Inhibition of HCV subgenomic replicons by siRNAs derived from plasmids with opposing U6 and H1 promoters

Hepatitis C virus (HCV) is a main cause of chronic liver disease, which may lead to the development of liver cirrhosis and hepatocellular carcinoma. Therapeutic options are still limited in a significant proportion of patients. Small interfering RNAs (siRNAs) are an efficient tool to inhibit gene expression by RNA interference. As HCV RNA replicates in the cytoplasm of liver cells without integration into the genome, RNA-directed antiviral strategies are likely to successfully block its replication cycle. In this study, a panel of siRNAs was used to target various important regions of the HCV genome [5' untranslated region (UTR), NS3, NS4A, NS4B, NS5B, 3' UTR]. Convergent opposing human H1 and U6 polymerase III promoters were used to generate siRNAs. Target genes in sense and antisense orientation were attached to a luciferase reporter system to test the inhibitory efficiency of both siRNA strands. Our data revealed effective RNA interference against the HCV(+)-strand, the HCV(-)-strand or both strands simultaneously up to 65%. Subsequently, active siRNAs were tested in HCV subgenomic replicon cells and suppression of HCV RNA and NS5B protein levels up to 75% was confirmed. Interestingly, siRNAs that were effective against the sense as well as the antisense strand revealed the greatest inhibitory effects on HCV subgenomic replicons. Additionally, combinations of siRNAs induced a greater inhibition of HCV subgenomic replication of up to 90% proving the potential of this combined antiviral approach.     

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.     

含EGFP报告基因单顺反子HCV亚基因组复制子载体的构建和表达

目的构建含增强型绿色荧光蛋白(EGFP)报告基因的HCV复制子表达载体,并实现其在细胞中的复制表达。方法用分子生物学基因克隆技术对HCV 2a型复制子的基因进行改造,用EGFP基因替代HCV基因组中的包膜基因(E1和E2)体外构建重组单顺反子HCV亚基因组复制子真核表达质粒pcDNA-JFH1-EGFP,经限制性内切酶酶切分析和测序鉴定;脂质体介导转染人肝癌细胞系Huh-7细胞,用荧光显微镜观察EGFP表达,采用半定量RT-PCR方法检测重组复制子的HCV RNA负链,采用Western blot检测HCV NS3蛋白的复制表达,并观察IFN-α对重组质粒表达的HCV RNA复制的抑制作用。结果构建的4个重组质粒酶切分析与预期相符,HCV亚基因复制子表达载体中未发生EGFP和HCV编码区读码框架改变,转染重组载体Huh-7细胞检测到HCV负链及EGFP和HCV NS3蛋白表达。转染后48h,1 000IU/ml和2 000IU/ml IFN-α处理的细胞HCV RNA表达水平分别为未处理组的20.0%和7.6%。结论含EGFP报告基因的单顺反子HCV亚基因组复制子表达载体pcDNA-JFH1-EGFP构建成功,在Huh-7细胞中能有效复制表达,为进一步研究HCV提供了实验平台。     

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.     

An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain

Aim: The hepatitis C virus (HCV) strain JFH‐1 was cloned from a patient with fulminant hepatitis. A JFH‐1 subgenomic replicon and full‐length JFH‐1 RNA efficiently replicate in cultured cells. In this study, an infectious, selectable HCV replicon containing full‐length JFH‐1 cDNA was constructed. Methods: The full‐genome replicon was constructed using the neomycin‐resistant gene, EMCV IRES and wild‐type JFH‐1 cDNA. Huh7 cells were transfected with RNA synthesized in vitro, and then cultured with G418. Independent colonies were cloned to establish cell lines that replicate the full‐length HCV replicon. Results: HCV RNA replication was detected in each isolated cell line. HCV proteins and HCV RNA were secreted into culture medium, and exhibited identical density profiles. Interestingly, culture supernatants of the replicon cells were infectious for naïve Huh7 cells. Long‐term culture did not affect replication of replicon RNA in the replicon cells, but it reduced core protein secretion and infectivity of culture supernatant. Culture supernatant obtained after serial passage of replicon virus was infectious for Huh7 cells. Conclusions: Selectable infection was established using HCV replicon containing full‐length genotype 2a JFH‐1 cDNA. This system might be useful for HCV research.     

Roles for endocytic trafficking and phosphatidylinositol 4-kinase III alpha in hepatitis C virus replication

Hepatitis C virus (HCV) reorganizes cellular membranes to establish sites of replication. The required host pathways and the mechanism of cellular membrane reorganization are poorly characterized. Therefore, we interrogated a customized small interfering RNA (siRNA) library that targets 140 host membrane-trafficking genes to identify genes required for both HCV subgenomic replication and infectious virus production. We identified 7 host cofactors of viral replication, including Cdc42 and Rock2 (actin polymerization), EEA1 and Rab5A (early endosomes), Rab7L1, and PI3-kinase C2gamma and PI4-kinase IIIalpha (phospholipid metabolism). Studies of drug inhibitors indicate actin polymerization and phospholipid kinase activity are required for HCV replication. We found extensive co-localization of the HCV replicase markers NS5A and double-stranded RNA with Rab5A and partial co-localization with Rab7L1. PI4K-IIIalpha co-localized with NS5A and double-stranded RNA in addition to being present in detergent-resistant membranes containing NS5A. In a comparison of type II and type III PI4-kinases, PI4Ks were not required for HCV entry, and only PI4K-IIIalpha was required for HCV replication. Although PI4K-IIIalpha siRNAs decreased HCV replication and virus production by almost 100%, they had no effect on initial HCV RNA translation, suggesting that PI4K-IIIalpha functions at a posttranslational stage. Electron microscopy identified the presence of membranous webs, which are thought to be the site of HCV replication, in HCV-infected cells. Pretreatment with PI4K-IIIalpha siRNAs greatly reduced the accumulation of these membranous web structures in HCV-infected cells. We propose that PI4K-IIIalpha plays an essential role in membrane alterations leading to the formation of HCV replication complexes.     

Inhibition of subgenomic hepatitis C virus RNA replication in HeLa cells

BACKGROUND/AIMS: Antiviral therapy such as combination interferon and ribavirin can eradicate hepatitis C virus (HCV) RNA by up to 40-50%. However, many patients still remain non-responders to this treatment for various reasons. The aim of this study was to evaluate the effect of interferon or ribavirin treatment on subgenomic HCV RNA replication in 'non-hepatic' HeLa cells. METHODOLOGY: Huh-7 or HeLa cells harboring HCV replicon were constructed by using cellular RNA of Huh-7 harboring HCV replicon RNAs, named as C13-3 cells. We also tested whether interferon or ribavirin can suppress HCV RNA in HeLa cells. RESULTS: Huh-7 or HeLa cells harboring HCV replicon RNAs were constructed by using cellular RNA of C13-3 cells than using in vitro-transcribed RNA. Ribavirin at 1 microg/mL or 10 microg/mL did not suppress colony formation in HeLa cells, but at 100 microg/mL suppression was observed. Interferon-alpha 2b suppressed HCV replication even at 1 U/mL. CONCLUSIONS: HeLa cells harboring HCV replicon RNAs also might be useful for the development of antiviral drugs.     

Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy

Background

Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine.

Methods

Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-α (IFNα) was evaluated.

Results

The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNα enhanced the antiviral effect of IFNα and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNα but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine.

Conclusion

The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.     

CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity

The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.     

RNAi: a powerful tool to unravel hepatitis C virus-host interactions within the infectious life cycle

Cellular cofactors affecting hepatitis C virus infection and replication. Randall G, Panis M, Cooper JD, Tellinghuisen TL, Sukhodolets KE, Pfeffer S, Landthaler M, Landgraf P, Kan S, Lindenbach BD, Chien M, Weir DB, Russo JJ, Ju J, Brownstein MJ, Sheridan R, Sander C, Zavolan M, Tuschl T, Rice CM. Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target DICER, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target DICER inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh7.5 cells, and Huh7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV. [Abstract reproduced by permission of Proc Natl Acad Sci USA 2007;104:12884-12889]     

Griseofulvin, an oral antifungal agent, suppresses hepatitis C virus replication in vitro

Aim: Hepatitis C virus (HCV), which infects an estimated 170 million people worldwide, is a major cause of chronic liver disease. The current standard therapy for chronic hepatitis C is based on pegylated interferon (IFN)alpha in combination with ribavirin. However, the success rate remains at approximately 50%. Therefore, alternative agents are needed for the treatment of HCV infection. Methods: Using an HCV-1b subgenomic replicon cell culture system (Huh7/Rep-Feo), we found that griseofulvin, an oral antifungal agent, suppressed HCV-RNA replication and protein expression in a dose-dependent manner. We also found that griseofulvin suppressed the replication of infectious HCV JFH-1. A combination of IFNalpha and griseofulvin exhibited a synergistic inhibitory effect in Huh7/Rep-Feo cells. Results: We found that griseofulvin blocked the cell cycle at the G(2)/M phase in the HCV subgenomic replicon cells, but did not inhibit HCV internal ribosome entry site-dependent translation. Conclusion: Our results suggest that griseofulvin may represent a new approach to the development of a novel therapy for HCV infection.     

Cellular cofactors affecting hepatitis C virus infection and replication

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.     

Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of approximately 22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs. These data support the principle of siRNA-based HCV antiviral therapy.     

Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes

BACKGROUND & AIMS: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. METHODS: We transfected antagomir of miR-122, 2'-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). RESULTS: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2'-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 mumol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. CONCLUSIONS: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.     

Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response

BACKGROUND & AIMS: Persistent infection with hepatitis C virus (HCV) leads to chronic hepatitis and hepatocellular carcinoma (HCC). RNA interference (RNAi) may act as a host antiviral response against viral RNA. METHODS: The effects of RNAi on both the replicative intermediates and the internal ribosome entry site (IRES) of HCV were studied by using HCV-related short interfering RNA (siRNA) detection assay. The mechanism that permits HCV to escape RNAi was studied by using RNAi assay materials. RESULTS: These studies demonstrate that the Dicer, an RNase enzyme that generates short siRNA, can target and digest both the IRES and the replicative intermediate of HCV into siRNA of approximately 22 nucleotides. Further studies also show that Dicer can inhibit the replication of the HCV subgenomic replicon. However, the HCV core protein inhibits this RNAi and rescues the replication of the HCV subgenomic replicon through a direct interaction with Dicer. CONCLUSIONS: RNAi is a limiting factor for HCV infection, and the core protein suppresses the RNA silencing-based antiviral response. This ability of the core protein to counteract the host defense may lead to a persistent viral infection and may contribute to the pathogenesis of HCV.