HCV Core Protein Suppresses RNA Silencing Induced By Shrna In Mammal Cells

RNA silencing is a form of nucleic acid-based immunity against viruses in plants and invertebrate animals. Successful infection requires evasion or suppression of gene silencing. Here we report that the core protein of Hepatitis C virus is a potent silencing suppressor that antagonist RNA silencing. Core protein could inhibit RNA silencing induced by short hairpin RNA but not by synthetic siRNA in various mammal cells. Furthermore, a direct interaction between core protein and Dicer, an RNase enzyme that generates siRNA in host cells, was observed. In vitro, core protein inhibited the function of Dicer to process double-strained RNAs into siRNAs. Moreover, the N-terminal domain was found to be essential for core protein to abrogate RNA silencing by mapping study. Thus our study demonstrated that core protein abrogated cell's RNA silencing defense by subverting the ability of Dicer to process precursor dsRNA into siRNAs. This ability of core protein may contribute to the persistent viral infection and the pathogenesis of HCV. Objective: Establish a cell line stably expressing EGFP and construct the plasmid expressing short hairpin RNA targeting EGFP. Assess the down-regulation effect of intrinsic EGFP by vector-derived shRNA.Methods Transfected the HepG2 cells with pEGFP-N1 and selected the cells stably expressing EGFP by adding G418 into the culture medium. The cells with stringent green fluorescence were picked out and amplified. The integrity of EGFP gene into cell chromosome was confirmed by PCR using genome DNA as template. The interfering plasmid pshRNA-GFP was constructed by inserting the reverse repeated sequence corresponding to EGFP into pTZU6+1. The inserted sequence was confirmed by DNA sequencing. Cells of HepG2.GFP were transfected with pshRNA-GFP, pshRNA-HCV and pTZU6+1 by liposome, respectively. Fluorescences of transfected cells were observed with fluorescent microscope. The expression of GFP was assayed by western blot and RT-PCR, respectively.Results The EGFP gene was detected in the genome DNA of HepG2.GFP by PCR; pshRNA-GFP inhibieted the expression of intrinsic GFP significantly.Conclusion A cell line stably expressing EGFP was established successfully. The vector-derived shRNA inhibited the expression of intrinsic GFP efficiently. Objective: To establish a quantitive RNA interference system targeting luciferase gene that could be used to study RNA silence mechanism.Methods siRNA was designed and synthesized according to documents. shRNA expressing plasmid (pshRNA-Luc) was constructed by inserting the corresponding reverse repeated DNA sequence into an eukaryotic plasmid containing U6 promoter. Dual-luciferase vector (pLuc-FR) expressing firefly luciferase and renilla luciferase at the same time was constructed by inserting renilla luciferase sequence into PGL3 control plasmid. Co-transfected the siRNA or pshRNA-Luc with pLuc-FR into HepG2 cells. The activities of luciferase were assayed by dual luicferase assay system.Results Both the synthesized siRNA and vector-derived shRNA were shown to efficiently and specifically down-regulated the expression of the target gene.Conclusion A RNAi system targeting firefly luciferase was established successfully. It could be used to study the mechanism of RNAi.Ⅰc P19 of tomato bushy stunt virus suppresses RNA silencing induced by short hairpin RNA in mammal cellsobjective: RNA interference (RNAi) is a unique RNA-guided antiviral mechanism that operates in a wide range of eukaryotic organisms from plants to mammals. To counteract this immune system in parasitic host, some viruses encode proteins to suppress the RNAi effect. In this report, to test the ability for screening RNA silencing suppressors of the two RNAi systems in mammal cells established in our former work, P19 of tomato bushy stunt virus was studied.Methods The shRNA expressing vectors were co-transfected with plasmids containing target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by dual-luciferase assay system.Results when P19 was introduced into the RNAi systems, both the expression of GFP and the luciferase activity were mostly recovered compared with the control groups.Conclusion P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells. the two RNAi systems of GFP and luciferase were useful for screening RNA silencing suppressors.PartⅡHCV core protein inhibits RNA silencing induced by shRNA in mammal cellsObjective: To identify the potent RNA silencing suppressor encoded by Hepatitis C virus.Methods Plasmids expressing various proteins encoded by Hepatitis C virus were constructed by inserting the corresponding DNA sequence into pCDNA3.1-3FLAG. The expression of proteins in HepG2 cells were confirmed by immunofluoresnce staining. The various proteins were added into the RNA interference system targeting firefly luciferase gene. Luciferase activity was assayed by dual luciferase assay system. The potent RNA silencing suppressor was further confirmed using an RNA interference system targeting intrinsic EGFP. The suppressing effect was observed in other two non-liver cell lines and the dose dependent effect was studied. The essential domains were identified by deletion mutation mapping study. The effect on siRNA of the potent suppressor was studied using a RNA silence system targeting luciferase induced by presynthesized siRNA.Results The various proteins expressing plasmids were constructed successfully. The core proteins rescued both extrinsic luciferase activity and intrinsic EGFP expression level which were silenced by shRNA in HepG2 cells. This suppressing effect acted in a dose-dependent manner. When the N terminal of core protein was deleted, it could not rescue the luciferase activity whereas the deletion of C terminal did not impact its suppressing function. Furthermore, core protein did not rescue luciferase activity silenced by synthesized siRNA.Conclusions HCV core protein could act as a potent RNA silencing suppressor. The N terminal was essential for this suppressing function. The effect of suppressing was not due to counteract siRNA. This ability of core protein might contribute to the persistent viral infection and the pathogenesis of HCV. Objectives: To study the interaction between HCV core protein and Dicer enzyme.Methods Core protein was expressed in mammal cells and the cell lysis was subjected to perform Immunoprecipitation test. dsRNA was prepared by transcription in vitro and used to test the dicing function of recombinant Dicer enzyme. Later, core protein was added into the dicing system to test its influence on the function of Dicer.Results A Dicer bind was observed by specific antibody against Dicer in lysate from the mammal cells transfected with plasmid expressing core protein. dsRNA could be processed into～21nt siRNA by the recombinant Dicer enzyme. Adding of recombinant core protein abrogated the dicing function of Dicer and inhibited the production of siRNA.Conclusions Core protein could combine Dicer and inhibit its dicing function . In this way, core protein antagonisted RNAi in mammal cells.