Cleavage of intracellular hepatitis C RNA in the virus core protein coding region by deoxyribozymes

Hepatitis C virus (HCV) infection represents an important global health problem. Current antiviral therapeutics for HCV have proven inadequate in stemming the disease process. A novel therapeutic strategy involves the use of deoxyribozymes, also known as DNA enzymes or DNAzymes. These catalytic DNA molecules, designed to target and cleave specific RNA sequences, have shown promise in in vitro experimental models for various diseases and may serve as an alternative or adjunct to current HCV drug therapy. We designed and tested several deoxyribozymes that can bind and cleave highly conserved RNA sequences encoding the HCV core protein in in vitro systems. One of these deoxyribozymes reduced the level of our HCV RNA target by 32% and 48% after 24 h of cell exposure when tested in human hepatoma and epithelial cell lines, respectively. As this deoxyribozyme showed significant cleavage activity against HCV core protein target RNA in human cells, it may have potential as a therapeutic candidate for clinical trial in HCV infected patients.     

Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production

Hepatitis C virus (HCV) is a major human pathogen that causes serious illness, including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Using a mass spectrometry-based proteomics approach, we have identified 175 proteins from a cell culture supernatant fraction containing the HCV genotype 2a (JFH1) virus, among which fatty acid synthase (FASN), the multifunctional enzyme catalyzing the de novo synthesis of fatty acids, was confirmed to be highly enriched. Subsequent studies showed that FASN expression increased in the human hepatoma cell line, Huh7, or its derivative, upon HCV infection. Blocking FASN activity by C75, a pharmacological FASN inhibitor, led to decreased HCV production. Reduction of FASN by RNA interference suppressed viral replication in both replicon and infection systems. Remarkably, FASN appeared to be selectively required for the expression of claudin-1, a tight junction protein that was recently identified as an entry coreceptor for HCV, but not for the expression of another HCV coreceptor, CD81. The decrease in Claudin-1 expression resulting from FASN inhibition was accompanied by a decrease in transepithelial electric resistance of Huh7 cells, implying a reduction in the relative tightness of the cell monolayer. Consequently, the entry of human immunodeficiency virus-HCV pseudotypes was significantly inhibited in C75-treated Huh7 cells. CONCLUSION: As far as we know, this is the first line of evidence that demonstrates that HCV infection directly induces FASN expression, and thus suggests a possible mechanism by which HCV infection alters the cellular lipid profile and causes diseases such as steatosis.     

Impact of human immune deficiency virus infection on hepatitis C virus infection and replication

Human immune deficiency virus (HIV) and human hepatitis C virus (HCV) infection are frequent in patients who have been exposed to blood or blood-derived products. It has been suggested that HIV infection increases HCV replication altering the course of HCV-related disease. However, it is not known if HIV directly enhances HCV replication or if its effect is the consequence of HIV infection of other cell types that control HCV replication (lymphocytes, macrophages). While the main cell targets for HIV infection are mononuclear leukocytes bearing CD4 and the chemokine receptors CCR5 and CXCR4, HCV was originally thought to be strictly hepatotropic, but it is now known that HCV can also replicate in peripheral blood mononuclear cells (PBMC). Therefore, in co-infected individuals, these two different viruses could share cell targets and interact either directly or indirectly. Some membrane receptors can be used by both HCV and HIV for entry into target cells, but the intracellular mechanisms shared by these viruses are not known. Lack of experimental systems providing suitable methods for the study of HCV replication in the presence or absence of HIV co-infection has hampered advances in this research area, but recent investigations are currently going on in order to answer these questions. This is an important issue, as knowledge of HIV/HCV interactions is required for the design of effective antiviral therapies.     

Hepatitis C virus enters human peripheral neuroblastoma cells – evidence for extra‐hepatic cells sustaining hepatitis C virus penetration

Summary. Patients with chronic hepatitis C virus (HCV) infection show an increased incidence of nervous system disorders such as chronic fatigue syndrome, depression and cognitive dysfunction. It is unclear whether this is because of HCV replication in the brain and in peripheral neuronal cells or to more indirect effects of HCV infection on the central or peripheral nervous system. The aim of this study was to investigate whether cells originating from these tissues are permissive for HCV cell entry, RNA replication and virus assembly. Among eight cell lines analysed, the human peripheral neuroblastoma cell line SKNMC expressed all HCV entry factors and was efficiently infected with HCV pseudoparticles (HCVpp) independent of the HCV genotype. All remaining cell types including human neuroblastoma and glioblastoma cell lines and microglial cells lacked expression of at least one host factor essential for HCV entry. When transfected with HCV luciferase reporter virus RNA, inoculated with HCV reporter viruses or challenged with high‐titre cell culture–derived HCV, none of these cells supported detectable HCV RNA replication. Thus, in conclusion, this comprehensive screening did not reveal evidence directly strengthening the notion that HCV enters and replicates in the central nervous system. However, productive viral entry into the peripheral neuroblastoma cell line SKNMC indicates that HCV may penetrate into certain nonhepatic cell types which may serve as viral reservoirs and could modulate viral pathogenesis.     

Impact of interleukin-28B genotype on in vitro and in vivo systems of hepatitis C virus replication

Identification of the relationship between the interleukin (IL)-28B genotype and the effect of peginterferon plus ribavirin treatment has had a great impact on the study of antiviral therapy for patients with chronic hepatitis C virus (HCV) infection. Differential expression levels of interferon-stimulated genes (ISG) in the liver and white blood cells based on the IL-28B genotype, which may in turn lead to differences in outcome of therapy, indicate that previous studies should be re-evaluated taking the effect of the IL-28B single nucleotide polymorphism (SNP) into consideration, although the exact mechanism of how variation in IL-28B SNPs affect HCV eradication remains unknown. These results suggest that the genotypes of multiple cell types, including liver and immune cells, contribute to the efficacy of therapy. Studies using human hepatocyte chimeric mice, in which effector cells of the human adaptive immune response are absent, showed that viral load, ISG expression levels and reduction of HCV RNA by interferon are affected by the IL-28B genotype. Genetic differences among hepatocytes may, therefore, contribute to differences in baseline viral loads and response to interferon therapy. Further studies should be done to clarify the mechanism of action of IL-28B SNP on viral load and effect of interferon treatment. Advances in cell culture systems and human hepatocyte chimeric mice, as well as upcoming in vitro and in vivo experimental systems, provide an effective platform to examine the effects of host and viral genetic variation on infection and response to interferon.     

Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.     

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.     

Unexpected host range of hepatitis C virus replicons

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known if extrahepatic tissues are also infected by the virus nor if nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and other species. We have tested this hypothesis and found that subgenomic HCV RNA can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.     

Molecular biology of hepatitis C virus

Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes persistent, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. For many years, the characterization of the HCV genome and its products has been done by heterologous expression systems because of the lack of a productive cell culture system. The development of the HCV replicon system is a highlight of HCV research and has allowed examination of the viral RNA replication in cell culture. Recently, a robust system for production of recombinant infectious HCV has been established, and classical virological techniques are now able to be applied to HCV. This development of reverse genetics-based experimental tools in HCV research can bring a greater understanding of the viral life cycle and pathogenesis of HCV-induced diseases. This review summarizes the current knowledge of cell culture systems for HCV research and recent advances in the investigation of the molecular virology of HCV.     

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury

Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.     

Hepatitis C virus entry: molecular biology and clinical implications

With an estimated 170 million infected individuals, hepatitis C virus (HCV) has a major impact on public health. A vaccine protecting against HCV infection is not available, and current antiviral therapies are characterized by limited efficacy, high costs, and substantial side effects. Binding of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between virus and the target cell that is required for the initiation of infection. Because this step represents a critical determinant of tissue tropism and pathogenesis, it is a major target for host cell responses such as antibody-mediated virus-neutralization-and a promising target for new antiviral therapy. The recent development of novel tissue culture model systems for the study of the first steps of HCV infection has allowed rapid progress in the understanding of the molecular mechanisms of HCV binding and entry. This review summarizes the impact of recently identified viral and host cell factors for HCV attachment and entry. Clinical implications of this important process for the pathogenesis of HCV infection and novel therapeutic interventions are discussed.     

丙型肝炎病毒感染人端粒酶逆转录酶基因转导后细胞的研究

用丙型肝炎病毒阳性血清感染人端粒酶逆转录酶(hTERT)基因转导后人外周血单个核细胞(PBMC),以 期建立长期存活的HCV体外复制细胞模型。将hTERT基因导入正常人PBMC,丙型肝炎阳性血清直接感染转导后 的细胞,RT-PCR检测细胞内HCV RNA正、负链的表达。hTERT基因转入PBMC内,RT-PCR测定其在mRNA水平 有表达,丙肝阳性血清感染后,检测到细胞内HCV RNA正链持续出现,负链问断出现。hTERT基因转导后的。PBMC, 用丙型肝炎阳性血清感染后,病毒在细胞内持续存在并复制,初步建立了长期存活的HCV体外复制细胞模型。     

HCV受体及入胞相关分子的研究进展

HCV是丙型肝炎的致病体，在被感染组织和血清中浓度较低，病毒高度变异及缺乏稳定的体外细胞培养系统及理想的实验动物模型等问题，阻碍了对HCV的进一步研究。近年来各种HCV体外培养系统的建立，为HCV的研究提供了强有力的工具，使人们对HCV的认识提升到新的层次。本文就CD81、低密度脂蛋白受体、清道夫受体B1及claudin—1等受体及入胞相关分子研究进展作一简要综述。     

Tissue culture and animal models for hepatitis C virus

In recent years, significant advances have been achieved both in the development of animal- and tissue-culture models for HCV. Among all the new systems, the small animal model based on transgenic mice with chimeric mouse-human livers and the replicon system will presumably have the most profound impact on future HCV research. Yet, in spite of this progress, much more work will be required to optimizse both systems. In case of the mouse model, breeding homozygous Alb-uPa animals is difficult because of the toxicity of the transgene, and the transplantation of primary human hepatocytes into mice a few days after birth is technically challenging. These are immunodeficient, and, therefore, it will be desirable to furnish them with components of the human immune system in order to expand the applicability of this in vivo model to questions related to pathogenesis. Advances in cryopreservation techniques are urgently needed, moreover, as this would improve the availability of primary hepatocytes and in turn also the accessibility of this small animal model. As regards the replicon system, a number of open questions remain that will hopefully be answered by future research. Why, for instance, has replication in cell culture so far been achieved only with genotype 1b isolates, whereas an isolate with proven infectivity derived from genotype 1a failed to replicate in Huh-7 cells? And why can replicons so far be propagated only in this particular cell line? Is this attributable to the lack of certain inhibitory factors, or the presence of specific activators? What are the mechanisms underlying cell-culture adaptation. and what determines whether a certain Huh-7 cell replicates HCV RNA more efficiently? Finally, the replicon system may also lead the way to the development of systems for efficient virus production in cell culture, and ultimately also a permissive cell line. These developments would at last allow us to model the complete viral life cycle, something researchers have been struggling with ever since the first identification of HCV.     

Claudin‐1, miR‐122 and apolipoprotein E transductions improve the permissivity of SNU‐182, SNU‐398 and SNU‐449 hepatoma cells to hepatitis C virus

Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU‐182, SNU‐398 and SNU‐449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin‐1 (CLDN1) expression limited the viral entry process in SNU‐182 and SNU‐398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR‐122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU‐182 and SNU‐398 cells but not in SNU‐449 cells. Nevertheless, the supplementation of SNU‐182, SNU‐398 and SNU‐449 cells with CLDN1, miR‐122 and ApoE was not sufficient to render these cells as permissive as HuH‐7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR‐122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection.     

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture-derived HCV (HCVcc). Anti-SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI-specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81-HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV-CD81 interaction.     

The influence of T cell cross-reactivity on HCV-peptide specific human T cell response

Detection of hepatitis C virus (HCV)-specific T cell response after exposure to hepatitis C in anti-HCV-positive or anti-HCV-negative patients has been associated with an ability to successfully control the infection. However, cross-reactivity between common human pathogens and HCV sequences has been demonstrated. The aim of this study was to investigate the impact of T cell cross-reactivity on HCV-specific T cell responses and their detection in HCV infected and non-infected subjects. The magnitude, function, and cross-reactivity of HCV peptide reactive T cells were studied in non-HCV-infected newborns and adults using a broad array of HCV peptides (601 peptides) spanning the entire HCV sequence. Comparisons were made with responses present in recovered and in chronically HCV-infected patients. HCV peptide reactive T cells are detectable in adults irrespective of previous HCV exposure and cross-reactivity between HCV peptides, and sequences of common pathogens, such as human herpes virus 1, can be demonstrated. Furthermore, the comprehensive magnitude of HCV-peptide reactive T cells present in chronically HCV-infected patients is similar and in some cases even lower than that of HCV-peptide reactive T cell response found in HCV-negative adults. In conclusion, the presence of oligo-specific HCV-peptide reactive T cells in humans does not always reflect a demonstration of previous HCV contact, whereas cross-reactivity with other common pathogens can potentially influence the HCV-specific T cell profile. The conspicuous deficit of HCV-peptide-specific T cells found in chronically HCV-infected patients confirms the profound collapse of virus-specific T cell response caused by HCV persistence.     

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.