Construction Of A Novel Cell System To Evaluate The HCV NS5B Polymerase Activity Using The Minigenome

Hepatitis C virus (HCV) is grouped in the genus Hepacivirus within the familyFlaviviridae, and it is one of the pathogens of chronic liver disease. HCV infection is themain cause of liver cirrhosis and hepatocellular carcinomas and it has a tendency of globalprevalence. There is an estimated number of infected people exceeding130millionworldwide. HCV is a single positive-stranded RNA virus. It’s genome is approximately9.6kb which is comprised of5′-untranslated region (5′UTR), an open reading frame (ORF)and3′-untranslated region (3′UTR). The open reading frame encodes a polyproteinprecursor which would resolve into three structural proteins and seven non-structuralproteins by the host and viral proteases. Structural proteins include core protein (Core),envelope glycoproteins E1and E2, and non-structural proteins include p7, NS2, NS3,NS4A, NS4B, NS5A and NS5B. The non-structural protein NS5B, a RNA-dependent RNA polymerase (RdRp), is the key enzyme during the synthesis of HCV RNA, and alsothe key target for anti-HCV therapy. With the understanding of the mechanism of viralgenome replication, the minigenome system of HCV which was established by reversegenetic manipulation provides a new tool for studying HCV replication, interaction withhost cell, HCV pathogenic mechanism and the screening of inhibitors. It also providessupport for the high throughput screening (HTS) of HCV NS5B inhibitors. Therefore, theobjectives of this study is to construct a novel cell model to evaluate the HCV NS5Bpolymerase activity and to provide a new experimental system for high throughputscreening of HCV NS5B inhibitors using the RdRp activity of HCV NS5B and the reversegenetic manipulation.Based on the latest studies of HCV replication mechanism and the preliminary studiesof our laboratory, the eukaryotic expression plasmid pCMV-NS3-5B-IRES-EGFP,minigenome (-)5UIRrG(-)3U and the eukaryotic expression plasmid pcDNA3.1(+)-(-)5UIRrG(-)3U were designed and constructed. Then the pCMV-NS3-5B-IRES-EGFPand pCMV/5UrFI3Urz plasmids were cotransfected into BHK-21cells, the activity offirefly luciferase (FLuc) in cells was detected to verify the NS5B activity ofpCMV-NS3-5B-IRES-EGFP. The pcDNA3.1(+)-(-)5UIRrG(-)3U and pCMV-NS3-5B-IRES-EGFP plasmids were cotransfected into BHK-21cells, the activity of Gaussialuciferase (GLuc) from cell culture medium and cells were detected respectively toidentify the function of minigenome (-)5UIRrG(-)3U. The main results of the study arelisted as bellow:1. The plasmid expressing HCV NS3-5B replication complex was successfully constructedand the RdRp activity of NS5B was identified.(1) The HCV NS3-5B gene was amplified by PCR and then the eukaryotic expressionplasmid pCMV-NS3-5B-IRES-EGFP was successfully constructed. Enzyme restrictionanalysis and BLast showed that the fragment size and the amino acid sequence werecorrect. Results of fluorescence detection showed that the green fluorescent protein wasexpressed in the BHK-21cells. Western Blot showed that the protein NS3-4A and NS5Bwere expressed in the cells. (2) The pCMV-NS3-5B-IRES-EGFP and pCMV/5UrFI3Urz (constructed by ourlaboratory) plasmids were cotransfected into BHK-21cells. FLuc activity assay showedthat the NS5B provided by the plasmid pCMV-NS3-5B-IRES-EGFP not only had RdRpactivity, but also the activity of the NS5B located in the replication complex wassignificantly higher than the individual NS5B activity.2. The minigenome (-)5UIRrG(-)3U was successfully constructed and the function of theminigenome was identified.(1) The minigenome (-)5UIRrG(-)3U was designed, which contains the negative-strandof HCV5’UTR sequences, the3’UTR sequence, the internal ribosome entry site (IRES)sequence of the encephalomyocarditis virus (EMCV), DsRed2gene and the reversecomplemented sequence of Gaussia luciferase (rvGLuc). The upstream and downstreamprimers were designed for each gene fragment. Then the minigenome(-)5UIRrG(-)3U wasconstructed by overlap extension PCR which could splice each gene fragment. Results ofagarose gel electrophoresis showed that the length of each gene fragment was correct.(2) The eukaryotic expression plasmid pcDNA3.1(+)-(-)5UIRrG(-)3U was successfullyconstructed. Enzyme restriction analysis and BLast showed that the fragment size and theamino acid sequence were correct. When the plasmid was transfected into the cells, redfluorescent protein was expressed in the BHK-21cells. This result showed that thefunction of minigenome (-)5UIRrG(-)3U was normal in cells.3. The cell system which could be used to evaluate the HCV NS5B polymerase activitywas preliminarily established.When the recombinant plasmids pCMV-NS3-5B-IRES-EGFP and pcDNA3.1(+)-(-)5UIRrG(-)3U were cotransfected into BHK-21cells, green fluorescent protein and redfluorescent protein could be simultaneously expressed in the cells and the minigenome(-)5UIRrG(-)3U could replicate and transcript with the help of NS5B protein which wasprovided by the plasmid pCMV-NS3-5B-IRES-EGFP. Then GLuc were expressed in theBHK-21cells. So the HCV NS5B polymerase activity could be evaluated by the GLucactivityIn summary, the eukaryotic expression plasmid expressing HCV NS3-5B replication complex and the minigenome containing a reporter gene were successfully constructed inthis study, and then the cell system which could be used to evaluate the HCV NS5Bpolymerase activity was preliminarily established. This system could provide a novel wayto establish a high throughput screening system for screening HCV NS5B inhibitors, andprovide the experiment basis to further establish transgenic animal model for screeningand evaluating HCV NS5B inhibitors.