Investigation On The Construction Of HCV Minigenome Expressing Transgene And Its Primary Application

Virus genome replication plays an important role in its life cycle. It is known that the replication of plus-stranded RNA virus divides into two steps: genomic RNA is firstly transcribed into a minus-strand intermediate, which in turn serves as the template for producing progeny plus-strand RNA. During this process, RNA dependent RNA polymerase(RdRP)is the key replicase; Untranslated regions(UTRs) are major regulatory sequences in which the 3′UTR is regarded as a main binding region of replicase to direct the de novo synthesis of viral genome. Based on deep understanding of virus replication mechanism and its details, the construction of RNA virus minigenome has been a focus in molecular virology research recently. It can be used to elucidate the molecular mechanism of viral replication and identify the trans-acting proteins involved in virus maturation and packaging. Furthermore, it can provide new strategy against HCV infection.Hepatitis C virus (HCV) is a member of the Flaviviridae family with a positive-sense RNA genome of about 9600 nucleotides in length. It contains a single open-reading frame (ORF) flanked by two untranslated regions (UTR). HCV genome has a high degree of sequence variation which leads to the production of viral quasispecies. There are six different genotypes with more than 100 subtypes worldwide. The analysis of HCV genome sequence indicates that cis-acting elements functioned in the RNA replication are highly conserved, including 5′UTR, 3′UTR, and part sequence of core and ns5b. NS5B is RdRP of HCV and playes a key role in RNA replication. Replicase complex formed by NS5B along with other NS protein (such as NS3, NS4A, NS4B) are always anchored on endocytoplasmic reticulum. These progresses on understanding HCV RNA replication make it possible to construct HCV minigenome.The discovery of new anti-HCV drugs are main aim for the scientists in the worldwide. Potential small molecular inhibitors of NS3/4A protease, helicase, and RdRP NS5B always lead to drug resistant mutants in clinical trial. The present treatment for HCV infection is IFN-αin combination with ribavirin (RBV), having limited effectiveness and great side effects. Therefore, the targeting HCV infected cells'therapy is promising.In this study we firstly use cis-acting elements to construct HCV minigenome, in which the antisense sequence of firefly luciferase (flu) and IRES gene from EMCV were inserted. RdRP replicase complex bind 3'UTR to synthesize minus strand RNA, in which flu and IRES gene were in the sense direction and expressed. Then several different truncated minigenomes were designed. The transcribed minus strand RNAs from them had different mutant stem loops at 3′end. The levels of luciferase activity of different truncated minigenomes were compared. Finally, HCV minigenomes were used to express IFN-βfor targeting gene therapy against HCV. This study can be divided into three parts:1. Construction and functional identification of HCV minigenome Designed and amplified HCV 5UTR1-377 , 3UTR sequences from HCV 1b gene sequence of Genbank,with the T7 promoter directly coupled at the 5′-end; Designed and amplified IRES sequence (from EMCV), firefly luciferase gene; Synthesized HDV Ribozyme sense, antisense sequence and annealed; Cloned these fragments into pMD18T vector separately to identify sequences, then cloned them into pUC18 vector to construct pUC18-T7/5UTR-rIRES-3UTR-HDV Rz, labeled to pT7/5UrI3URz. Then the luciferase gene was inserted into Xba I digested pT7/5UrI3URz, generating pT7/5UrFI3URz,which was the HCV minigenome. HCV replicon RNA was synthesized in vitro by T7 RNA polymerase after the plamid BB7 was linearized by Sca I. Huh7.5BB7 stable cell line was obtained by eletroperation of BB7 RNA,and selected with G418, then confirmed by RT-PCR and Western blot. Plasmid pT7/5UrFI3URz was linearized with EcoR ?, and transcripted into minigenome RNA which was transfected into Huh7.5BB7 cell and Huh7.5 cell via eletroperation. After 48 hrs, cells were harvested and luciferase activities were assessed. The results showed luciferase was expressed selectively in Huh7.5BB7 cell but not in Huh7.5 cell, which suggested the minigenome was constructed successfully.2. Comparison of different truncated HCV minigenomes HCV replication is the de novo synthesis from the 3′-end of plus or minus strand. The secondary structure of the 3′-end at the minus strand HCV RNA has been determined. With the exception of the short SL-A1 stem loop located at the very 3′-end, other structures differe from its antisense sequence corresponding to 5′UTR. Therefore, their respective roles in the RNA synthesis initiation also differe from 5′UTR. In this study, we constructed several truncated minigenomes (?45,?124,?227,?131-315) which produced the minus strand RNAs with deletion of different stem loops at the 3′UTR. These minigenomes were transcribed into the corresponding RNAs in vitro, then transfected into Huh7.5BB7 cell. After 48 hrs, luciferase activities were assessed. The result shows that ?131-315 exhibited the higher luciferase activity compared with that of the full minigenome (p<0.01),whereas that of ?45 minigenome exhibited lower (p<0.01). ?125, ?227 minigenomes didn't show any luciferase activity. RT-PCR also suggested the same results.3. Exploiting HCV minigenome to express IFN-βfor HCV targeting gene therapyPEG-IFN in combination with ribavirin has been the standard treatment for HCV infection currently. But sustained virological response (SVR) varies between different genotypes. The SVR rates ranged from 38% to 52% among genotype 1 and 4. One important reason is that HCV proteins could block IFN inducing anti-virus response. In this study, IFN-βgene was invertedly inserted into minigenome and transfected into the cells harboring HCV replicase in which the minus strand minigenome RNA was synthesized and IFN-βcould be expressed and inhibit HCV RNA replication. First, we extract lymphocytes from peripheral blood and stimulated by poly I:C to express the IFN gene. Total RNA extracted from lymphocytes was reversely transcripted into cDNA. IFN-βgene was amplified by PCR and was sequenced, and then the correct gene was cloned into pT7/5UrI3URz in antisense direction to obtain pT7/5UrIFNI3URz. The plasmid was linearized by EcoR I and transcripted into RNA in vitro, then transfected into Huh7.5BB7. After 48hrs, cell was harvested and total proteins were extracted to do Western-blot, IFN-βantibody as the first antibody. The result showed that IFN-βwas specifically expressed in replicon cell. To apply the targeting strategy to gene therapy which require expression from polymerase II RNA polymerase rather than T7-directed transcription. So we clone the minigenome 5UrIFNI3URz into the plasmid pcDNA3.1 by positioning the minigenome immediately adjacent to the transcription start site of the cytomegalovirus (CMV) promoter. Then the pCMV-5UrIFNI3URz was constructed. The results of Western-blot analysis showed that IFN-βcould be specifically expressed after the pCMV-5UrIFNI3URz was trasnfected into Huh7.5BB7 cell lines. By the same way as the establishment of Huh7.5BB7 cell, the Huh7.5JFH-1 cell line was constructed in which virus RNA can replicate autonomously and virus particle can be packed successfully. When the pCMV-5UrIFNI3URz was transfected into Huh7.5JFH-1 cell,the real-time PCR assay showed that the level of HCV RNA replication was distinctly decreased.In conclusion, the HCV minigenome was successfully constructed and optimized for expression of the foreign gene. The targeting minigenome carrying IFN-βcould specifically expressed in HCV infected cell lines,the replication level was inhibited in a certain extent.