%0 Generic
%A Quinkert, Doris
%D 2007
%F heidok:7755
%K Hepatitis C Virus , Replikation , Replikationskomplex , Co-FaktorenHepatitis C Virus , replication , replication complex , co-factors
%R 10.11588/heidok.00007755
%T Quantitative analysis of the Hepatitis C Virus replication complex and identification of associated cellular factors
%U https://archiv.ub.uni-heidelberg.de/volltextserver/7755/
%X Hepatitis C virus (HCV) has a positive-strand RNA genome and is grouped into the family of Flaviviridae. Similar to other positive-stranded RNA viruses, HCV RNA replication takes place in the cytoplasm. The sites of viral replication are designated “membranous web” and represented by an accumulation of vesicular structures, which are induced by the viral non-structural proteins and probably originate from membranes of the Endoplasmic Reticulum. The aim of this work was to purify and characterize these viral replication complexes (RCs) in vitro and to identify potential host factors of viral replication.  First a purification strategy for enzymatically active viral replication complexes was developed to determine associated cellular proteins by proteomics. Thereby, several potential host factors of viral replication were identified and the most reproducible, Annexin II (ANXA2) was further characterized.  In immunofluorescence analyses, ANXA2 strongly colocalized to the sites of viral replication in all applicable cell lines supporting HCV replication, in HCV-transfected as well as in infected cells. In contrast, we found no obvious colocalization of HCV proteins with Annexin I, IV or V or with p11 (also denoted S100A10), a common cellular ligand of Annexin II. Specificity of the ANXA2-HCV interaction was further indicated by the lack of colocalization with replication sites of other positive-strand RNA viruses, namely Dengue virus and Semliki-Forest-Virus. By individual expression of the viral non-structural (NS) proteins we found that NS5A colocalized with Annexin II, indicating that NS5A might be involved in the recruitment of ANXA2. SiRNA-mediated silencing clearly reduced Annexin II levels but failed to block HCV replication. However, FACS analyses revealed a strong correlation of intracellular HCV and ANXA2 levels even in presence of ANXA2 siRNA, suggesting that Annexin II expression was induced by HCV, thereby counteracting siRNA-mediated knockdown. Still, ANXA2 silencing moderately reduced the number of HCV positive cells. Interestingly, the presence of replicating HCV sequences in HepG2 cells, harboring very little endogenous ANXA2, clearly induced Annexin II expression to detectable levels perfectly colocalizing with the viral NS proteins. However, the role and function of ANXA2 in the HCV life cycle has yet to be defined. In a second line of investigations, a detailed stoichiometric analysis of HCV RCs was performed. Thus, the ratio of non-structural proteins to RNA that is required for HCV RNA replication could be determined. Almost the entire negative- and positive-strand RNA but <5% of the non-structural proteins present in HCV-harboring cells were protected against nuclease and protease treatments. Nevertheless, this protease-resistant portion of NS proteins accounted for the full in vitro replicase activity. Therefore, only a minor fraction of the HCV non-structural proteins was actively involved in RNA synthesis. However, due to the high amounts present in replicon cells, this still represented a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, an active HCV replication complex probably consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred non-structural protein copies. These might be required as structural components of the vesicular compartments that are the site of HCV replication.