chemokines secreted by cells transduced with HCV core, interleukin-8 was identified as the strongest inducer of alpha-smooth muscle actin expression in LX-2 and primary hepatic stellate cells. This effect was accompanied by a decrease in cell migration and increased focal contact organisation. The expression of the HCV core in hepatocytes may contribute to the establishment of a profibrogenic microenvironment.

Hepatitis C virus core protein enhances Telomerase activity in Huh7 cells

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.

Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

BackgroundHepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells.ResultsWe constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector.ConclusionThese findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing highly effective therapeutic agents that can be further developed for HCV gene therapy applications.

Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

BackgroundThe pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV) infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver.MethodsLiver biopsy samples from transgenic mice with tetracycline(tet)-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement), which inhibits activation of C3.ResultsTransgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation) and coagulation pathways (fibrinogen B up-regulation). Administration of CD55 reduced hepatic inflammation.ConclusionTransgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

Differential Expression of HCV Core Protein from Two Different Quasispecies

Abstract － Hepatitis C virus (HCV) has genetic diversity like most of RNA viruses. HCV major genotypes are classified into several subtypes which are further divided into quasispecies having, genetically different but closely related variants. The HCV core that is a nucleocapsid protein located at the amino terminus of the viral polyprotein is relatively a conserved protein among the HCV isolates and thus it has been one of plausible targets for anti-HCV drug development. However, different quasispecies of HCV core gene have also been found. In this study, we compared the expression level of core protein between two different quasispecies of HCV genotype 1b. Our data demonstrate that a little differences of amino acid sequence lead to substantial difference of expression level. It might be another important reason of different pathogenesis among HCV infected patients.Keywords: Hepatitis C virus, Core protein, Quasispecies INTRODUCTION Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver transplants and over the 170 million per-sons chronically are infected with it world widely. It is a en-veloped virus with a 9.6 kb positive-sense single-stranded RNA genome encoding a single open reading frame (ORF), which is processed by cellular and viral proteases to produce at least 10 structural and nonstructural proteins. It has been known now that there are at least six major genotypes and a number of subtypes of HCV which are generated due to high error rates in the viral RNA repli-cation, (Choo

Gene expression profile of Huh‐7 cells expressing hepatitis C virus genotype 1b or 3a core proteins

The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype. To identify some hepatitis C virus (HCV) genotype-specific virus-host interactions potentially leading to clinically significant consequences. We compared the gene expression profile of Huh-7 cells transiently expressing the core protein of HCV genotype 1b and 3a using microarray technology. Thirty-two genes were overexpressed in Huh-7 transfected with the HCV genotype 1b core protein and 57 genes in cells transfected with the genotype 3a core protein. On the other hand, we found 20 genes downregulated by core 1b and 31 genes by core 3a. These included genes involved in lipid transport and metabolism, cell cycle, immune response and insulin signalling. The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.

612 WEEK 8 HCV-RNA IS THE OPTIMAL PREDICTOR OF RELAPSE IN NON-RVR PATIENTS WITH GENOTYPE 1/4 RANDOMISED TO 48 OR 72 WEEKS PEG-IFN ALFA-2A PLUS RBV

The mechanism of innate immune response to the hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We examined the innate immune response to the HCV structural proteins using HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching expression system. The level of HCV core protein was 15-47 pg/mg of total protein in the HCV Tg mice livers at 12 h after the HCV transgene expression. In contrast, we found a high level of expression of HCV core protein (1597 pg/mL) in transgenic mice with a large depletion of natural killer (NK) cells. Cre-mediated genomic DNA recombination efficiency in the HCV Tg mice was was strongly observed in NK cell depleted mice, compared to non-treated mice between 0.5 and 1 day.These data indicated that in the early naive immune responses the NK cells have important roles in initial recognition of HCV protein and the later elimination of core expressed hepatocytes.

609 BASELINE CHARACTERISTICS AND WEEK-4 RESPONSE AMONG 1546 CHC PATIENTS INFECTED WITH HCV GENOTYPE 1, 2, 3 AND 4: INTERIM RESULTS OF THE PROPHESYS COHORT

The mechanism of innate immune response to the hepatitis C virus (HCV) has not been fully elucidated, largely due to the lack of an appropriate model. We examined the innate immune response to the HCV structural proteins using HCV transgenic (Tg) mice, which express core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching expression system. The level of HCV core protein was 15-47 pg/mg of total protein in the HCV Tg mice livers at 12 h after the HCV transgene expression. In contrast, we found a high level of expression of HCV core protein (1597 pg/mL) in transgenic mice with a large depletion of natural killer (NK) cells. Cre-mediated genomic DNA recombination efficiency in the HCV Tg mice was was strongly observed in NK cell depleted mice, compared to non-treated mice between 0.5 and 1 day.These data indicated that in the early naive immune responses the NK cells have important roles in initial recognition of HCV protein and the later elimination of core expressed hepatocytes.

Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression

Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. Here, we report on the inhibition, by baculovirus-based vector-derived shRNAs, of core-protein expression in full-length hepatitis C virus (HCV) replicon cells. shRNAs were designed to target the highly conserved core region of the HCV genome. In particular, the core-shRNA452 containing nucleotides 452–472, as the target in the HCV core gene, dramatically inhibited the expression of the HCV core protein in replicon cells. Furthermore, HCV core-protein expression was inhibited more strongly by the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped baculovirus vector than by the wild-type baculovirus vector.

The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression

Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an ‘alternate reading frame’ (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named 86/88ARFP. Interestingly, 86/88ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle.

Molecular basis for the synergy between alcohol and hepatitis C virus in hepatocarcinogenesis

Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). In addition, heavy alcohol use has been linked with earlier progression to HCC in chronic hepatitis C patients. However, in the pathogenesis of HCV-associated HCC, it still remains controversial as to whether the virus plays a direct or an indirect role, and as to how alcohol operates in the acceleration of HCC development. Several studies using transgenic mouse models, in which the core protein of HCV has an oncogenic potential, indicate that HCV is directly involved in hepatocarcinogenesis, although other factors such as continuous inflammation or environmental factors seem also to play a role. The downstream events of the HCV core protein expression in the transgenic mouse HCC model are segregated into two pathways. One is the augmented production of oxidative stress in the absence of inflammation along with the attenuation of some scavenging systems in the putative preneoplastic stage with steatosis in the liver. The other pathway is the alteration in cellular gene expression and intracellular signaling, including the mitogen-activated protein kinase cascade. The combination of these pathways would explain the unusually high incidence and multicentric nature of HCC development in HCV infection. In addition, alcohol feeding in this animal model further activated the two pathways synergistically with HCV, leading to an earlier development of HCC. Such a synergy would reveal the molecular basis for the acceleration of HCC development by alcohol in HCV infection.

Hepatitis C virus replication is inhibited by 22β-methoxyolean-12-ene-3β, 24(4β)-diol (ME3738) through enhancing interferon-β

A derivative of soyasapogenol, 22beta-methoxyolean-12-ene-3beta, 24(4beta)-diol (ME3738), ameliorates liver injury induced by Concanavalin A in mice. We examined whether ME3738 has independent antiviral effects against hepatitis C virus (HCV) using an established HCV replication model that expresses the full-length genotype 1a HCV complementary DNA plasmid (pT7-flHCV-Rz) under the control of a replication-defective adenoviral vector expressing T7 polymerase. Hepatocellular carcinoma (HepG2) cells, human hepatoma (Huh7) cells, or monkey kidney (CV-1) cells were transfected with pT7-flHCV-Rz, and infected with adenoviral vector expressing T7 polymerase. ME3738 or interferon-alpha (IFN-alpha) was added thereafter and then protein and RNA were harvested from the cells at 9 days after infection. HCV-positive and HCV-negative strands were measured by real-time reverse-transcription polymerase chain reaction and HCV core protein expression was measured using an enzyme-linked immunosorbent assay. The messenger RNA levels of innate antiviral response-related genes were assessed using real-time reverse-transcription polymerase chain reaction. ME3738 dose-dependently reduced HCV-RNA and core protein in hepatocyte-derived cell lines. The antiviral effect was more pronounced in HepG2 than in Huh7 cells. ME3738 increased messenger RNA levels of interferon-beta (IFN-beta) and of IFN-stimulated genes (2'-5' oligoadenylate synthetase, myxovirus resistance protein A [MxA]). Interferon-beta knockdown by small interfering RNA abrogated the anti-HCV effect of ME3738. Moreover, the anti-HCV effects were synergistic when ME3738 was combined with IFN-alpha. ME3738 has antiviral effects against HCV. The enhancement of autocrine IFN-beta suggests that ME3738 exerts antiviral action along the type I IFN pathway. This anti-HCV action by ME3738 was synergistically enhanced when combined with IFN-alpha. ME3738 might be a useful anti-HCV drug either with or without IFN-alpha.

Acute expression of hepatitis C core protein in adult mouse liver: Mitochondrial stress and apoptosis

Objective. In infection with hepatitis C virus (HCV), spontaneous clearance of the virus occurs in 30–40% of cases. By contrast, in chronic infection, this is rare. The basis for viral clearance in acute disease is unknown. Whereas cellular immune responses have been studied in detail, few data exist on the role of viral structural proteins, such as the core protein. The purpose of this study was to investigate the effects of core produced de novo within adult mouse hepatocytes by using a new transgenic mouse line in which expression of HCV core is regulated by tetracycline (tet-off). Material and methods. In this work, transgenic mice with conditional HCV core were created, to study the acute expression of HCV core protein in the context of the mature liver. The subcellular distribution of the core, hepatocellular oxidative stress and apoptosis were monitored. Results. Core protein is readily detectable and strongly associated with cytoplasmic lipid vesicles, endoplasmic reticulum and mitochondria. Mitochondrial oxidative stress was evidenced by a reduction in thioredoxin-2 (trx2). Concurrently, caspase-3 activity and TUNEL increased and, over time, the level of core protein in the liver declined. Conclusions. Mice that are conditionally transgenic for HCV core protein, which is readily detected and morphologically associated with steatosis in individual hepatocytes, were developed. Acute expression of core protein causes mitochondrial stress, as demonstrated by a reduction in trx2 and in the apoptosis of core-positive hepatocytes. We speculate that these events could be involved in the clearance of virus during acute hepatitis C, by both reducing the burden of virus in the liver and effectively priming the immune response.