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Abstract

Hepatitis C virus is a major causative agent of liver-associated diseases including liver cirrhosis and liver cancer. A prophylactic vaccine is not available and highly efficient antiviral therapy that can eliminate the virus in infected individuals is not affordable in many high-prevalence countries. Therefore, more efforts are required regarding the global eradication of HCV infection. The HCV life cycle has been extensively studied since the discovery of HCV in 1989 and it has become an excellent research model for the studying of other pathogenic positive-strand RNA viruses. However, important gaps in the understanding of the HCV life cycle remain, especially the mechanism of HCV assembly, the site within the cells where virions are forming, and involved viral and host cell factors. Recently, it has been suggested that HCV possibly coordinates viral RNA replication and particle assembly by generating and using double-membrane vesicles (DMVs) that are tightly connected to the endoplasmic reticulum (ER), which wraps around lipid droplets. However, thus far it has not been possible to visualize assembling HCV particles. In the first part of my thesis, I describe my efforts to faithfully identify HCV assembly sites. To this end, I established a triple-label live-cell imaging approach by fluorescently tagging Apolipoprotein E (ApoE), a host cell component of infectious HCV particles, the viral envelope glycoprotein E2, and the HCV replicase factor NS5A. Based on the available literature, I originally hypothesized that nascent HCV particles bud into the ER lumen at the sites where NS5A, E2, and ApoE are enriched and colocalize. Therefore, I investigated HCV assembly events by time-lapse live-cell confocal imaging coupled with light and electron microscopy (CLEM). I found that the triple-positive signals of ApoE, NS5A, and E2 exist, but only at a very low frequency. Unfortunately, ultrastructural analysis by CLEM did not allow the unambiguous detection of nascent or mature intracellular HCV particles. In the course of these studies, I observed that ApoE associates with NS5A to a large extent and this occurs independent of HCV assembly. Therefore, I focused my project to study the role of the association between ApoE and NS5A in processes other than HCV assembly, which is described in the second part of my thesis. Since NS5A also is involved in the formation of HCV-produced extracellular vesicles (EVs), reported to contribute to de novo infection and virus-induced pathogenesis, I employed HCV as a model system to investigate the intracellular association, co-secretion and co-transmission of ApoE-containing lipoproteins with EVs generated in and released from infected cells. EVs and lipoproteins are two essential ways exploited by cells and viruses for intercellular communication. The former is responsible for cell-to-cell transmission of encapsulated nucleic acids, proteins, lipids, and metabolites while the latter is crucial for the transport of cholesteryl esters and triglycerides. Several lines of evidence suggest an association of lipoproteins with EVs but the role of this interplay is not well known. Moreover, the intercellular co-transmission of these two vesicle species has not been documented. Distinct from a variety of viruses that modify the host endosomal pathway and release virus-produced EVs, HCV generates, in addition to classical virus particles, EVs containing the complete viral genome and supporting the dissemination of infection in addition to the virus particle route. Moreover, HCV also induces drastic changes in the lipid homeostasis of infected host cells, including the lipoprotein pathway. In this study, I found that in HCV-replicating cells, NS5A is enriched in ApoE and CD63 double-positive late endosomes. There, ApoE interacts with NS5A-positive intraluminal vesicles (IVs) which are the precursors of EVs. In addition, I found that infected cells release EVs containing NS5A and viral RNA. These EVs interact with ApoE either in infected cells or after release from cells into the extracellular medium. ApoE-associated EVs are taken up by non-infected bystander cells, thus transmitting viral RNA. Importantly, ApoE-NS5A interaction appeared to be important for efficient EV-mediated secretion of HCV RNA. In the third part of my thesis, I describe the characterization of the association of ApoE-containing lipoproteins with EVs in the context of non-infected cells. I found that ApoE and CD63 double-positive IVs traffic along the pathway of late endosomes. Importantly, secreted ApoE associates with a fraction of EVs and co-enters neighboring bystander cells. These results suggest a more general role of ApoE in EV-mediated cell-to-cell communication and reveal genuine intercellular co-transmission of EVs in association with ApoE.