Luciferase Cells Were Infected With The Hepatitis C Virus As A Reporter Gene System Establishment And Application Of

Hepatitis C Virus (HCV) is the major causative agent of Hepatitis C, which now is an major infectious disease worldwide. It is reported that a global burden of 170 million people is infected with HCV, and nearly 80% infected person develop chronic hepatitis, and some patients progress to liver cirrhosis and hepatocellular carcinoma. Hepatitis C Virus infection has already been an important public health challenge in the world. However, the preventive measures and treatment strategies are quite limited, and a protective vaccine does not currently exist. At present, the standard therapeutic option is the combination of PEG-IFNa and Ribavirin. The sustain virus response rate can reach to about 50%, but HCV various genotypes present different therapeutic effect. Besides, the side effects are so severe that some people can not tolerate. Hence, the development of more effective and safer antiviral drugs is urgent.Since the HCV replicon system was established in 1999, it has been widely applied in the study of HCV replication, which is useful to develop and evaluate the antivirals. However, this mode can not produce infectious virus particles, hence it can not be used to study HCV infection. In 2005, the HCV cell-culture (HCVcc) system based on the genotype 2a strain JFH1 was a great breakthrough. This system facilitates the research on HCV whole life cycle including viral entry, assembly reasese and infection. Hence, HCVcc system provides an efficient tool to search novel antiviral targets and strategies.However, both the replicon system and the HCVcc infection system themselves have some limitations, and there are specific drawbacks in relevant experiment operations, such as real time q-PCR, the traditional test for detecting HCV RNA, is laboursome and time-consuming. In this work, reporter gene-Luciferase gene was introduced into HCV 2a chimeric to construct a series of HCV reporter plasmids. Then these plamids were used as templates to synthetize corresponding HCV RNA. The recombinant HCV RNAs were electroporated into hepatocellular carcinoma cell line Huh7.5. HCV RNA replication and viral protein expression were tested by real-time qPCR, Luciferase Activity Assay and Westernblotting, respectively. Virus infection experiments to test whether the luciferase gene- tagged HCV RNAs can produce infectious progeny virus. The results showed that Gaussia luciferase tagged HCV 2a chimeric Gluc-Jcl can efficiently replicate in Huh7.5 cells, and the progeny virus has the capacity to infect naive Huh7.5 cells. Moreover, luciferase activity assay showed that the change of luciferase activity in cells and in the supernate can respectively reflect HCV RNA level and the producted virus infectivity. All the results demonstrated that Gaussia luciferase tagged HCV 2a infectious system was established. Then, we tested whether this system can be use to evaluate antivirals. Gluc-Jcl infected Huh7.5 cells were treated with PEG-IFN a-2a, we found that Gaussia luciferase activity decreased in a PEG-IFN a-2a dose-dependent manner in contrast to the untreated cells, this is consistent with HCV RNA and protein level in cells after the treatment.Another luciferase tagged HCV 2a chimeric Fluc-J6/JFH1, we demonstrated this recombinant HCV RNA also has the ability to complete its virus life cycle in vitro by performing the above experiments. Then, we used it to research the role of stem-loop 1 in HCV IRES -mediated translation of genotype 2a. We found that deletion this structure in HCV 5'UTR significantly impired HCV IRES-mediated translation. And the change of luciferase acivity in the reporter gene Firefly luciferase-tagged HCV infectious system confirmed this finding sensitively,simply and intuitively.To sum up, luciferase reporter gene tagged HCV infectious systems were established. Compared with replicon system and HCVcc system, in these systems it is more sensitive, convenient and timely to detect HCV RNA replication level and progeny virus production by the luciferase activity assay, and our results showed these systems can provide a more efficient tool for the study of HCV replication and pathopoiesis, and the development of antivirals.