Study On The Inhibition Of HCV With HIRF Expressed By Recombinant Baculovirus

HCV is an enveloped positive-strand virus which belongs to the genus Hepacivirus in the Flaviviridae family and is a serious human pathogen that infects 3% of the world population. Approximately 20% of HCV-infected individuals recover from acute infection, and the cell-mediated immune response has been recognized as the major factor allowing this recovery. In contrast, a defective immune response is associated with persistent infection in 80% of infected individuals, leading to the development of chronic hepatitis infection and the occurrence of cirrhosis in 0.5 to 30% of cases and development of hepatocellular carcinoma at a rate of 1 to 3% per year .It has markedly improved in the treatment of HCV in recent years.The current standard treatment is combination therapy with pegylated IFNα-2a and ribavirin, although positive results are obtained in only 40 to 60% cases, depending on the HCV genotype. A significant fraction of HCV-infected patients are therefore resistant to IFN treatment, and particular attention has focused on the ability of HCV to interfere with the IFN system, with the goal of improving therapeutic intervention.The induction of type I interferons (IFNs) and expression of interferon- stimulated gene(ISG) is crucial for innate immunity, induced by different viral or bacterial components it is mediated by the activation of pattern-recognition receptors, such as Toll-like receptors and cytosolic receptors such as RIG-I and MDA5. The type I IFN induction is primarily controlled at the gene transcriptional level, wherein a family of transcription factors, interferon regulatory factors (IRFs), plays central roles. Among the IRFs, IRF3 and IRF7, which are highly homologous, have gained much attention as the key regulators of type I IFN gene expression induced by viruses. It has been shown that hepatitis C virus (HCV) nonstructural proteins 3 and 4A (NS3/4A) cleave IPS-1, thereby inhibiting the RIG-I- and MDA5-mediated activation of IRF3 and/or IRF7 and induction of IFN during HCV infection.It is one of the mechanism of escape the host antiviral response of HCV. The objective of the present study is to evoluate the potential antiviral effect of the overexpression of the IRF3 and IRF7 in the replicon system and HCV-infected cells.One of critical technology of the gene therapy is the gene transfer. In this study, we chose the baculovirus as the gene therapy vector. As a gene therapy vector, baculovirus possesses the following advantages: 1. the genome is large ,thus rendering the virus flexible to carry multiple genes or large insert; 2. recombinant baculovirus are easy to construct and produce to high titers simply by infecting insect cells; 3. the purification of baculovirus can be readily performed by ultracentrifugation; 4. baculovirus can entry into mammalian cells and non-replication; 5. the transfergene can express under the control of the promoter in mammalian cells; 6. biosafety. In summary, baclovirus can be used as an efficient vector for gene delivery into insect and mammanlian cells for a wide variety of applications.The development of efficient therapies for hepatitis C has been hampered by the lack of a reliable cell-culture system, as well as by the absence of a non- primate animal model. The HCV replicon consists of an antibiotic selection marker and a genotype 1b HCV RNA, which replicates autonomously in the intracellular compartments in a human hepatoma cell line, Huh7. This replicon system has functioned as an important tool in the investigation of HCV replication and it has served as a cell-based assay system for the evaluation of antiviral compounds. Recently, cell culture systems for in vitro replication and infectious viral production were established based on the full-length HCV genome of genotype 2a, which was isolated from an HCV-infected patient who developed fulminant hepatitis. However, no robust in vitro culture systems for the 1a and 1b genotypes, which are the most prevalent HCV genotypes in the world, have been established to date.In the present study, we chosed the above-mentioned two cell culture system to investigate the effect of overexpression of IRFs on the replication of HCV.We used Bac to Bac expression system to construct IRF recombinant baculovirus and transducted the recombinant baculovirus to the above-mentioned cell line. We explored the its possibility as a novle gene therapeutic drug.The results as the following:1. The effects of IRF7 and its deletion mutation (MR3) on the activity of IFNα6 promotor in Huh7 and replicon cells: seven plasmids that express mutant wild type IRF7 were constructed by overlap extension PCR. The expression of the desired mutant IRF7 was confirmed by western blot analysis. The mutant- expressing plasmids, pIFNα6-luc and pRL-TK reporter plasmids were cotransfected into Huh7 and replicon cells and luciferase activities were measured 20h after the transfection. The results showed that among the seven mutants, the effect of MR3 on the activity of IFNα6 promotor is the strongest. So we chosed MR3, wild type IRF3 and IRF7 in the following study. 2. Overexpression of MR3, wild type IRF3 and IRF7 and effects on HCV replication in the HCV subgenomic replicon: The expression plasmid of MR3, wild type IRF3 and IRF7 were transfected into replicon cells by FuGene6. 48h after the transfection, protein and RNA was extracted and the samples were processed for western blot and real time PCR respectively. The results showed that in contrast to the empty vector and wild type IRF3 and IRF7 expressing plasmid, the overexpression of MR3 resulted in a significant inhibition of the expression of HCV NS5A. in replicon cells. At the same time. there is a significantly higher IFN-β, TNF-αand IL-8 induction ratio by MR3 overexpression than that of wild type IRF3 and IRF7.3. Overexpression of MR3, wild type IRF3 and IRF7 and effects on HCV replication in the HCV-infected cells: 3 days after the viral RNA of JFH1 introduced into Huh7.5.1, according to the above-mentioned methods ,the expressing plasmids were transfected into HCV-infected cells. As positive control, we uses the IFN-α(200ng/ml) to treat the HCV-infected cells. 48h after the transfection, protein and RNA was extracted and the samples were processed for western blot and real time PCR respectively. The results showed that as negative control, NS5A was not detected in the HCV-uninfected cells. The expressioin of NS5A was significantly inhibited in the IFN-treated cells. Although the antiviral effect of overexpression of MR3 was weaker than that of IFN, the expression of NS5A was descreased in compare with that of wild type IRF3 and IRF7-transfected cells and untreated HCV-infected cells.4. Overexpression of MR3, wild type IRF3 and IRF7 and effect on IFN promoter activity: cotransfection of pCDNA3.1-IRF3/IRF7/MR3myc-His with IFNα6/IFNβ-luciferase reporter plasmids, pRL-TK-luc into Huh7 and replicon cells resulted in a significant increase of IFN activity in cells in which MR3, wild type IRF3 and IRF7 was overexpression. IRF3 is more potent in activating the IFN-βgene than the IFN-αgenes, whereas IRF7 and MR3 efficiently activates both IFN-βand IFN-α.The effect of MR3 is stronger than that of wild IRF7.5. The expression of the recombinant baculovirus containing MR3 coding sequence in Huh7 cells: the recombinant baculovirus was constructed by Bac-to-Bac recombinant baculovirus expression system. 48h after absorption infection, the expression of MR3 in Huh7 cells was detected by western blot. The results showed that six clones can efficiently express MR3 in Huh7 cells. We chosed the highest expression clone to produce large-scale expansion.6. The induction of innate immunity in Huh7 cell by baculovirus: To investigate wether bacurovirus is capable of stimulating antiviral activity in Huh7 cells, we used bacurovirus(AcCAGGFP) to infect Huh7 cells and detected the activity of IFN and associated cytokines by real-time PCR. The results showed that there was not obvious baculovirus-induced IFN and TNF-α, IL-8 expression in Huh7 cells.As control, the gene of GFP can be transducted into Huh7 cell and expressed.7. The trandsduction of recombinant baculovirus (AcVSVGCMV/ AcVSVGCMV-MR3) and effects on HCV replication in the HCV subgenomic replicon: 48h after the absorption infection of recombinant baculovirus, protein and RNA was extracted and the samples were processed for western blot and real time PCR respectively. The results showed that the overexpression of MR3 resulted in a significant dose-dependent inhibition of the expression of HCV NS5A in the replicon cells. At control, there was not obvious effect of AcVSVGCMV on the expression of HCV NS5A .8. The trandsduction of recombinant baculovirus (AcVSVGCMV/ AcVSVGCMV-MR3) and effects on HCV replication in the HCV-infected cells: 3 days after the viral RNA of JFH1 introduced into Huh7.5.1, according to the above-mentioned methods, recombinant baculovirus was absorption infected the HCV-infected cells. As positive control, we uses the IFN-α(200ng/ml) to treat the HCV-infected cells. 48h after the infection, protein and RNA was extracted and the samples were processed for western blot and real time PCR respectively. Similar to the results of above experiment, the overexpression of MR3 resulted in a significant dose-dependent inhibition of the expression of HCV NS5A in HCV-infected cells.9. The effect of MR3 recombinant baculovirus on IFN promoter activity: 24h after cotransfection of ISER/ IFNα6/IFNβ-luciferase reporter plasmids with pRL-TK-luc into Huh7 and replicon cells, cells were infected with recombinant baculovirus. Luc assay was preformed after 20h. the result showed that there was a significant dose-dependent increase of IFN activity in cells in which MR3 was overexpression.In summary, in this study, we firtst used IRF expressing plasmids to transfect HCV cells. The results showed tha the overexpression of IRF has effect on inhibiting the expression HCV NS5A and inducing the activity of IFN promotor. Then, we successfully constructed the recombinant baculovirus. With the introduction of baculovirus, we proved that the protein of IRF can express in the HCV cell and the overexpression of MR3 resulted in a significant dose-dependent inhibition of the expression of HCV NS5A and induction of activity of IFN promotor in HCV cells. Thus, the results indicate that IRF overexpression is able to bypass the HCV-mediated inhibition and restore the innate antiviral response and suggest that IRF recombinant baculovirus-based anti-HCV strategy may open a path toward to improve HCV therapy, and open a new avenue for treating chronic HCV infection.