Cell-free replication of the hepatitis C virus subgenomic replicon

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.     

Characterization of hepatitis C virus subgenomic replicon resistance to cyclosporine in vitro

Treatment of hepatitis C virus (HCV) infection has been met with less than satisfactory results due primarily to its resistance to and significant side effects from alpha interferon (IFN-alpha). New classes of safe and broadly acting treatments are urgently needed. Cyclosporine (CsA), an immunosuppressive and anti-inflammatory drug for organ transplant patients, has recently been shown to be highly effective in suppressing HCV replication through a mechanism that is distinct from the IFN pathway. Here we report the selection and characterization of HCV replicon cells that are resistant to CsA treatment in vitro, taking advantage of our ability to sort live cells that are actively replicating HCV RNA in the presence of drug treatments. This resistance is specific to CsA as the replicon cells most resistant to CsA were still sensitive to IFN-alpha and a polymerase inhibitor. We demonstrate that the resistant phenotype is not a result of general enhanced replication and, furthermore, that mutations in the coding region of HCV NS5B contribute to the resistance. Interestingly, a point mutation (I432V) isolated from the most resistant replicon was able to rescue a lethal mutation (P540A) in NS5B that disrupts its interaction with its cofactor, cyclophilin B (CypB), even though the I432V mutation is located outside of the reported CypB binding site (amino acids 520 to 591). Our results demonstrate that CsA exerts selective pressure on the HCV genome, leading to the emergence of resistance-conferring mutations in the viral genome despite acting upon a cellular protein.     

Proteome analysis of human liver carcinoma Huh7 cells harboring hepatitis C virus subgenomic replicon

Chronic infection by hepatitis C virus (HCV) is closely correlated with serious liver diseases. Although considerable progress has been made during recent years, the mechanism of replication and pathogenesis of HCV infection are still elusive. We have applied proteomic techniques in this work to globally analyze the protein expression profiles of a human liver cell lines Huh7 in absence and presence of HCV replication, aiming at elucidating the components of HCV replication and the cellular responses to HCV replication. The protein mixtures of three subcellular fractions from Huh7 and Huh7-HCV were separated by 2-DE under various pH gradients. Differentially expressed spots were identified by MALDI-TOF MS, followed by database searching. A total of 179 comparative proteins were identified unambiguously, including proteins associated with host cytoskeleton, intracellular traffic, oxidative and ER stress, proteasome degradation, translation, apoptosis, proliferation, etc. Host proteins known to interact with HCV proteins, such as HSP27, alpha-actinin, nucleolin and eukaryotic initiation factor 4A-I, were elevated in Huh7-HCV cells. Our study provides the global information of proteomic alteration of Huh7 cells in the presence of HCV replication and the clues for further understanding of the mechanism of HCV replication and pathogenesis.     

Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins

A trans-packaging system for hepatitis C virus (HCV) subgenomic replicon RNAs was developed. HCV subgenomic replicon was efficiently encapsidated by the HCV structural proteins that were stably expressed in trans under the control of a mammalian promoter. Infectious HCV-like particles (HCV-LPs), established a single-round infection, were produced and released into culture medium in titers of up to 10³ focus forming units/ml. Expression of NS2 protein with structural proteins (core, E1, E2, and p7) was shown to be critical for the infectivity of HCV-LPs. Anti-CD81 treatment decreased the number of infected cells, suggesting that HCV-LPs infected cells in a CD81-dependent manner. The packaging cell line should be useful both for the production of single-round infectious HCV-LPs to elucidate the mechanisms of HCV assembly, particle formation and infection to host cells, and for the development of HCV replicon-based vaccines.     

Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro

The hepatitis C virus (HCV) replicon system is a potent tool for understanding the mechanisms of HCV replication and proliferation, and for the development of treatments for patients with HCV. Recently, we established an HCV subgenomic replicon (50-1) using HCV genome RNA obtained from the cultured human T cell line MT-2C infected with HCV (isolate 1B-1) in vitro. In order to further obtain other HCV replicons without difficulty, we generated a replicon RNA library derived from human non-neoplastic hepatocytes infected with HCV (isolate 1B-2) in vitro. Upon transfection of the generated RNA library to "cured cells," from which the 50-1 subgenomic replicon was eliminated by prolonged treatment with interferon-alpha, we successfully established a new HCV subgenomic replicon, 1B-2R1. We characterized 1B-2R1 replicon in terms of efficiency of replication, HCV sequence, and sensitivity to interferons. The results revealed that the replication level of the 1B-2R1 replicon was comparable to that of the 50-1 replicon. We also found that the 1B-2R1 replicon possessed an HCV sequence distinct from those of other replicons established to date, and that the 1B-2R1 replicon was sensitive to interferon-alpha, interferon-beta, and interferon-gamma. Taken together, present results indicate that the replicon RNA library generated using an in vitro HCV infection system is useful for the establishment of an HCV subgenomic replicon.     

Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon

The hepatitis C virus (HCV) genotype 2a subgenomic replicon can replicate in two human non-hepatocyte-derived cell lines, HeLa and 293, with in vitro-transcribed replicon RNA. Sequencing analysis revealed that mutations in HCV-derived regions were not essential for replication in these cells, as some clones displayed no mutations.     

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.     

Replication of a hepatitis C virus replicon clone in mouse cells

Background

The duration of treatment for HCV infection is partly indicated by the genotype of the virus. For studies of disease transmission, vaccine design, and surveillance for novel variants, subtype-level classification is also needed. This study used the Shimodaira-Hasegawa test and related statistical techniques to compare phylogenetic trees obtained from coding and non-coding regions of a whole-genome alignment for the reliability of subtyping in different regions.

Results

Different regions of the HCV genome yield inconsistent phylogenies, which can lead to erroneous conclusions about classification of a given infection. In particular, the highly conserved 5' untranslated region (UTR) yields phylogenetic trees with topologies that differ from the HCV polyprotein and complete genome phylogenies. Phylogenetic trees from the NS5B gene reliably cluster related subtypes, and yield topologies consistent with those of the whole genome and polyprotein.

Conclusion

These results extend those from previous studies and indicate that, unlike the NS5B gene, the 5' UTR contains insufficient variation to resolve HCV classifications to the level of viral subtype, and fails to distinguish genotypes reliably. Use of the 5' UTR for clinical tests to characterize HCV infection should be replaced by a subtype-informative test.     

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.     

Mathematical modeling of subgenomic hepatitis C virus replication in Huh-7 cells

Cell-based hepatitis C virus (HCV) replicon systems have provided a means for understanding HCV replication mechanisms and for testing new antiviral agents. We describe here a mathematical model of HCV replication that assumes that the translation of the HCV polyprotein occurs in the cytoplasm, that HCV RNA synthesis occurs in vesicular-membrane structures, and that the strategy of replication involves a double-stranded RNA intermediate. Our results shed light on the intracellular dynamics of subgenomic HCV RNA replication from transfection to steady state within Huh-7 cells. We predict the following: (i) about 6 x 10(3) ribosomes are involved in generating millions of HCV NS5B-polymerase molecules in a Huh-7 cell, (ii) the observed 10:1 asymmetry of plus- to minus-strand RNA levels can be explained by a higher-affinity (200-fold) interaction of HCV NS5B polymerase-containing replication complexes with HCV minus-strand RNA over HCV plus-strand RNA in order to initiate synthesis, (iii) the latter higher affinity can also account for the observed approximately 6:1 plus-strand/minus-strand ratio in vesicular-membrane structures, and (iv) the introduction of higher numbers of HCV plus-strand RNA by transfection leads to faster attainment of steady-state but does not change the steady-state HCV RNA level. Fully permissive HCV replication systems have been developed, and the model presented here is a first step toward building a comprehensive model for complete HCV replication. Moreover, the model can serve as an important tool in understanding HCV replication mechanisms and should prove useful in designing and evaluating new antivirals against HCV.     

Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons

Hepatitis C virus (HCV) is the only known positive-stranded RNA virus that causes persistent lifelong infections in humans. Accumulation of HCV RNA can be inhibited with alpha interferon (IFN-alpha) in vivo and in culture cells. We used cell-based assay systems to investigate the mechanisms responsible for the cytokine-induced inhibition of HCV replication. The results showed that IFN-alpha could suppress the accumulation of viral RNA by a noncytopathic pathway and could also induce apoptosis of virally infected cells in a concentration- and cell line-dependent fashion. Whereas the noncytopathic IFN-alpha response depended on a functional Jak-STAT signal transduction pathway, it did not appear to require double-stranded RNA-dependent pathways. Moreover, we found that functional proteasomes were required for establishment of the IFN-alpha response against HCV. Based on the results described in this study we propose a model for the mechanism by which IFN-alpha therapy suppresses HCV replication in chronic infections by both cytopathic and noncytopathic means.     

Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase

Mutations were introduced into the NS3 helicase region of a hepatitis C virus (HCV) Con1 subgenomic replicon to ascertain the role of the helicase in viral replication. One new replicon lacked two-thirds of the NS3 helicase (Deltahel), and six others contained one of the following six amino acid substitutions in NS3: R393A, F438A, T450I, E493K, W501A, and W501F. It has been previously reported that purified R393A, F438A, and W501A HCV helicase proteins do not unwind RNA but unwind DNA, bind RNA, and hydrolyze ATP. On the other hand, previous data suggest that a W501F protein retains most of its unwinding abilities and that purified T450I and E493K HCV helicase proteins have enhanced unwinding abilities. In a hepatoma cell line that has been cured of HCV replicons using interferon, the T450I and W501F replicons synthesized both negative-sense and positive-sense viral RNA and formed colonies after selection with similar efficiencies as the parent replicon. However, the Deltahel, R393A, F438A, and W501A replicons encoded and processed an HCV polyprotein but did not synthesize additional viral RNA or form colonies. Surprisingly the same phenotype was seen for the E493K replicon. The inability of the E493K replicon to replicate might point to a role of pH in viral replication because a previous analysis has shown that, unlike the wild-type NS3 protein, the helicase activity of an E493K protein is not sensitive to pH changes. These results demonstrate that the RNA-unwinding activity of the HCV NS3 helicase is needed for RNA replication.     

Replication studies using genotype 1a subgenomic hepatitis C virus replicons

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.     

Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells

Background  

Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is disparately needed.     

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.     

Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells

Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005-->I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.