Anti-Hepatitis C Virus Antikörper [B2] (A32738)

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.

Both viral and host factors are thought to influence the pathogenesis of hepatitis C virus (HCV) infection. We studied strain HC-TN (genotype 1a), which caused fulminant hepatic failure in a patient and, subsequently, severe hepatitis in a chimpanzee (CH1422), to analyze the relationship between disease severity, host immune response, viral evolution, and outcome. A second chimpanzee (CH1581) was infected from CH1422 plasma, and a third chimpanzee (CH1579) was infected from RNA transcripts of a consensus cDNA of HC-TN (pHC-TN). RNA transcripts of pHC-TN did not replicate in Huh7.5 cells, which were recently found to be susceptible to infection with another fulminant HCV strain (JFH1). The courses of viremia were similar in the three animals. However, CH1581 and CH1579 developed a less severe acute hepatitis than CH1422. CH1579 and CH1422 resolved the infection, whereas CH1581 became persistently infected. CH1579 and CH1581, despite their differing outcomes, both developed significant intrahepatic cellular immune responses, but not antibodies to the envelope glycoproteins or neutralizing antibodies, during the acute infection. We analyzed the polyprotein sequences of virus recovered at five and nine time points from CH1579 and CH1581, respectively, during the first year of follow-up. High mutation rates and high proportions of nonsynonymous mutations suggested immune pressure and positive selection in both animals. Changes were not selected until after the initial decrease in virus titers and after the development of immune responses and hepatitis. Subsequently, however, mutations emerged repeatedly in both animals. Overall, our results indicate that disease severity and outcome of acute HCV infection depend primarily on the host response.

Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response.

Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single- and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4+ T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4+ T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection.

Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic alpha-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-alpha. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.

The hepatitis C virus (HCV) core protein is among the most conserved proteins in HCV and is known to induce sensitization of cytotoxic T lymphocytes (CTL). Therefore, it is a prime candidate for a component of a potential HCV vaccine. The HCV core protein has, however, been reported to exert multiple effects on cell functions, raising questions as to its suitability for this purpose. This question was investigated here with mice into which replication-deficient adenoviruses expressing core protein of an HCV genotype 1b isolate were injected. We show that induction of cytokines in response to the infection, infiltration of lymphocytes into the infected liver, priming of virus-specific CTL, and liver injury are not modulated by expression of the core protein in the liver. Moreover, no changes in the sensitivity to tumor necrosis factor alpha- or Fas-mediated liver injury are demonstrable. A similar lack of demonstrable effects of the core protein on immune functions has also been obtained using transgenic mice expressing another HCV genotype 1b core protein. It is concluded that the HCV core protein of genotype 1b has no modulatory effects on induction of virus-specific immune responses and may therefore be a suitable component of an HCV vaccine.

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.

Hepatitis C virus genotype-3a (HCV-3a) is directly linked to the development of steatosis. We previously showed that, through sterol regulatory element binding protein-1 (SREBP-1), HCV-3a core protein upregulates the promoter activity of fatty acid synthase, a major enzyme involved in de novo lipid synthesis. In this study, we investigated whether HCV-3a core can activate SREBP-1 and studied the role of phosphoinositide 3-kinase (PI3K)-Akt-2 pathway in modulating SREBP-1 activity by HCV-3a core. To determine whether HCV-3a core could activate SREBP-1, the level of mature SREBP-1 was analysed by Western blotting. Our results showed that the level of mature SREBP-1 was enhanced by HCV-3a core protein after transient expression and in the chimeric HCV-3a core/1b replicon cells in comparison to controls. To investigate the role of the PI3K-Akt-2 pathway in SREBP-1 activation by HCV-3a core, PI3K and Akt-2 activity was inhibited by using the chemical inhibitor LY294002, a dominant-negative Akt-2 plasmid, or knockdown of Akt-2 by small hairpin RNA. Our results showed that inhibition of PI3K and Akt-2 was associated with reduced SREBP-1 activation by HCV-3a core. These results indicate a role for PI3K and Akt-2 in increasing SREBP-1 activity by HCV-3a core protein and provide a mechanism of steatosis caused by HCV.

Hepatitis C virus (HCV) infection causes acute and chronic liver disease often leading to liver cirrhosis and hepatocellular carcinoma. Numerous studies have shown that despite induction of virus specific immunity, a curative response is often not attained; this has led to the hypothesis that HCV genes modulate immunity, thereby enabling chronic infections. This study examined the effects on immune-mediated liver injury in transgenic mice expressing core protein throughout the body and bone marrow chimeras expressing core protein in either the lymphoid compartment or liver parenchyma. Presence of core protein in the liver parenchyma but not in lymphoid cells protects from autoimmune hepatitis induced by mitogen concanavalin A (ConA). Consistent with this observation, core transgenic hepatocytes are relatively resistant to death induced by anti-Fas antibody and tumor necrosis factor alpha (TNFalpha). This protective effect is associated with preferential activation of signal transducer and activation of transcription factor 3 (STAT3) versus STAT1 in livers of ConA-injected animals. In agreement with this effect of core protein on the Janus kinase (JAK)-STAT signaling pathway, transgenic mice accelerate liver regeneration after partial hepatectomy but are not protected from hepatocyte death. In conclusion, HCV core inhibits STAT1 and stimulates STAT3 activation, which protects infected hepatocytes from attack by the cell-mediated immune system and promotes their proliferation.

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca(2+) uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C.

Hepatic steatosis and hepatocellular carcinoma (HCC) are common and serious features of hepatitis C virus (HCV) infection, and the core protein has been shown to play distinct roles in the pathogenesis. Here we report the direct interaction of HCV core protein with retinoid X receptor alpha (RXRalpha), a transcriptional regulator that controls many aspects of cell proliferation, differentiation, and lipid metabolism. The core protein binds to the DNA-binding domain of RXRalpha, leading to increase the DNA binding of RXRalpha to its responsive element. In addition, RXRalpha is activated in cells expressing the core protein as well as in the livers of the core-transgenic mice that would develop hepatic steatosis and HCC later in their lives. Using promoter genes of cellular retinol binding protein II (CRBPII) and acyl-CoA oxidase as reporters, we also show that the expression of the core protein enhances the transcriptional activity regulated by the RXRalpha homodimer as well as by the heterodimer with peroxisome proliferator activated receptor alpha. Furthermore, expression of the CRBPII gene is also up-regulated in the livers of HCV core-transgenic mice. In conclusion, these results suggest that modulation of RXRalpha-controlled gene expression via interaction with the core protein contributes to the pathogenesis of HCV infection.

BACKGROUND & AIMS:

Autophagy is a lysosome-mediated catabolic process that mediates degradation and recycling of all major components of eukaryotic cells. Different stresses, including viral and bacterial infection, induce autophagy, which can promote cell survival by removing the stress inducer or by attenuating its dangerous effects. High levels of autophagy occur during infection of cells with hepatitis C virus (HCV), but the clinical relevance of this process is not clear.

METHODS:

Levels of autophagy were analyzed in liver biopsy samples from 22 patients with HCV infection using microtubule-associated protein-1 light chain 3 immunoblotting; associations with histological and metabolic parameters were evaluated by Pearson correlation analysis. We investigated the role of HCV-induced autophagy in lipid degradation in cells infected with the virus or replicons, and analyzed autophagosome contents by confocal microscopy and by measuring lipid levels after inhibition of autophagy by Beclin 1 knockdown or lysosome inhibitors.

RESULTS:

In liver biopsy samples from patients with HCV, there was an inverse correlation between microvesicular steatosis and level of autophagy (r = -0.617; P = .002). HCV selectively induced autophagy of lipids in virus-infected and replicon cells. In each system, autophagosomes frequently colocalized with lipid deposits, mainly formed by unesterified cholesterol. Inhibition of the autophagic process in these cells significantly increased the induction of cholesterol accumulation by HCV.

CONCLUSIONS:

Autophagy counteracts the alterations in lipid metabolism induced by HCV. Disruption of the autophagic process might contribute to development of steatosis in patients with HCV.

Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

BACKGROUND & AIMS:

Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates of the 7 major HCV genotypes, and determining their sensitivity to a specific NS5A inhibitor and to interferon-α.

METHODS:

Huh7.5 hepatoma cells were transfected with RNA of genotype 1-7 NS5A recombinants. Viability was determined by measuring HCV replication and infectivity titers. Putative adaptive mutations were analyzed by reverse genetics. The activity of antiviral agents was determined in high-throughput infection assays.

RESULTS:

Cells infected with viable HCV that expressed NS5A of genotypes 1-7 produced relatively high viral titers; most NS5A recombinants required introduction of specific adaptive mutations. The efficacy of the NS5A inhibitor BMS-790052 varied greatly, based on NS5A isolate, with median effective concentration (EC(50)) values ranging from 0.009 nmol/L to 14 nmol/L; the high sensitivity of genotype 1b NS5A to BMS-790052 reflected observations from clinical studies. Specific residues in NS5A domain I were associated with >100-fold variations in sensitivity between isolates of the same HCV subtype. The Y/T2065H mutation conferred resistance to BMS-790052 that varied among NS5A isolates. When infected cultures were incubated with interferon-α, all NS5A recombinants had EC(50) values of ∼0.2 IU/mL, including an NS5A genotype 1b mutant with a putative sensitive-type, interferon sensitivity determining region.

CONCLUSIONS:

We developed efficient in vitro systems in which recombinant viruses express HCV genotypes 1-7 NS5A; these permit genotype- and isolate-specific analyses of NS5A and the effects of antiviral compounds and resistance mutations. These culture systems will facilitate development of specific inhibitors against NS5A of different HCV variants.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

The hepatitis C virus (HCV) core protein is involved in viral pathogenesis such as oxidative stress induction and lipid metabolism disturbance, and is primarily located in the cytoplasm and endoplasmic reticulum in association with lipid droplets as well as in the mitochondria. To clarify the impact of the core protein on mitochondria, we analyzed the expression pattern of mitochondrial proteins in core protein-expressing cells by two-dimensional polyacrylamide gel electrophoresis. Several proteins related to the mitochondrial respiratory chain or protein chaperons were identified by mass spectrometry. Among the identified proteins with consistently different expressions, prohibitin, a mitochondrial protein chaperon, was up-regulated not only in core-expressing cells but also in full-genomic replicon cells and livers of core-gene transgenic mice. The stability of prohibitin was increased through interaction with the core protein. Further analysis demonstrated that interaction of prohibitin with mitochondrial DNA-encoded subunits of cytochrome c oxidase (COX) was disturbed by the core protein, resulting in a significant decrease in COX activity.

CONCLUSION:

The HCV core protein affects the steady-state levels of a subset of mitochondrial proteins including prohibitin, which may lead to an impaired function of the mitochondrial respiratory chain with the overproduction of oxidative stress.

BACKGROUND:

Recently, a hepatitis C virus (HCV) cell-culture system was developed that employed strain JFH1 (genotype 2a), and JFH1-based intra- and intergenotypic recombinants now permit functional studies of the structural genes (Core, E1, and E2), p7, and NS2 of genotypes 1-4. The goal was to adapt the system to employ genotype 5.

METHODS:

Huh7.5 cells infected with SA13/JFH1, containing Core-NS2 of strain SA13 (genotype 5a), were monitored for Core expression and for supernatant infectivity and HCV-RNA titers. Adaptive mutations of SA13/JFH1 were identified by sequence analysis of recovered genomes and reverse-genetic studies. Receptor blockage was performed with anti-CD81 and anti-SR-BI. For neutralization experiments, SA13/JFH1 or JFH1-based viruses of other genotypes were incubated with patient sera.

RESULTS:

SA13/JFH1 with NS2 and NS3 mutations yielded infectivity titers >10(5) TCID50/mL. Infection with SA13/JFH1 was inhibited by CD81 blocking and SR-BI blocking, respectively, and by preincubation with genotype 5a chronic-phase patient sera. Such sera had varying cross-genotype neutralization potential. However, preincubation and treatment with homologous neutralizing antibodies could not control SA13/JFH1 infection in culture.

CONCLUSION:

The SA13/JFH1 culture permits genotype 5a-specific studies of Core-NS2 function and interfering agents. The ability of HCV to spread in vivo during treatment with neutralizing antibodies was confirmed in vitro.

BACKGROUND & AIMS:

Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide.

METHODS:

Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies.

RESULTS:

S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids.

CONCLUSIONS:

We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.