Generation of a system enabling the study of hepatitis C entry factors in vivo and characterisation of HCV entry factor claudin-1

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Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. The standard therapeutic regime consists of pegylated interferon 2a/2b and ribavirin and depending on the genotype the protease inhibitors telaprevir or boceprevir.Despite all progress that has been done, treatment of HCV is rich of side effects and does not lead to a sustained virological response for a significant number of patients.An elemental prerequisite for the development of new therapeutic agents is the understanding of the viral life cycle. A crucial step is the virus entry into the hepatocyte.Until now seven (co-)receptors, that are involved in HCV cell entry (CD81, SCARB1, OLCN, NPC1L1, EGRF, EphA2 and CLDN1), have been identified.The proposed project aims to further elucidate the role of CLDN1 in HCV cell entry by using a genetically humanized mouse model. This model is based on the previous observation, that mice, which are naturally not susceptible for HCV, can be rendered permissive to cell culture derived HCV (HCVcc-FlpE) by adenoviral expression of human CD 81 and OCLN. The genome of HCVcc-FlpE encodes for a FlpE-recombinase and activates a yellow fluorescent protein reporter in Rosa26-FSF-F2AY reporter mice, which is used as a read-out for infection.This genetically humanized mouse model, which is well established in a modified form will now be combined with the technique of conditional gene ablation. The gerneration of mice with floxed exons in the CLDN1 locus is currently under progress. These mice will be crossed to an already existing deleter strain expressing CRE recombinase under an albumin promoter and leading to a liver-specific knockout of the CLDN1 gene. This step is necessary as a complete homozygous CLDN1 deficiency results in a neonatally lethal phenotype in mice.In the first instance we aim to characterize the hepatic CLDN1 deficiency. We will particularly address the question, how the expression profile of other members of the claudin family is altered and whether they can adopt the physiological role of CLDN1.The next step will be to cross the reporter mouse line with the liver-specific knockout of CLDN1. The resulting progeny will be transduced with human CD81 and OCLN and also with human or murine CLDN1. After injection of HCVcc-FlpE we will quantify reporter activity.This approach will allow to examine the impact of every given gene on HCV infection, even if the corresponding knockout would lead to a lethal phenotype.