SiRNA Effectively Inhibit Hepatitis C Virus Core Protein Expression

Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma. The HCV genome is a positive-stranded 9.6 kb RNA molecule consisting of a single ORF, which is flanked by 5'and 3'UTR. The HCV ORF encodes a single large precursor polyprotein that is 3,008-3,037 aa in length which is cleaved by both cellular and viral proteases into structural and nonstructural proteins and is posttranslationally modified to produce at least ten different proteins: core, envelope proteins El and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Now the combination therapy with IFN and ribavirin is used effectively on most chronically infected patients.Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.Core protein is encoded at the 5' -terminal region of the ORF and is composed of a basic, RNA-binding amino-terminal domain and a highly hydrophobic carboxy-terminal region. Mature 21 kDa HCV core protein is produced by cleavage between aa 191 and 192 of the precursor polyprotein. HCV core protein products of 19 and 16 kDa have further been described. The core protein is associated with the cytoplasmic side of the endoplasmic reticulum, but has also been detected in the nucleus. In addition to encapsidation of viral RNA, recent studies have suggested a possible role of the HCV core protein in host cell growth, apoptosis, and carcinogenesis. The transforming potential of the HCV core protein has been demonstrated in rat embryo fibroblasts and transgenic mice. Although it is likely thatthe HCV core protein has an important role in the regulation of cell growth, apoptosis, or carcinogenesis, the mechanisms by which the HCV core protein influences these processes remain to be determined.Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. MAPK is mitogen-activated protein kinase including MAPK/ERK, JNK, ERK5/BMK1 and p38. MAPK signal pathway plays an important role in mediating the intracellular and extrocelluar stimulation, the activation of different subfamily will present different effection such as cell proliferation, differentiation. The uncontrolled activation will lead to human hepatocellular carcinoma. MAPK/ERK play a role in cell proliferation and HCV core protein can activate it to influence cell proliferation.RNAi (RNAinterference) , originally discovered by Fire in 1998, is induced by double-strand RNA (dsRNA). In the process, dsRNA is cleaved into 21-23 nucleotide dsRNA molecules, known as small interfering RNA or siRNA, by an RNase Ill-like enzyme known as Dicer. These siRNA molecules, with the terminal of 5'-phosphate and 3'-hydroxy with two or three protruded ir-pairing bases, associate with a multiprotein complex known as the RNA-induced silencing complex (RISC) and targets homologous mRNA for degradation to silence the target gene, belongs to post-transcriptional gene silencing. Therefore, RNAi has been employed in the study of gene function and complex signal transduction pathways, and in the gene therapy of human diseases including cancers, neurogenerative diseases and infectious diseases. RNAi, as a new technology of silencing gene expression, has been successfully employed in inhibiting viral duplication in cultured cells such as HIV, HBV/HCV. Therefore, hepatitis C virus (HCV), single-stranded plus-sense RNA virus, can become the target of RNAi and resist the viral infection by the degradation of viral RNA. In this study, we cloned and expressed HCV core gene. Then we designed two siRNAs specific to core gene, EGFP-siRNA and scramble siRNA as positive and negative control respectively, and synthesized them with T7 RiboMAX? expression system in vitro. Our results demonstrated that the specific siRNAs corresponding to HCV core gene specifically degraded core mRNA, and significantly inhibited core protein expression.1* Expression of HCV core gene in HEK 293T cellsThe expression plasmid pEGFP-C was constructed which contains the full length of HCV core gene. Then HEK 293T cells were transfected with the plasmid pEGFP-C. After 48 hours, quantity and fluorescence intensity in the transfected cells were observed by fluorescence microscope, the expression of core protein but not the C-EGFP fusion protein was detected using Western blot. The possible reason is that the product was cleaved by some sensitive protein enzyme. The possible reason remain to be determined.2, Silencing of core gene by siRNA in HEK 293T cellsWe adopted transcription in vitro with T7 RNA polymerase to acquire siRNAs. Firstly, the sense and antisense of seven siRNAs (C-siRNAl, C-siRNA2, C-siRNA3, C-siRNA4, C-siRNA5, scramble siRNA and EGFP-siRNA) templates and T7 promoter primer template were synthesized. The sense and antisense of siRNA were separately transcribed in vitro with T7 RNA polymerase and annealed to form double-strand siRNA. The 5' overhanging leader sequence of the generated dsRNA and DNA template remainders were digested with single-strand specific nuclease (SI nuclease) and RNase-free DNase I, respectively. The double stranded siRNAs of 21 nt in length were obtained. Then the siRNAs and pEGFP-C were co-transfected into HEK 293T cells. After 48 hours, green fluorescence was observed using fluorescence microscope, C-siRNAl and C-siRNA2 were primary screened;To detect the quantity and fluorescence intensity of EGFP in the transfected cells, samples were counted and analyzed with CellQuest software, using non-transfected HEK 293T cells as control. The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population;Total RNA from the transfected or non-transfected cells was extracted and semi-quantitative RT-PCR and Real-time PCR were performed using HCV core and GAPDH gene specific primers after being reverse transcribed into cDNA. The expression of HCV core protein was detected by Western blot. The results demonstrated that compared with the cells transfected with pEGFP-C alone (the percentage of fluorescence cells is 43.35% and mean fluorescence intensity is 110.68), the cells co-transfected scramble siRNA gave no significant reduction of EGFP expression, while the EGFP-siRNA gave an about 3.9-and 2.5-fold reduction in percentage of fluorescence cell population and meanfluorescence intensity respectively. Percentage of fluorescence cell population and mean fluorescence intensity reduced about 3.5- and 1.8-fold for C-siRNAl and 2.9-and 3.2-fold for C-siRNA2. Compared with the cells transfected with pEGFP-C alone, the transcript levels of HCV core gene were decreased about 13- and 9-fold in cells co-transfected with C-siRNAl and C-siRNA2 respectively. Correspondingly, HCV core transcript levels were decreased about 15-fold in cells co-transfected with EGFP-siRNA but not significantly changed in cells co-transfected with scramble siRNA, while GAPDH transcript level was not affected by the four siRNAs. HCV core protein was expressed at high level in cells transfected with pEGFP-C alone, similar to those co-transfected with scramble siRNA. The HCV core protein expression levels were obviously decreased in cells co-transfected with EGFP-siRNA, C-siRNAl and C-siRNA2 for their weak bands, while the siRNAs had no effect on the expression of GAPDH. These results demonstrated that siRNA can inhibit the target protein expression effectively and specifically through down-regulating the transcript level of target gene.We detected the MAPK/ERK phosphorylation by Western blotting after transfection of pEGFP-Nl and pEGFP-C in HEK 293T cells. The results showed that pEGFP-Nl had no effection but pEGFP-C activate the phosphorylation of MAPK/ERK. It is to say that using transient transfection we can detect the activated effection of HCV core. We combine the RNAi and signal transduction detecting the change of MAPK/ERK phosphorylation before and after RNAi. RNAi can decrease the MAPK/ERK phosphorylation after the interference of HCV core protein by siRNA.Summary:1 > In this study, the HCV core expression plasmid pEGFP-C was constructed which contains the full length of HCV core gene, while EGFP gene was reporter gene. HEK 293T cells were transfected with this plasmid. Western blot analysis showed that the plasmid could be expressed in the cells and the core protein was detected2> In this study, five siRNAs specific to HCV core gene, EGFP-siRNA (positive control) and scramble siRNA (negative control) were designed and synthesized with T7 RiboMAX? expression system in vitro. The cells were co-transfected with the plasmid of pEGFP-C and siRNAs.The results demonstrated that two of the fivesiRNAs could inhibit the expression of HCV core protein effectively and specifically. HCV core protein can activate the MAPK/ERK phosphorylation. This effection was decreased after RNAi by the siRNAs which specific to HCV core.