Bedeutung der Hepatitis C Virus Hüllproteine für die Assemblierung und Freisetzung infektiöser Viren
Zusammenfassung der Projektergebnisse
Main goal of this project was to characterize the role of the HCV glycoproteins E1 and E2 during viral morphogenesis. By investigating intra- and inter-genotypic glycoprotein chimeras in the HCV assembly process, we could demonstrate that the concerted action of core and p7 facilitates efficient virion production in the context of GT2a chimeric genomes. Glycoprotein sequences, however, had only minimal impact on this process. In contrast, in the context of inter-genotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT 1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within inter-genotypic chimeras glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2 and NS2. However, it caused an accumulation of non-enveloped core protein, and increased abundance of nonenveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment which likely depends on a genotype-specific interplay with additional viral factors. As another goal we aimed at developing a cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in depth mechanistic studies of envelope gene function. Therefore, we constructed a packaging cell line expressing core, p7 and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single round infectious particles by way of transcomplementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolatedependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Furthermore, replacement of J6 (GT 2a) core, p7 and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients thus opening up new opportunities to dissect envelope gene function in an individualized fashion.
Projektbezogene Publikationen (Auswahl)
- (2011). Inactivation and survival of hepatitis c virus on inaminate surfaces. J Infect. Dis. Dec 15;204(12):1830-8
Juliane Doerrbecker, Martina Friesland, Sandra Ciesek, Thomas J. Erichsen, Pedro Mateu-Gelabert, Jörg Steinmann, Jochen Steinmann, Thomas Pietschmann, Eike Steinmann
- (2011). The green tea polyphenol epigallocatechin-3-gallate (EGCG) inhibits hepatitis C virus (HCV) entry. Hepatology Dec;54(6):1947-55
Sandra Ciesek, Thomas von Hahn, Che C. Colpitts, Luis M Schang, Martina Friesland, Jörg Steinmann, Michael P. Manns, Michael Ott, Heiner Wedemeyer, Philip Meuleman, Thomas Pietschmann, Eike Steinmann
- (2012). Transmission of hepatitis C virus among people who inject drugs: viral stability and association with drug preparation equipment. J Infect. Dis. Jan;207(2):281-7
Juliane Doerrbecker, Patrick Behrendt, Pedro-Mateu-Gelabert, Sandra Ciesek, Nina Riebesehl, Corinne Wilhelm, Joerg Steinmann, Thomas Pietschmann, Eike Steinmann
(Siehe online unter https://doi.org/10.1093/infdis/jis677) - (2013) Hepatitis C virus p7 is critical for capsid assembly and envelopment. PloS Pathog. 9(5):e1003355
Juliane Gentzsch, Christiane Brohm, Eike Steinmann, Martina Friesland, Nicolas Menzel, Gabrielle Vieyres, Paula Perin, Anne Frentzen, Lars Kaderali, Thomas Pietschmann
(Siehe online unter https://doi.org/10.1371/journal.ppat.1003355) - (2013). Cell culture systems for hepatitis C virus. Curr Top Microbiol Immunol. 369:17-48
Eike Steinmann, Thomas Pietschmann
- (2013). Characterization of intra- and intergenotypic chimeras reveals a role of the glycoproteins in virus envelopment. J Virol Dec;87(24):13297-306
Eike Steinmann, Juliane Doerrbecker, Martina Friesland, Nina Riebesehl, Corinne Ginkel, Julia Hillung, Juliane Gentzsch, Chris Lauber, Richard Brown, Anne Frentzen, Thomas Pietschmann
- (2013). Entry and replication of recombinant hepatitis C viruses in cell culture. Methods Feb;59(2):233-48
Gabrielle Vieyres, Thomas Pietschmann
(Siehe online unter https://doi.org/10.1016/j.ymeth.2012.09.005) - (2013). Inactivation of hepatitis C virus infectivity by human breast milk. J Infect. Dis. Dec 15;208(12):1943-52
Stephanie Pfaender, Julia Heyden, Martina Friesland, Sandra Ciesek, Asim Ejaz, Joerg Steinmann, Jochen Steinmann, Andrea Malarski, Heribert Stoiber, Giorgos Tsiavaliaris, Werner Bader, Georg, Jahreis, Thomas Pietschmann, Eike Steinmann
(Siehe online unter https://doi.org/10.1093/infdis/jit519) - (2013). Tumeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells. Gut. 2014 Jul;63(7):1137-49
Anggakusuma, Che Colpitts, Luis M. Schang, Heni Rachmawati, Anne Frentzen, Stephanie Pfaender, Patrick Behrendt, Richard Brown, Dorothea Bankwitz, Joerg Steimann, Meichael Ott, Philip Meuleman, Charles M. Rice, Alexander Ploss, Thomas Pietschmann, Eike Steinmann
(Siehe online unter https://doi.org/10.1136/gutjnl-2012-304299) - (2014). Incoporation of primary patient-derived glycoproteins into infectious hepatitis C virus particles. Hepatology Aug;60(2):508-20
Juliane Doerrbecker, Martina Friesland, Nina Riebesehl, Corinne Ginkel, Patrick Behrendt, Richard Brown, Sandra Ciesek, Heiner Wedemeyer, Christoph Sarrazin, Lars Kaderali, Thomas Pietschmann, Eike Steinmann
(Siehe online unter https://doi.org/10.1002/hep.27190) - (2014). Incorporation of hepatitis C virus E1 and E2 glycoproteins: the keystones on a peculiar virion. Viruses Mar 11;6(3):1149-87
Gabrielle Vieyres, Jean Dubuisson, Thomas Pietschmann
(Siehe online unter https://doi.org/10.3390/v6031149)
