Hepatitis C Virus Replicon RNA Research Articles

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

MiR-122 does not modulate the elongation phase of hepatitis C virus RNA synthesis in isolated replicase complexes

miR-122 is an abundant, liver-specific microRNA that is required for efficient amplification of hepatitis C virus (HCV) RNA. Recent studies with a miR-122-specific locked nucleic acid antagomir have shown it to be an important host target for therapeutic intervention. However, considerable controversy exists concerning the mechanisms underlying the dependence of HCV replication on miR-122. We studied the impact of miR-122 on the rate of [ 32P]-incorporation into positive-strand viral RNA by membrane-bound replicase complexes isolated from cells containing HCV RNA replicons. [ 32P]-incorporation in this cell-free system represents primarily the elongation phase of RNA synthesis, with little or no de novo initiation, and was not affected by the addition of either excess miR-122 or a miR-122-specific antisense oligonucleotide that suppresses replication in vivo. We also found no evidence that detectable quantities of miR-122 are specifically associated with replicase complexes in vivo. These results are consistent with miR-122 acting at an alternative step in the viral life cycle, promoting cap-independent viral translation, enhancing viral RNA stability, or facilitating de novo initiation of viral RNA synthesis.

PSI-7851, a Pronucleotide of β- d -2′-Deoxy-2′-Fluoro-2′- C -Methyluridine Monophosphate, Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

293 cells over‐expressing human ADI1 and CD81 are permissive for serum‐derived hepatitis C virus infection

Human aci-reductone dioxygenase 1 (ADI1) is a member of the Cupin superfamily. It binds to and inhibits the activities of membrane-type 1 matrix metalloproteinase, a protein known to interact with the tight junction protein, claudin-1. Previously, a variant protein, named submergence-induced protein-like factor (Sip-L), consisting of ADI1 amino acids 64-179, was found to support hepatitis C virus (HCV) infection and replication in 293 cells. In the present study, it was discovered that over-expression of human ADI1 in 293 cells (293-ADI1 cells) also supported HCV infection and replication. Using serum-derived HCV as an infectious source, enhanced cell uptake of HCV to a Northern blot detectable level was found in 293 cells over-expressing both CD81 and ADI1 (293-ADI1-CD81 cells). The enhanced cell entry was confirmed by the use of the vesicular stomatitis virus-based HCV pseudotype particles. However, transfection of HCV replicon RNA by electroporation into naïve 293 and 293-ADI1 cells revealed no difference in replication efficiency. Using the infectious J6/JFH chimera as an infectious source, the infectivity was compared between 293-ADI1-CD81 and Huh-7.5 cells. More infection foci were formed in the 293-ADI1-CD81 cells in the first round of infection. In conclusion, human ADI1 over-expression in 293 cells enhances cell entry but not replication of HCV. 293-ADI1-CD81 cells are permissive for serum-derived HCV infection.

Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway

An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains. Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) and structured sequence elements within the 3'UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5'cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3'UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5'UTR and 3'UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5'cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5'UTR and HCV 3'UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5'UTR and/or HCV 3'UTR, recruits eIF3 and enhances HCV IRES-mediated translation.

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

Antiviral Potency Analysis and Functional Comparison of Consensus Interferon, Interferon-α2a and Pegylated Interferon-α2b against Hepatitis C virus Infection

Background Current treatments for chronic hepatitis C virus (HCV) employing pegylated interferon (PEG-IFN) plus ribavirin are successful in approximately 50% of patients. Consensus IFN (CIFN) is a recombinant type I IFN that has demonstrated efficacy where conventional therapy has failed. We evaluated the host cell antiviral response and anti-HCV actions induced by IFN-α2a, PEG-IFN-α2b or CIFN on cultured immortalized human hepatocytes, Huh7 human hepatoma cells and Huh7 cells that harboured genetically distinct HCV RNA replicons or were infected with HCV 2a. Methods Cultured cells were treated with each IFN at relevant dosing based upon the pharmacological attainable in vivo serum maximum IFN concentrations. Gene expression and antiviral properties were measured using protein, RNA and virus quantification assays. Results CIFN treatment maximally triggered Janus kinase signal transducer and activator of transcription signalling in association with enhanced IFN-stimulated gene (ISG) expression. Increased antiviral potency of CIFN was associated with enhancement of IFN-induced blockade upon viral protein synthesis, protection of the cellular IFN promoter stimulator-1 (IPS-1) protein from HCV proteolysis and reduced replication of an IFN-resistant HCV replicon variant. Microarray analyses revealed that CIFN treatment induced a distinct pattern of ISG expression in cultured hepatocytes compared with other IFNs. Conclusions CIFN exhibits increased anti-HCV potency over IFN-α2a and PEG-IFN through maximal and distinct induction of ISG expression and enhanced intracellular innate antiviral response, while protecting IPS-1 from HCV proteolysis. CIFN might offer a treatment regimen imparting translational control programmes and restoration of the retinoic acid-inducible gene-1/IPS-1 pathway and could be considered for previous treatment failures.

Preclinical Characteristics of the Hepatitis C Virus NS3/4A Protease Inhibitor ITMN-191 (R7227)

Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.

Regulation of the hepatitis C virus genome replication by miR-199a

Hepatitis C virus (HCV) infection causes chronic hepatitis and hepatocellular carcinoma. Current anti-HCV therapies are based on interferon therapy, which is insufficiently effective. microRNAs (miRNAs) are non-coding RNAs that regulate gene expression, and they have recently been shown to play an important role in viral replication. An algorithm-based search for miRNAs that target the HCV genome yielded one miRNA, miR-199a, with a sequence similar to the HCV genome that is conserved among HCV genotypes. Over expression of miR-199a inhibited HCV genome replication in two cells bearing replicons (replicon cell) HCV-1b or -2a, however, miRNA inhibition by specific antisense oligonucleotide (ASO) accelerated viral replication. Prior transfection of immortalized hepatocytes which were infected with serum of HCV genotype 1b and 2a-infected patients, with miR-199a reduced HCV RNA replication activity. Mutation in the miR-199a target site in the replicon reduced the effect of the miR-199a. HCV replicon RNA is accumulated to the RNA-induced silencing complex (RISC) when miR-199a was over-expressed to the replicon cell. This antiviral effect by miR-199a was independent of the interferon pathway. The results of this study suggest that miR-199a directly regulates HCV replication and may serve as a novel antiviral therapy.

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.

New efficient replication system with hepatitis C virus genome derived from a patient with acute hepatitis C

We report for the first time a new RNA replication system with a hepatitis C virus (HCV) strain (AH1) derived from a patient with acute hepatitis C. Using an HCV replicon RNA library constructed with the AH1 strain (genotype 1b), we first established a cloned cell line, sAH1, harboring the HCV replicon. Cured cells obtained with interferon treatment of sAH1 cells were used for transfection with genome-length HCV RNA possessing four mutations found in sAH1 replicon. Consequently, one cloned cell line, AH1, supporting efficient replication of genome-length HCV RNA was obtained. By the comparison of AH1 cells with the O cells supporting genome-length HCV RNA (HCV-O strain) replication, we found different anti-HCV profiles of interferon-γ and cyclosporine A between AH1 and O cells. Reporter assay analysis suggests that the diverse effects of interferon-γ are due to the difference in HCV strains, but not the cellular environment.

Isolation and gene analysis of interferon α-resistant cell clones of the hepatitis C virus subgenome

Hepatitis C virus (HCV) proteins appear to play an important role in IFN-resistance, but the molecular mechanism remains unclear. To clarify the mechanism in HCV replicon RNA harboring Huh-7 cells (Huh-9-13), we isolated cellular clones with impaired IFNα-sensitivity. Huh-9-13 was cultured for approximately 2 months in the presence of IFNα, and 4 IFNα-resistant cell clones showing significant resistances were obtained. When total RNA from clones was introduced into Huh-7 cells, the transfected cells also exhibited IFNα-resistance. Although no common mutations were present, mutations in NS3 and NS5A regions were accumulated. Transactivation of IFNα and IFNα-stimulated Stat-1 phosphorylation were reduced, and the elimination of HCV replicon RNA from the clones restored the IFNα signaling. These results suggest that the mutations in the HCV replicon RNA, at least in part, cause an inhibition of IFN signaling and are important for acquisition of IFNα resistance in Huh-9-13.

Combinations of 2'-C-Methylcytidine Analogues with Interferon-α2b and Triple Combination with Ribavirin in the Hepatitis C Virus Replicon System

Hepatitis C virus (HCV) polymerase is an essential enzyme for HCV replication and has multiple inhibitor binding sites making it a major target for antiviral intervention. It is apparent that no single drug can inhibit HCV replication in humans. Hence, combinations of nucleoside analogues beta-D-2'-C-methylcytidine (2'-C-MeC; NM-107) or beta-D-2'-deoxy-2'-fluoro-2'-C-methyleytidine (2'-F-C-MeC; PSI-6130) with interferon-alpha2b (IFN-alpha2b) or triple combination with ribavirin (RBV) were evaluated. Huh-7 cells containing the self-replicating subgenomic HCV replicon (Clone B) were used for drug combination studies. After drug treatment for 5 days, total cellular RNA was then extracted and both ribosomal RNA and HCV replicon RNA were amplified in a single-step multiplex real-time PCR assay. Drug interaction analyses were performed using the CalcuSyn program. Double combinations of 2'-C-MeC or 2'-F-C-MeC with IFN-alpha2b at all ratios tested had weighted average combination index (Cl(wt)) values <1 indicating synergistic inhibition of HCV replication in the replicon system. For the triple combinations of IFN-alpha2b plus RBV with either 2'-C-MeC or 2'-F-C-MeC, the Cl(wt) values at 1:1:1 ratio tested were 0.5 and 0.8, respectively, indicating synergistic antiviral effects. No apparent cytotoxicity effects were observed with any of the combinations tested. These promising in vitro data warrant clinical investigation of the nucleosides analogues such as 2'-C-MeC or 2'-F-C-MeC in their prodrug forms, together with IFN-alphac2b and RBV, for successful treatment of HCV infections.

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

P21-activated Kinase 1 Is Activated through the Mammalian Target of Rapamycin/p70 S6 Kinase Pathway and Regulates the Replication of Hepatitis C Virus in Human Hepatoma Cells

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.

Evaluation of chemical labeling strategies for monitoring HCV RNA using vibrational microscopy

Raman and coherent anti-Stokes Raman scattering (CARS) microscopies have the potential to aid in detailed longitudinal studies of RNA localization. Here, we evaluate the use of carbon-deuterium and benzonitrile functional group labels as contrast agents for vibrational imaging of hepatitis C virus (HCV) replicon RNA. Dynamic light scattering and atomic force microscopy were used to evaluate the structural consequences of altering HCV subgenomic replicon RNA. Modification with benzonitrile labels caused the replicon RNA tertiary structure to partially unfold. Conversely, deuterium-modified replicon RNA was structurally similar to unmodified replicon RNA. Furthermore, the deuterated replicon RNA provided promising vibrational contrast in Raman imaging experiments. The functional effect of modifying subgenomic HCV replicon RNA was evaluated using the luciferase gene as a genetic reporter of translation. Benzonitrile labeling of the replicon RNA prevented translation in cell-based luciferase assays, while the deuterated replicon RNA retained both translation and replication competency. Thus, while the scattering cross-section for benzonitrile labels was higher, only carbon-deuterium labels proved to be non-perturbative to the function of HCV replicon RNA.

In vitro replication models for the hepatitis C virus

Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.

Hepatitis C virus RNA replication is regulated by FKBP8 and Hsp90

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.