Reconstitution of the Entire Hepatitis C Virus Life Cycle in Nonhepatic Cells

Costa, Daniel Da; Turek, Marine; Felmlee, Daniel J.; Girardi, Erika; Pfeffer, Sébastien; Long, Gang; Bartenschlager, Ralf; Zeisel, Mirjam B.; Baumert, Thomas F.

doi:10.1128/jvi.01066-12

Cited by 84 publications

(95 citation statements)

References 41 publications

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“…We thus conducted experiments in 293T and Huh7.5 cells and revealed in general a significant correlation between the results for the two cell types. In line with this, it was recently demonstrated that the exogenous expression of HCV entry receptors, the microRNA miR122, and apolipoprotein E is sufficient to achieve completion of the whole HCV life cycle in 293T cells (30). We therefore postulate that major differences in FRET, which we observed occasionally between 293T and Huh7.5 cells (compare Fig.…”

Section: Hcv Protein Interactions Determined Via Facs-fret-tosupporting

confidence: 63%

The Intraviral Protein Interaction Network of Hepatitis C Virus

Hagen

Bayer

Rösch

et al. 2014

Molecular & Cellular Proteomics

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Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometrybased FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Hepatitis C virus (HCV)1 belongs to the family of Flaviviridae and is the only member of the genus Hepacivirus. The ϳ9.5-kB positive-strand RNA genome is directly translated via an internal ribosomal entry site into a polyprotein. This is proteolytically processed by cellular and viral proteases into structural (Core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A/B, and NS5A/B) proteins (1). In recent decades, light was shed on the importance and biological relevance of most HCV proteins, which ultimately led to the development of the first specific antiviral therapy involving inhibition of the NS3 serine protease (2). However, because HCV is highly variable and because of the rapid emergence of drug resistance, additional therapeutic approaches are urgently needed (2). An impressive body of data was derived from protein interaction or siRNA screens investigating the interplay of HCV proteins with cellular factors (3-5). Although these screens are essential in order for researchers to understand how HCV manipulates the host cell, their potential benefit for novel therapeutic approaches could be limited. HCV is a chronic viral infection, and targeting host factors might result in drugs with severe adverse effects. Thus, a promising strategy would be to specifically inhibit interactions among viral proteins. Surprisingly, until now, a comprehensive analysis of the putative interactions and the interplay of HCV proteins with each other in living human cells has been lacking.In the present work, we did an extensive and thorough analysis of intra-HCV protein interactions. We used our novel flow-cytometry-based FRET assay that allows rapid assessment of the interplay between proteins in thousands of living cells (6). Therefore, this experimental approach enables quantification and statistical evaluation of all results. From the total of 20 interactions established by FACS-FRET, we chose to further investigate three that were not yet described in the literature. The putative HCV viroporin p7 binds to the structural proteins, and this was verified via biochemical methods in cells expressing fully infectious HCV.The established network of intra-HCV protein interactions in living mammalian cells provides new insights into the biology of this important human pathogen. Furthermore, we identified several HCV protein interactions that could be targeted for an...

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Section: Hcv Protein Interactions Determined Via Facs-fret-tosupporting

confidence: 63%

The Intraviral Protein Interaction Network of Hepatitis C Virus

Hagen

Bayer

Rösch

et al. 2014

Molecular & Cellular Proteomics

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“…This new role of apoE is consistent with our previous observations showing that nonhepatic cell lines lacking very-low-density lipoproteinÀproducing components and engineered to express apoE can sustain the entire HCV life cycle and produce viruses with buoyant density profiles similar to virus produced in hepatoma cells. 37 Collectively, we demonstrate that apart from lipoprotein association, apoE mediates escape from nAbs. This finding reveals a novel strategy contributing to HCV's remarkable capacity to establish chronic infection.…”

Section: Discussionmentioning

confidence: 99%

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

et al. 2016

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BACKGROUND & AIMS:Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. METHODS: We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell cultureÀderived HCVÀproducing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell cultureÀderived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein 2 and HCV virions by immunoprecipitation. RESULTS: Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein 2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. CONCLUSIONS: In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.

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“…The FLAG peptide (100 g/ml) or cell culture supernatants from mock-electroporated cells were used for control experiments. HCV infection was assessed by reverse transcription-quantitative PCR (RT-qPCR) analysis of intracellular HCV RNA, immunostaining using the HCV E2-specific AP33 antibody, or determination of luciferase activity as described previously (25,27,28).…”

Section: Methodsmentioning

confidence: 99%

“…To study the effect of miR-146a-5p on HCV translation, cells were transfected with the indicated siRNAs, miRNAs, and anti-miRNAs and then, 48 h later, with HCV RNA (JcR2a) bearing a Renilla luciferase reporter gene sequence. HCV RNA translation was assessed by measuring intracellular luciferase activity after 4 h (25,28,36). To assess the relevance of miR-146a-5p for viral replication, cells were electroporated with JFH1/⌬E1E2 RNA carrying a firefly luciferase reporter gene (23).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C Virus-Induced Upregulation of MicroRNA miR-146a-5p in Hepatocytes Promotes Viral Infection and Deregulates Metabolic Pathways Associated with Liver Disease Pathogenesis

Bandiera

Pernot

Saghire

et al. 2016

J Virol

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Hepatitis C virus (HCV)-induced chronic liver disease is a leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in homeostasis within the liver, and deregulation of miRNAs has been associated with liver disease, including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here we combined a hepatocyte-like cell-based model system, high-throughput small RNA sequencing, computational analysis, and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least 2-fold, including miRNAs that were previously described to target genes associated with inflammation, fibrosis, and cancer development. Further investigation demonstrated that the miR-146a-5p level was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes, as well as in liver tissue from HCV-infected patients. Genomewide microarray and computational analyses indicated that miR-146a-5p overexpression modulates pathways that are related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p has a positive impact on late steps of the viral replication cycle, thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease. IMPORTANCEHCV is a leading cause of chronic liver disease and cancer. However, how HCV induces liver cancer remains poorly understood. There is accumulating evidence that a viral cure does not eliminate the risk for HCC development. Thus, there is an unmet medical need to develop novel approaches to predict and prevent virus-induced HCC. miRNA expression is known to be deregulated in liver disease and cancer. Furthermore, miRNAs are essential for HCV replication, and HCV infection alters miRNA expression. However, how miRNAs contribute to HCV-driven pathogenesis remains elusive. Here we show that HCV induces miRNAs that may contribute to liver injury and carcinogenesis. The miR-146a-5p level was consistently increased in different cell-based models of HCV infection and in HCV patient-derived liver tissue. Furthermore, miR-146a-5p increased HCV infection. Collectively, our data are relevant to understanding viral pathogenesis and may open perspectives for novel biomarkers and prevention of virus-induced liver disease and HCC. H epatitis C virus (HCV) infection is a leading cause of chronic liver disease and hepatocellular carcinoma (HCC) worldw...

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Reconstitution of the Entire Hepatitis C Virus Life Cycle in Nonhepatic Cells

Cited by 84 publications

References 41 publications

The Intraviral Protein Interaction Network of Hepatitis C Virus

The Intraviral Protein Interaction Network of Hepatitis C Virus

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

Hepatitis C Virus-Induced Upregulation of MicroRNA miR-146a-5p in Hepatocytes Promotes Viral Infection and Deregulates Metabolic Pathways Associated with Liver Disease Pathogenesis

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