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.     

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.     

Interference of hepatitis C virus RNA replication by short interfering RNAs

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFNalpha in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.     

Antiviral therapy: inhibition of Hepatitis C Virus expression by RNA interference directed against the NS5B region of the viral genome

Background: Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-a) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells). Methods: We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay. Results: Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells. Conclusions: Synthetic siRNA against NS5B-HCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.     

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.     

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.     

Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors

BACKGROUND/AIMS: Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR). METHODS: In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells. RESULTS: PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells. CONCLUSIONS: A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.     

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury

Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.     

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]     

Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA

Background and Aim:  We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro . In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo .
Methods:  We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo .
Results:  Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by ∼10⁻³. Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/ lox P switching system resulted in specific suppression of virus protein synthesis in the liver.
Conclusion:  Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.     

A cell-based, high-throughput screen for small molecule regulators of hepatitis C virus replication

BACKGROUND & AIMS: Only half of patients with chronic hepatitis C virus (HCV) infection experience sustained virologic response to pegylated-interferon and ribavirin, which cause numerous side effects. Thus, the identification of more effective and better tolerated agents is a high priority. We applied chemical biology to screen small molecules that regulate HCV. METHODS: We first optimized the Huh7/Rep-Feo replicon cell line for the 384-well microplate format and used this line to screen a large library of well-characterized, known biologically active compounds using automated technology. After identifying several molecules capable of either stimulating or inhibiting HCV replication in this primary screen, we then validated our hit compounds using a full-length HCV replicon cell line in secondary screens. RESULTS: We identified and validated a number of antiviral and proviral agents, including HMG-CoA reductase inhibitors (antiviral) and corticosteroids (proviral). The finding of increased replication associated with corticosteroids suggests that these agents directly promote viral replication independent of their suppressive effects on the immune response. The finding of antiviral activity associated with the HMG-CoA reductase inhibitors implies an important role for lipid metabolism in the viral life cycle. CONCLUSIONS: We have developed a simple, reproducible, and reliable cell-based high-throughput screening assay system using an HCV replicon model to identify small molecules that regulate HCV replication. This method can be used to identify not only putative antiviral agents, but also cellular regulators of viral replication.     

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.     

Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line

Interferon alfa (IFN-alpha)-based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN-alpha antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN-induced direct antiviral activity in hepatocytes. In this report, we show that IFN-alpha can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN-alpha can induce a number of responsive genes in the replicon cells. One of the genes, 6-16 (G1P3), can enhance IFN-alpha antiviral efficacy. In addition, we demonstrate that IFN-alpha can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN-alpha signaling. In conclusion, our results indicate that IFN-alpha antiviral activity is associated with activation of STAT3-signaling pathway and intracellular gene activation. Our results also suggest that IFN-alpha-induced target genes may play an important role in IFN-alpha anti-HCV activity.     

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.     

Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.