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Bedeutung der Hepatitis C Virus Hüllproteine für die Assemblierung und Freisetzung infektiöser Viren

Subject Area Virology
Term from 2010 to 2016
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 162577101
 
Final Report Year 2014

Final Report Abstract

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.

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