Construction Of Hepatitis C Virus Cell Culture System And Observation Of The Ultrastructure Of HCV-like Particles In Infected Cells And Exploration Of Receptor-mediated HCV Endocytosis

Hepatitis C Virus (HCV) is a major cause of chronic liver disease, with over 170 million persistently infected individuals worldwide. HCV-associated liver diseases frequently progress to cirrhosis, which can lead to liver failure and hepatocellular carcinoma. HCV has been a serious threat to global health. The mechanism of HCV infection is not completely clear. There are no effective vaccines and current medical treatments are limited.HCV is an enveloped, positive-sense RNA virus that belongs to the Hepacivirus genus in the Flaviviridae family.The first step of the enveloped virus infecting cells is the interaction between viral envelope proteins and their receptors distributing on the surface of the target cellular membrane. Many cellular attachment factors and specific receptors are involved in the HCV entry, such as low-density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-BI), CD81 and claudin-1. However, the detailed mechanisms of the interaction between these molecules and HCV entry are unclear. A better understanding the mechanisms of HCV endocytosis, is needed to identify novel therapeutic targets. After HCV entering host cells, the processes of producing integrated HCV particles include replication, synthesis, assembly and release. The HCV particle admittedly consists of a nucleocapsid surrounded by a lipid bilayer where the two envelope glycoproteins. It is currently unsure about the ultrastructure of HCV particles since there are remarkable differences about the sizes and morphological characteristics of HCV particles from different sources. In order to find new methods inhibiting HCV infecting, an intensive study on the characteristics of morphogenesis and distribution of HCV particles in infected cells and the cytopathic effect of the hosts are needed to better understand the viral life cycle and the host's structures involved in the HCV production.Researchers have been hampered by the lack of a robust cell-culture system yielding infectious virus for a long time until 2005, cell culture model of HCV was established in the lab of HCV center led by Charles M. Rice, Rockefeller University. With the help of the staff of the Rice's lab, we constructed the HCV cell-culture system and profermed some primary studies based on this system. Our studies were as follows:1 Construction and identification of the HCV cell-culture system (HCVcc)Plasmid J6/JFH encoding the full length HCV chimeric genome was transcribed to HCV RNA in vitro and the RNA was transfected into Huh-7.5 cells by electroporation. The expression of HCV mRNA and protein were detected in posttransfected Huh7.5 cells, HCV copies in the supernatant of transfected cells were quantitated. The cell-culture supernatant was collected to incubate with na?ve Huh7.5 cells and the infectivity of HCV was assayed and calculated by Reed﹠Muench method. The results showed that HCV mRNA and protein expressed in the transfected cells. High level virus copies in supernatant of transfected cells were quantitated. TCID50/ml of the supernatant was 6.98×104. In conclusion, the HCV cell culture system was successfully constructed and the infectivity of HCVccs was identified.2 Observation of HCVcc infected na?ve Huh7.5 cell through transmission electron microscopy.The cell-culture supernatant was collected to incubate with na?ve Huh7.5 cells, then the infected cells was used to made ultrathin sections. Transmission electron microscopy was used to observe morphological characteristics of virus particles and changes of intracellular ultrastructure in infected Huh7.5 cells. Large numbers of spherical virus-like particles (VLPs) were observed in infected Huh7.5 cells. The diameters of the VLPs ranged from 33-52nm and most of the VLPs distributed near by the rough endoplasmic reticulum close to the nucleus. We also found hyperplasia of some membrane- enclosed organelles in the cytoplasm, and the internal diameter of the cisternae of endoplasmic reticulum dilated remarkably. Several features characterizing flavivirus infected cells such as vacuoles and electron-dense inclusion bodies were also observed. The study on morphology not only proved the reliability of the HCVcc, but also intuitively demonstrated the HCV particles'ultrastructural characteristics in human hepatoma cell lines and their correlation with endoplasmic reticulum.3 SiRNA inhibiting the expression of CD81 or SR-BI affected HCVcc infection.Chemically synthesized siRNA targeting CD81 or SR-BI was transfected into Huh-7.5 cells to silence the expression of CD81 or SR-BI respectively and the susceptibility to HCVcc was tested. The results showed that CD81 or SR-BI were remarkably reduced by chemically synthesized siRNA in Huh-7.5 cells. Susceptibility to HCVcc was obviously reduced in CD81 or SR-BI silenced Huh-7.5 cells compared with cells treated with an irrelevant siRNA. The experiment indicated that both CD81 and SR-BI played important roles in HCV endocytosis.