HCV Replicon Research Articles

Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line

The Hepatitis C virus (HCV), a member of the family Flaviviridae, is a major cause of chronic liver disease. Patients are currently treated with alpha interferon (IFN-alpha) that is given alone or in combination with ribavirin. Unfortunately, this treatment is ineffective in eliminating the virus in a large proportion of individuals. IFN-induced antiviral activities have been intensively studied in the HCV replicon system. It was found that both IFN-alpha and IFN-gamma inhibit HCV replicons, but the underlying mechanisms have not yet been identified. Of note is that nearly all of these studies were performed with the human hepatoma cell line Huh-7. Here, we report that genotypes 1b and 2a replicons also replicate in the human hepatoblastoma cell line HuH6. Similar to what has been described for Huh-7 cells, we observed that efficient HCV replication in HuH6 cells depends on the presence of cell culture-adaptive mutations and the permissiveness of the host cell. However, three major differences exist: in HuH6 cells, viral replication is (i) independent from ongoing cell proliferation, (ii) less sensitive to certain antiviral compounds, and (iii) highly resistant to IFN-gamma. The latter is not due to a general defect in IFN signaling, as IFN-gamma induces the nuclear translocation of signal transducer and activator of transcription 1 (STAT1), the enhanced transcription of several IFN-regulated genes, and the inhibition of unrelated viruses such as influenza A virus and Semliki Forest virus. Taken together, the results establish HuH6 replicon cells as a valuable tool for IFN studies and for the evaluation of antiviral compounds.

Trans-complementation of HCV replication by non-structural protein 5A

Although establishment of the subgenomic replicon system has considerably facilitated genetic analysis of HCV replication, many details remain largely unknown. To initially test whether HCV replication could be affected in trans, complementation studies were conducted in which defective replicon RNAs carrying a luciferase reporter were introduced into stable cells bearing functional replicons. The NS3 protease and the NS5B viral polymerase genes on the transfected replicons were rendered null by active site mutations and shown not to be complemented in trans by functional proteins expressed from the endogenous replicons. A new strategy was also developed to examine whether adapted copies of NS4B and NS5A could enhance the replication of transfected replicons carrying non-adapted genes. The replication efficiency of a replicon carrying two adaptive mutations in NS3 (E1202G and T1280I) had previously been shown to be greatly enhanced by the presence of a third mutation in either NS4B (K1846T) or NS5A (S2197P). A partially adapted luciferase replicon carrying only the two NS3 mutations was used to transfect cells containing replicons bearing the adapted NS4B or NS5A. Using this approach, NS5A, but not NS4B, was found to trans-complement. In a final confirmatory study, ectopically expressed NS5A also complemented the HCV replicon genome bearing the non-adapted NS5A. These studies strongly suggest that HCV non-structural proteins, with the exception of NS5A, can only act in cis on the RNA from which they were translated.

Suppression of HCV RNA replication by baculovirus-mediated shRNA expression

We describe a HCV core-short-hairpin RNA (shRNA) expressed from a human U6 Pol II promoter, which synergistically enhanced the inhibition of HCV replication in HCV replicon. We also describe the construction of recombinant baculovirus expressing shRNAs of the HCV core gene under the control of the U6 promoter. The anti-HCV shRNAs may provide a practical basis for applying shRNA-based gene therapy to the treatment of HCV.

Design, Synthesis, and Antiviral Activity of 2‘-Deoxy-2‘-fluoro-2‘-C-methylcytidine, a Potent Inhibitor of Hepatitis C Virus Replication

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.

Synthesis of 5-haloethynyl- and 5-(1,2-dihalo)vinyluracil nucleosides: Antiviral activity and cellular toxicity

In this article, we report the synthesis of hitherto unknown 5-haloethynyl and 5-(1,2-dihalo)vinyluracil nucleosides in the 2′-deoxy, 3′-deoxy- and ribosyl series, and we discuss their in vitro anti-HIV and anti-HCV activities and cellular toxicitites. As a result, on the basis of their selectivity index (SI) obtained with the HCV replicon system, but also on their cytotoxicity on peripheral blood mononuclear, CEM and VERO cell lines, the best compounds were the 5-bromoethynyluridine (SI = 3.2) and the 5-(1-chloro-2-iodo)vinyluridine (SI > 2.8).

983. Inhibition of Hepatitis C Virus Replication by In Vitro Selected Specific RNA Aptamers

Top of pageAbstract Hepatitis C virus (HCV) is the main etiological agent of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Although the HCV infection causes worldwide health problem, efficient and specific antiviral therapy has not yet been developed. Thus, development of more specific and effective therapeutics against HCV is warranted. The RNA dependent RNA polymerase (NS5B) of HCV plays a central role in the viral replication. Therefore, NS5B is a good target for anti-HCV agents. Characteristics of RNAs which can adopt complex structures to bind target proteins with high affinities and encode amplificable genetic information make RNA a potentially very useful diagnostic and/or therapeutic lead compounds. Such short RNA ligands, termed RNA aptamers, have been identified from a random RNA library to bind several proteins with high affinity and specificity using in vitro selection techniques, called systemic evolution of ligands by exponential enrichment (SELEX). In the present study, we isolated RNA aptamers to NS5B from a combinatorial RNA library using SELEX techniques and analyzed the biochemical and biological functions of the RNA aptamers. The RNA aptamers were found to specifically and avidly bind NS5B with an apparent Kd of 1.3|[sim]|4 nM. Through enzymatic probing and footprinting analysis, critical regions of the RNAs for NS5B binding have been identified. Moreover, in vitro RNA synthesis by NS5B was significantly inhibited by the RNA aptamers. Interestingly, the RNA aptamers can be utilized as substrates for de-novo RNA synthesis by NS5B. Specific interaction between the RNAs and NS5B was confirmed by Maldi-Tof analysis of the aptamer-bound proteins in liver cells with HCV replicon. Importantly, the replication of HCV replicon in liver cells was efficiently inhibited by cytoplasmic expression of the RNA aptamers, but not by nuclear expression of the RNAs. In addition, we showed that these aptamers could function as decoys in HCV replicon cells, explaining the mechanism underlying inhibition phenomena of HCV replicon replication observed in cells by the specific RNA aptamers against HCV NS5B. These results suggest that the RNA aptamers could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV replication mechanism.

997. Loop Composition as Well as Orientation of the Guide Strand in shRNA May Dramatically Modulate Activity of the shRNA: Effect on the Development of RNAi-Based Inhibitors

RNA interference (RNAi) is a new promising technology for therapeutic drug development. It can be initiated either by chemically synthesized double-stranded RNA delivered to a cell, or by transcription of a short hairpin RNA in the cell nucleus from a DNA template. It has been realized that many RNA sequences although |[ldquo]|active|[rdquo]| as siRNAs, are often inactive as shRNAs. The composition of the loop sequence in shRNA has been suggested to play a role in determining its activity by affecting shRNA transport from the cell nucleus to the cytoplasm (Kawasaki and Taira, Nucleic Acids Res. 2003, 31(2):700-7). In the present study we identified several siRNA sequences that efficiently inhibit HCV replication in the replicon system. In subsequent attempts to generate active shRNAs spanning these validated sequences, we tested the effect of two different loop sequences, as well as the guide strand orientation on the shRNA activity. Functional tests were performed in the HCV replicon systems, as well as in a model luciferase-HCV fusion systems. We demonstrate that the context of the loop within the shRNA has a dramatic effect on shRNA activity, and a loop that is |[ldquo]|inactive|[rdquo]| in one shRNA can be fully active in the context of another shRNA. We also demonstrate that the orientation of the guide strand in the shRNAs (sense vs antisense) may dramatically influence the shRNA activity. Changes in the extent and precision of the shRNA processing by Dicer will be presented. The effect of these findings on the development of ddRNAi-based inhibitors will be discussed.

441 Combinations of hairpin ribozymes and siRNAs targeting highly conserved HCV regions strongly inhibit RNA replication and translation from subgenomic HCV replicons

441 Combinations of hairpin ribozymes and siRNAs targeting highly conserved HCV regions strongly inhibit RNA replication and translation from subgenomic HCV replicons

462 Inhibition of JAK/STAT3 signalling leads to apoptosis of human hepatoma cells expressing a HCV replicon through ER stress-induced mechanisms

462 Inhibition of JAK/STAT3 signalling leads to apoptosis of human hepatoma cells expressing a HCV replicon through ER stress-induced mechanisms

SYNTHESIS AND IN VITRO ANTI-HCV ACTIVITY OF β-D- and L-2′-DEOXY-2′-FLUORORIBONUCLEOSIDES

Based on the discovery of β-D-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of β-D- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2′-fluoro group was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely β-D-2′-deoxy-2′,5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As β-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2′-fluoro were combined into one molecule, yielding β-D-2′-deoxy-2′-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

Background & Aims Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV. Methods HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined. Results pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture. Conclusions These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.

Synthesis and anti-viral activity of a series of d- and l-2′-deoxy-2′-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2′ R)- d-2′-deoxy-2′-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2′-deoxy-2′-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2′-fluoro group, was achieved by either fluorination of 2,2′-anhydronucleosides with hydrogen fluoride–pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2′ R)- d-2′-deoxy-2′,5-difluorocytidine ( 13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue ( 25) exhibited inhibition of ribosomal RNA. As N 4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N 4-hydroxyl and the 2′-fluoro into one molecule, resulting (2′ R)- d-2′-deoxy-2′-fluoro- N 4-hydroxycytidine ( 23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.

Analysis of the 5’ End Structure of HCV Subgenomic RNA Replicated in a Huh7 Cell Line

Objective: Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5′ end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate. Methods: HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5′ end. Results: Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase. Conclusion: It is suggested that the 5′ end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5′ end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5′ end. This result indicates that the 5′ end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.

Establishment of authentic HCV replication systems for drug screening

Abstract Although the interferon‐based therapeutics for chronic hepatitis C has improved remarkably over the decades, in some groups of patients the current therapeutic efficacy is far from satisfactory. Although new therapeutics is vigorously sought, the lack of appropriate cell culture systems and small animal models greatly hindered the progress of drug development. By introducing a selection marker into the HCV subgenomes, several successful replicon constructs were established. HCV replicons harboring adaptive mutations confer markedly increased efficiencies of replication. Furthermore, Huh‐7 cells carrying replicon that contained the whole genome were established but these cells were unable to secrete infectious viral particles. By use of SCID mice carrying a plasmogen activator transgene, an animal model carrying a chimeric human liver was established. These mice are permissive for HCV infection and the viral passages can be achieved successfully. However, the accessibility of this system is limited. By use of a molecular screening strategy, a hepatic factor, Sip‐L, is found to be capable of supporting HCV replication in an otherwise non‐permissive cell line. Potentially, this factor may be used to promote the infectivity and replication in other known permissive cell lines. An ex vivo assay using human liver biopsy slices has been established. This method can be used to test new anti‐HCV agents. In conclusion, although much progress has been made, culture cells permissive for highly efficient HCV infection are still lacking. An easily accessible small animal model is still in need. Much effort is still needed to establish an efficient and easily accessible authentic HCV replication system.

Synthesis of 9-(2-β- C-methyl-β- d-ribofuranosyl)-6-substituted purine derivatives as inhibitors of HCV RNA replication

A series of 9-(2′-β- C-methyl-β- d-ribofuranosyl)-6-substituted purine derivatives were synthesized as potential inhibitors of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was used to further improve the potency of these compounds by increasing the intracellular levels of 5′-monophosphate metabolites. These nucleotide prodrugs showed much improved inhibitory activities of HCV RNA replication.

Enhancement of antiviral activity against hepatitis C virus in vitro by interferon combination therapy

Alpha, beta, and gamma interferons (IFN-α, IFN-β, IFN-γ) have been shown to be effective inhibitors of HCV replication in human cell lines carrying HCV replicons. To help define the divergent cellular processes involved in the control of intracellular HCV replication by these agents, we have characterized the activity of monotherapies and combination therapies with the major types of human interferons against HCV replication in the HCV replicon-containing cell line, AVA5. IFN-α, IFN-β, and omega interferon (IFN-ω) were equally effective at inhibiting HCV replication, while IFN-γ was approximately 10-fold more potent. In kinetic experiments, IFN-β and IFN-γ inhibited HCV replication more rapidly, and for a more prolonged period following the removal of treatment, than IFN-α. Combination interferon therapies produced enhanced anti-HCV activity in most cases, and displayed a diverse range of interactions. Mixtures of IFN-α and IFN-β exhibited generally additive to slightly antagonistic interactions, IFN-α or IFN-β combined with IFN-ω were strongly antagonistic, while IFN-α/IFN-γ and IFN-β/IFN-γ combinations displayed the most enhanced and strongly synergistic antiviral effects. Simultaneous administration of interferons in the combination treatments was found to be superior to sequential administration. Ribavirin did not exhibit any selective anti-HCV activity in cell culture, consistent with in vivo monotherapies, and did not influence the effectiveness of IFN-α in combination treatments. A panel of human cytokines and immune response modifiers induced by interferon and ribavirin therapies in vivo did not demonstrate anti-HCV activity in HCV replicon-containing cultures. Combination therapy can be effectively modeled using HCV replicon technology yielding potentially more effective treatment regimens. HCV replicon technology has potential utility in designing combination therapies to significantly enhance the anti-HCV activity of IFN-α.

RNAi expression mediated inhibition of HCV replication

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Synthesis of New 2′‐β‐C‐Methyl Related Triciribine Analogues as anti‐HCV Agents.

Ten new beta-D-ribofuranosyl and 2'-beta-C-methyl-beta-D-ribofuranosyl triciribine derivatives 4-13 with various N4 and 6-N substituents on the tricyclic ring were synthesized from the corresponding toyocamycin and new 2'-beta-C-methyl toyocamycin derivatives. The inhibitory studies of these compounds in the HCV replicon assay reveal that some of them possess interesting anti-HCV properties with low cytotoxicity.

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Synthesis of new 2 ′-β- C-methyl related triciribine analogues as anti-HCV agents

Ten new β- d-ribofuranosyl and 2 ′-β- C-methyl-β- d-ribofuranosyl triciribine derivatives 4– 13 with various N4 and 6- N substituents on the tricyclic ring were synthesized from the corresponding toyocamycin and new 2 ′-β- C-methyl toyocamycin derivatives. The inhibitory studies of these compounds in the HCV replicon assay reveal that some of them possess interesting anti-HCV properties with low cytotoxicity.