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The role of cellular lipid homeostasis in the hepatitis C virus replication cycle

Stoeck, Ina Karen

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Abstract

With approximately 160 million people being persistently infected with hepatitis C virus (HCV), which bears a high risk of serious liver damage (fibrosis, cirrhosis, hepatocellular carcinoma), infections with HCV represent a major global health burden. Recent advances in the development of HCV-specific direct acting antiviral drugs have substantially improved the success rates of antiviral therapy. Nevertheless major challenges such as high costs, high number of undiagnosed infections and the possibility of reinfection even after therapy-induced virus elimination remain. There is increasing evidence that infections with this hepatotropic virus are tightly linked to lipid metabolism. For instance, lipid droplets (LDs) play an important role in the replication cycle of HCV, but also of the related Dengue virus (DENV). Moreover, HCV induces profound upregulation of lipogenic genes and its replication is attenuated by certain statins, which are known inhibitors of the cholesterol synthesis pathway. With the overall goal to decipher the role of cellular lipid homeostasis for the HCV replication cycle I have studied two interrelated aspects. The first one aimed at gaining a better insight into the link between HCV and LDs, the cellular lipid storage organelles. By performing a RNAi screen targeting host factors implicated in LD homeostasis the aim was to identify novel candidates involved in the replication cycle of HCV, and for comparative purposes, DENV, as well as genes involved in the regulation of LD-linked pathways. Obtained results highlighted a central role of LDs in the replication cycle of both viruses and revealed the cellular DEAD box RNA helicase 3 (DDX3) as a key factor in the replication cycle of HCV and DENV. By using reverse genetics and biochemical assays I found that DDX3 is recruited to LDs via the HCV core protein, but loss of recruitment had no discernable effect on the viral life cycle. Nevertheless, our results identified a yet unexplored role of DDX3 in the production of infectious HCV. The second part of this PhD thesis focused on another aspect related to the link between HCV and lipid homeostasis. Similar to other positive-strand RNA viruses, HCV remodels endomembranes to generate viral replication factories. There is increasing evidence that HCV co-opts cellular pathways in order to generate a specific lipid microenvironment that harbors the viral replication machinery. Using a set of microscopy-based approaches I found that HCV reorganizes the distribution of free cholesterol and possibly recruits it from the plasma membrane to the sites of viral replication. To understand the molecular mechanisms responsible for the reshaping of the cholesterol distribution, I performed a small scale RNAi screen targeting a selection of lipid transfer proteins (LTPs) implicated in direct, non-vesicular lipid transport. In this way I identified several LTPs (i.e. Niemann-Pick disease type C1 (NPC1), Oxysterol-binding protein-related protein 1A, StAr-related lipid transfer domain protein 3, membrane associated phosphatidylinositol transfer protein 1) required for HCV replication, highlighting the critical role of endosomal cholesterol transport for efficient virus propagation. Pharmacological inhibition of endosomal cholesterol export induced lipid accumulation in lysosomal vesicles and impaired the transfer of free cholesterol to potential sites of viral replication. Concomitantly, viral RNA replication was impaired, implying that HCV RNA replication depends on the redistribution of plasma membrane unesterified cholesterol through the endosomal pathway. Thus, HCV might usurp LTPs such as NPC1 to allow recruitment of free cholesterol to the viral replication factories.

Item Type: Dissertation
Supervisor: Bartenschlager, Prof. Dr. Ralf
Date of thesis defense: 20 June 2016
Date Deposited: 15 Jul 2016 12:11
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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