Juliane Gentzsch

Publications (4)27.27 Total impact

• Article: Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment.

  Juliane Gentzsch, Christiane Brohm, Eike Steinmann, Martina Friesland, Nicolas Menzel, Gabrielle Vieyres, Paula Monteiro Perin, Anne Frentzen, Lars Kaderali, Thomas Pietschmann
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  ABSTRACT: Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.
  PLoS Pathogens 05/2013; 9(5):e1003355. · 9.13 Impact Factor
• Article: Subcellular localization and function of an epitope-tagged p7 in hepatitis C virus-producing cells.

  Gabrielle Vieyres, Christiane Brohm, Martina Friesland, Juliane Gentzsch, Benno Wölk, Philippe Roingeard, Eike Steinmann, Thomas Pietschmann
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  ABSTRACT: The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood in part due to poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive HA-epitope tags at its N-terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild type virus. However, HA- p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which co-localized with the HCV envelope glycoprotein 2 (E2), but also partially with viral non-structural proteins 2, 3 and 5A. Using co-immunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2 and NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle.
  Journal of Virology 11/2012; · 5.40 Impact Factor
• Source

  Available from: Friedemann Weber

  Article: Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

  Anne Frentzen, Kathrin Hüging, Julia Bitzegeio, Martina Friesland, Sibylle Haid, Juliane Gentzsch, Markus Hoffmann, Dirk Lindemann, Gert Zimmer, Florian Zielecki, Friedemann Weber, Eike Steinmann, Thomas Pietschmann
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  ABSTRACT: Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.
  PLoS Pathogens 04/2011; 7(4):e1002029. · 9.13 Impact Factor
• Article: Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.

  Juliane Gentzsch, Bettina Hinkelmann, Lars Kaderali, Herbert Irschik, Rolf Jansen, Florenz Sasse, Ronald Frank, Thomas Pietschmann
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  ABSTRACT: Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.
  Antiviral research 02/2011; 89(2):136-48. · 3.61 Impact Factor