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1.  Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes 
World Journal of Gastroenterology : WJG  2014;20(48):18284-18295.
AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.
METHODS: The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.
RESULTS: The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 108 ± 2.747 × 107 vs 5.368 × 107 ± 1.016 × 107, P < 0.05; 1.049 × 108 ± 2.747 × 107 vs 5.243 × 107 ± 1.194 × 107, P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.
CONCLUSION: Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.
PMCID: PMC4277965  PMID: 25561795
Hepacivirus; Replicon; Internal ribosome entry site; Tricistronic expression
2.  A Small-Molecule Inhibitor of Hepatitis C Virus Infectivity 
One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.
PMCID: PMC3910743  PMID: 24165192
3.  Identification of Novel Anti-Hepatitis C Virus Agents by a Quantitative High Throughput Screen in a Cell-Based Infection Assay 
Antiviral research  2015;124:20-29.
Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high-throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ~ 350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ~ 3,000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells.
PMCID: PMC4684727  PMID: 26515788
antiviral; HCV inhibitors; high throughput screening; cell-based assay; viral life cycle
4.  A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry 
PLoS Pathogens  2010;6(9):e1001086.
Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.
Author Summary
Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is a leading cause of chronic liver disease. Current treatments are not optimal; however, several molecules that inhibit HCV replication are in development. However, resistance to individual antivirals is likely to develop, requiring therapy consisting of a combination of drugs targeting different stages of the viral life cycle. The entry of HCV into hepatocytes is a multistep process, involving at least four cellular receptors, leading to virion endocytosis and fusion of the viral and cellular membranes. Unlike the HCV replication process, these steps have not been thoroughly exploited as targets for antiviral intervention. Therefore, we screened a small molecule library for inhibitors of HCV entry and identified a compound, EI-1, that potently blocked genotype 1a and 1b HCV infection. Importantly, EI-1 also prevented direct cell-to-cell spread of HCV, a potentially significant route of transmission in infected livers. In addition, our studies suggest that EI-1 susceptibility is mediated by the viral E2 envelope glycoprotein, as resistance in E2 can overcome inhibition. The antiviral activity of EI-1 is potentiated by combinations with other HCV inhibitors, demonstrating the value of entry inhibitors in potential combination antiviral regimens.
PMCID: PMC2936744  PMID: 20838466
5.  Cost-effective Screening for Acute Hepatitis C Virus Infection in HIV-Infected Men Who Have Sex With Men 
We investigated the effectiveness and cost-effectiveness of screening for acute hepatitis C virus (HCV) infection in human immunodeficiency virus-infected men who have sex with men. One-time screening at enrollment in care was never optimal. Depending on HCV infection incidence, regular screening with liver function tests was cost-effective.
Background. We used a Monte Carlo computer simulation to estimate the effectiveness and cost-effectiveness of screening for acute hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)–infected men who have sex with men.
Methods. One-time screening for prevalent HCV infection was performed at the time of enrollment in care, followed by either symptom-based screening, screening with liver function tests (LFTs), HCV antibody (Ab) screening, or HCV RNA screening in various combinations and intervals. We considered both treatment with pegylated interferon and ribavirin (PEG/RBV) alone and with an HCV protease inhibitor. Outcome measures were life expectancy, quality-adjusted life expectancy, direct medical costs, and cost-effectiveness, assuming a societal willingness to pay $100 000 per quality-adjusted life-year (QALY) gained.
Results. All strategies increased life expectancy (from 0.49 to 0.94 life-months), quality-adjusted life expectancy (from 0.47 to 1.00 quality-adjusted life-months), and costs (from $1900 to $7600), compared with symptom-based screening. The incremental cost-effectiveness ratio of screening with 6-month LFTs and a 12-month HCV Ab test, compared with symptom-based screening, was $43 700/QALY (for PEG/RBV alone) and $57 800/QALY (for PEG/RBV plus HCV protease inhibitor). The incremental cost-effectiveness ratio of screening with 3-month LFTs, compared with 6-month LFTs plus a 12-month HCV Ab test, was $129 700/QALY (for PEG/RBV alone) and $229 900/QALY (for PEG/RBV plus HCV protease inhibitor). With HCV protease inhibitor–based therapy, screening with 6-month LFTs and a 12-month HCV Ab test was the optimal strategy when the HCV infection incidence was ≤1.25 cases/100 person-years. The 3-month LFT strategy was optimal when the incidence was >1.25 cases/100 person-years.
Conclusions. Screening for acute HCV infection in HIV-infected MSM prolongs life expectancy and is cost-effective. Depending on incidence, regular screening with LFTs, with or without an HCV Ab test, is the optimal strategy.
PMCID: PMC3403839  PMID: 22491339
6.  Attenuation of 40S Ribosomal Subunit Abundance Differentially Affects Host and HCV Translation and Suppresses HCV Replication 
PLoS Pathogens  2012;8(6):e1002766.
For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.
Author Summary
Hepatitis C virus (HCV) infection causes chronic liver diseases that threaten ∼2% of the world population. There is no effective vaccine, and the current standard therapy, the combination of interferon and ribavirin, is effective to less than 50% of genotype-1 infected patients. While antivirals targeting at specific HCV proteins might ultimately lose their effectiveness due to the emergence of resistance-associated mutations, an alternative strategy that explores the genetic stability of host factors indispensable for HCV replication may provide better therapeutic targets for anti-HCV medicine. Here, we employed a loss-of-function screen method to identify such potential targets and uncovered a potential novel anti-HCV mechanism by manipulating the biogenesis of 40S ribosomal subunit. We showed that inhibiting 40S ribosome biogenesis can selectively suppress HCV translation and thus effectively inhibit HCV replication. In contrast to the conventional thinking, the 40S ribosomal subunit can differentially affect different translational modes, and HCV translation is more sensitive to the amounts of 40S ribosomal subunit as compared to general translation in host cell. Since HCV is known to evade anti-viral effects including translational suppression elicited by interferon, our findings may help design a therapeutic strategy to supplement interferon-based therapy and minimize mutation-associated drug resistance problem.
PMCID: PMC3394201  PMID: 22792060
7.  Specific Sequence of a Beta Turn in Human La Protein May Contribute to Species Specificity of Hepatitis C Virus 
Journal of Virology  2014;88(8):4319-4327.
Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a β-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting β-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La β-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.
IMPORTANCE Hepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented the development of a small-animal model, which is important for development of new antiviral drugs. Although a number of human-specific proteins are responsible for this species selectivity and some of these proteins—-mostly entry factors—-have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence allows the mouse La to behave like its human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.
PMCID: PMC3993764  PMID: 24478427
8.  A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement 
PLoS Pathogens  2011;7(4):e1001333.
Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.
Author Summary
Two decades after its identification, hepatitis C virus (HCV) remains a leading cause of serious liver diseases worldwide. The poor in vitro propagation of patient isolates has impaired their study. Conversely, viral strains of the most prevalent (∼70% of total infections) and clinically problematic (∼45% cured with the standard of care) genotype 1 adapted for in vitro replication display mutations impairing yield and/or in vivo infectivity. We established a new cell culture model for producing infectious HCV in a cell line stably bearing a subgenomic replicon from West Nile virus (a flavivirus belonging to the same family as HCV) that circumvents the requirement for HCV RNA replication. To study viral infectivity in vitro, we devised several HCV genome-based constructs. This system produced wild type HCV particles of subtypes 1a, 1b, 2a and a 1b/2a chimera. All specifically infected permissive target cells, and HCV particles containing wild type genomes known to be infectious in vivo infected human liver slices ex vivo. The production of authentic HCV particles independent of HCV RNA replication represents a new paradigm to decipher requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant genotypes.
PMCID: PMC3077361  PMID: 21533214
9.  Novel Chimeric Genotype 1b/2a Hepatitis C Virus Suitable for High-Throughput Screening▿ †  
A major obstacle in hepatitis C virus (HCV) research has been the lack of a permissive cell culture system that produces infectious viral particles. Significant breakthroughs have been achieved lately in establishing such culture systems. Yet to date, there are no reports of the applications of any of these systems in HCV drug screening. Here, we report the generation of two monocistronic, chimeric genotype 1 full-length HCV genome molecules. These molecules, C33J-Y835C-UBI and C33J-Y835C-FMDV2A, both contain the structural protein region from genotype 1 (subtype 1b, Con1) and the remaining region from the genotype 2a (JFH1) clone. Both contain the humanized Renilla luciferase reporter gene which is separated from the rest of the HCV open reading frame by two different cleavage sites. The viral RNAs replicated efficiently in transfected cells. Viral particles produced were infectious in naïve Huh7.5 cells, and the infectivity could be blocked by monoclonal antibody against a putative HCV entry cofactor, CD81. A pilot high-throughput screen of 900 unknown compounds was executed by both the genotype 2a subgenomic replicon system and the infectious system. Thirty-one compounds were identified as hits by both systems, whereas 78 compounds were identified as hits only for the infectious system, suggesting that the infectious system is capable of identifying inhibitors targeting the viral structural proteins and steps involving them in the viral life cycle. The infectious HCV system developed here provides a useful and versatile tool which should greatly facilitate the identification of HCV inhibitors currently not identified by the subgenomic replicon system.
PMCID: PMC2224717  PMID: 18039917
10.  Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds 
Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.
PMCID: PMC3910886  PMID: 24277038
11.  Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site 
Journal of Virology  1999;73(6):4941-4951.
The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K+ concentrations, with a sevenfold difference evident at 130 to 150 mM K+. These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.
PMCID: PMC112538  PMID: 10233956
12.  Establishment of chronic hepatitis C virus infection: Translational evasion of oxidative defence 
Hepatitis C virus (HCV) causes a clinically important disease affecting 3% of the world population. HCV is a single-stranded, positive-sense RNA virus belonging to the genus Hepacivirus within the Flaviviridae family. The virus establishes a chronic infection in the face of an active host oxidative defence, thus adaptation to oxidative stress is key to virus survival. Being a small RNA virus with a limited genomic capacity, we speculate that HCV deploys a different strategy to evade host oxidative defence. Instead of counteracting oxidative stress, it utilizes oxidative stress to facilitate its own survival. Translation is the first step in the replication of a plus strand RNA virus so it would make sense if the virus can exploit the host oxidative defence in facilitating this very first step. This is particularly true when HCV utilizes an internal ribosome entry site element in translation, which is distinctive from that of cap-dependent translation of the vast majority of cellular genes, thus allowing selective translation of genes under conditions when global protein synthesis is compromised. Indeed, we were the first to show that HCV translation was stimulated by an important pro-oxidant-hydrogen peroxide in hepatocytes, suggesting that HCV is able to adapt to and utilize the host anti-viral response to facilitate its own translation thus allowing the virus to thrive under oxidative stress condition to establish chronicity. Understanding how HCV translation is regulated under oxidative stress condition will advance our knowledge on how HCV establishes chronicity. As chronicity is the initiator step in disease progression this will eventually lead to a better understanding of pathogenicity, which is particularly relevant to the development of anti-virals and improved treatments of HCV patients using anti-oxidants.
PMCID: PMC3961964  PMID: 24659872
Hepatitis C virus; Oxidative stress; Hydrogen peroxide; Translation; Internal ribosome entry site; Chronicity; Persistence
13.  Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase 
Antimicrobial Agents and Chemotherapy  2004;48(12):4813-4821.
A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.
PMCID: PMC529219  PMID: 15561861
14.  Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture 
PLoS ONE  2012;7(4):e35351.
Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection.
PMCID: PMC3335862  PMID: 22545104
15.  Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation 
Nucleic Acids Research  2003;31(2):734-742.
Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5′ untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2′-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14–17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC50s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2′-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.
PMCID: PMC140505  PMID: 12527783
16.  Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication 
PLoS ONE  2012;7(8):e43600.
Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.
PMCID: PMC3427374  PMID: 22937067
17.  Chemical combinations elucidate pathway interactions and regulation relevant to Hepatitis C replication 
SREBP-2, oxidosqualene cyclase (OSC) or lanosterol demethylase were identified as novel sterol pathway-associated targets that, when probed with chemical agents, can inhibit hepatitis C virus (HCV) replication.Using a combination chemical genetics approach, combinations of chemicals targeting sterol pathway enzymes downstream of and including OSC or protein geranylgeranyl transferase I (PGGT) produce robust and selective synergistic inhibition of HCV replication. Inhibition of enzymes upstream of OSC elicit proviral responses that are dominant to the effects of inhibiting all downstream targets.Inhibition of the sterol pathway without inhibition of regulatory feedback mechanisms ultimately results in an increase in HCV replication because of a compensatory upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression. Increases in HMGCR expression without inhibition of HMGCR enzymatic activity ultimately stimulate HCV replication through increasing the cellular pool of geranylgeranyl pyrophosphate (GGPP).Chemical inhibitors that ultimately prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced.
Chemical inhibition of enzymes in either the cholesterol or the fatty acid biosynthetic pathways has been shown to impact viral replication, both positively and negatively (Su et al, 2002; Ye et al, 2003; Kapadia and Chisari, 2005; Sagan et al, 2006; Amemiya et al, 2008). FBL2 has been identified as a 50 kDa geranylgeranylated host protein that is necessary for localization of the hepatitis C virus (HCV) replication complex to the membranous web through its close association with the HCV protein NS5A and is critical for HCV replication (Wang et al, 2005). Inhibition of the protein geranylgeranyl transferase I (PGGT), an enzyme that transfers geranylgeranyl pyrophosphate (GGPP) to cellular proteins such as FBL2 for the purpose of membrane anchoring, negatively impacts HCV replication (Ye et al, 2003). Conversely, chemical agents that increase intracellular GGPP concentrations promote viral replication (Kapadia and Chisari, 2005). Statin compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in the sterol pathway (Goldstein and Brown, 1990), have been suggested to inhibit HCV replication through ultimately reducing the cellular pool of GGPP (Ye et al, 2003; Kapadia and Chisari, 2005; Ikeda et al, 2006). However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. The use of statins for the treatment of HCV is likely to be complicated by the reported compensatory increase in HMGCR expression in vitro and in vivo (Stone et al, 1989; Cohen et al, 1993) in response to treatment. Enzymes in the sterol pathway are regulated on a transcriptional level by sterol regulatory element-binding proteins (SREBPs), specifically SREBP-2 (Hua et al, 1993; Brown and Goldstein, 1997). When cholesterol stores in cells are depleted, SREBP-2 activates transcription of genes in the sterol pathway such as HMGCR, HMG-CoA synthase, farnesyl pyrophosphate (FPP) synthase, squalene synthase (SQLS) and the LDL receptor (Smith et al, 1988, 1990; Sakai et al, 1996; Brown and Goldstein, 1999; Horton et al, 2002). The requirement of additional downstream sterol pathway metabolites for HCV replication has not been completely elucidated.
To further understand the impact of the sterol pathway and its regulation on HCV replication, we conducted a high-throughput combination chemical genetic screen using 16 chemical probes that are known to modulate the activity of target enzymes relating to the sterol biosynthesis pathway (Figure 1). Using this approach, we identified several novel antiviral targets including SREBP-2 as well as targets downstream of HMGCR in the sterol pathway such as oxidosqualene cyclase (OSC) and lanosterol demethylase. Many of our chemical probes, specifically SR-12813, farnesol and squalestatin, strongly promoted replicon replication. The actions of both farnesol and squalestatin ultimately result in an increase in the cellular pool of GGPP, which is known to increase HCV replication (Ye et al, 2003; Kapadia and Chisari, 2005; Wang et al, 2005).
Chemical combinations targeting enzymes upstream of squalene epoxidase (SQLE) at the top of the sterol pathway (Figure 4A) elicited Bateson-type epistatic responses (Boone et al, 2007), where the upstream agent's response predominates over the effects of inhibiting all downstream targets. This was especially notable for combinations including simvastatin and either U18666A or squalestatin, and for squalestatin in combination with Ro48-8071. Treatment with squalestatin prevents the SQLS substrate, farnesyl pyrophosphate (FPP) from being further metabolized by the sterol pathway. As FPP concentrations increase, the metabolite can be shunted away from the sterol pathway toward farnesylation and GGPP synthetic pathways, resulting in an increase in host protein geranylgeranylation, including FBL2, and consequently replicon replication. This increase in replicon replication explains the source of the observed epistasis over Ro48-8071 treatment.
Combinations between probes targeting enzymes downstream of and including OSC produced robust synergies with each other or with a PGGT inhibitor. Figure 4B highlights examples of antiviral synergy resulting from treatment of cells with an OSC inhibitor in combination with an inhibitor of either an enzyme upstream or downstream of OSC. A combination of terconazole and U18666A is synergistic without similar combination effects in the host proliferation screen. Likewise, clomiphene was also synergistic when added to replicon cells in combination with U18666A. One of the greatest synergies observed downstream in the sterol pathway is a combination of amorolfine and AY 9944, suggesting that there is value in developing combinations of drugs that target enzymes in the sterol pathway, which are downstream of HMGCR.
Interactions with the protein prenylation pathway also showed strong mechanistic patterns (Figure 4C). GGTI-286 is a peptidomimetic compound resembling the CAAX domain of a protein to be geranylgeranylated and is a competitive inhibitor of protein geranylgeranylation. Simvastatin impedes the antiviral effect of GGTI-286 at low concentrations but that antagonism is balanced by comparable synergy at higher concentrations. At the low simvastatin concentrations, a compensatory increase in HMGCR expression leads to increased cellular levels of GGPP, which are likely to result in an increase in PGGT enzymatic turnover and decreased GGTI-286 efficacy. The antiviral synergy observed at the higher inhibitor concentrations is likely nonspecific as synergy was also observed in a host viability assay. Further downstream, however, a competitive interaction was observed between GGTI-286 and squalestatin, where the opposing effect of one compound obscures the other compound's effect. This competitive relationship between GGTI and SQLE explains the epistatic response observed between those two agents. For inhibitors of targets downstream of OSC, such as amorolfine, there are strong antiviral synergies with GGTI-286. Notably, combinations with OSC inhibitors and GGTI-286 were selective, in that comparable synergy was not found in a host viability assay. This selectivity suggests that jointly targeting OSC and PGGT is a promising avenue for future HCV therapy development.
This study provides a comprehensive and unique perspective into the impact of sterol pathway regulation on HCV replication and provides compelling insight into the use of chemical combinations to maximize antiviral effects while minimizing proviral consequences. Our results suggest that HCV therapeutics developed against sterol pathway targets must consider the impact on underlying sterol pathway regulation. We found combinations of inhibitors of the lower part of the sterol pathway that are effective and synergistic with each other when tested in combination. Furthermore, the combination effects observed with simvastatin suggest that, though statins inhibit HMGCR activity, the resulting regulatory consequences of such inhibition ultimately lead to undesirable epistatic effects. Inhibitors that prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced.
The search for effective Hepatitis C antiviral therapies has recently focused on host sterol metabolism and protein prenylation pathways that indirectly affect viral replication. However, inhibition of the sterol pathway with statin drugs has not yielded consistent results in patients. Here, we present a combination chemical genetic study to explore how the sterol and protein prenylation pathways work together to affect hepatitis C viral replication in a replicon assay. In addition to finding novel targets affecting viral replication, our data suggest that the viral replication is strongly affected by sterol pathway regulation. There is a marked transition from antagonistic to synergistic antiviral effects as the combination targets shift downstream along the sterol pathway. We also show how pathway regulation frustrates potential hepatitis C therapies based on the sterol pathway, and reveal novel synergies that selectively inhibit hepatitis C replication over host toxicity. In particular, combinations targeting the downstream sterol pathway enzymes produced robust and selective synergistic inhibition of hepatitis C replication. Our findings show how combination chemical genetics can reveal critical pathway connections relevant to viral replication, and can identify potential treatments with an increased therapeutic window.
PMCID: PMC2913396  PMID: 20531405
chemical genetics; combinations and synergy; hepatitis C; replicon; sterol biosynthesis
18.  Antiviral Stilbene 1,2-Diamines Prevent Initiation of Hepatitis C Virus RNA Replication at the Outset of Infection▿ 
Journal of Virology  2011;85(11):5513-5523.
The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ∼300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a >1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen.
PMCID: PMC3094966  PMID: 21430055
19.  Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation 
Molecular and Cellular Biology  2001;21(13):4097-4109.
Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5′ m7GpppN cap and the 3′ poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3′ end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3′-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.
PMCID: PMC87071  PMID: 11390639
20.  Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation 
Molecular and Cellular Biology  2001;21(24):8357-8364.
Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3′X) targeting the HCV minus-strand replication intermediate at position 40 within the 3′X tail. Surprisingly, Rz3′X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3′X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3′X tail sequences, the results indicate that Rz3′X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome α-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3′X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.
PMCID: PMC100000  PMID: 11713272
21.  Hepatitis C Virus Controls Interferon Production through PKR Activation 
PLoS ONE  2010;5(5):e10575.
Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection.
PMCID: PMC2868028  PMID: 20485506
22.  Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening▿  
Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neor marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z′ value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 μM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals ≥50 and ≤40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.
PMCID: PMC1797688  PMID: 17060518
23.  Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro 
Antiviral Research  2015;115:39-47.
•Antiviral effects of 20 Brazilian natural compounds were investigated.•Four compounds with potent inhibitory activity on HCV replication were identified.•Antiviral activity was independent of HCV genotype.•Antiviral effect was not affected by variants described to confer resistance.•One of the four compounds inhibited HCV IRES-mediated translation.
Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50 = 2.3 μM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3∗43 (EC50 = 4.0 μM), 3∗20 (EC50 = 8.2 μM) and 5∗362 (EC50 = 38.9 μM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.
PMCID: PMC4329992  PMID: 25557602
Brazilian plants; Natural compounds; Antiviral; Hepatitis C virus; Replication
24.  Internal ribosome entry site within hepatitis C virus RNA. 
Journal of Virology  1992;66(3):1476-1483.
The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.
PMCID: PMC240872  PMID: 1310759
25.  Impaired Hepatitis C Virus-Specific T Cell Responses and Recurrent Hepatitis C Virus in HIV Coinfection 
PLoS Medicine  2006;3(12):e492.
Hepatitis C virus (HCV)-specific T cell responses are critical for spontaneous resolution of HCV viremia. Here we examined the effect of a lymphotropic virus, HIV-1, on the ability of coinfected patients to maintain spontaneous control of HCV infection.
Methods and Findings
We measured T cell responsiveness by lymphoproliferation and interferon-γ ELISPOT in a large cohort of HCV-infected individuals with and without HIV infection. Among 47 HCV/HIV-1-coinfected individuals, spontaneous control of HCV was associated with more frequent HCV-specific lymphoproliferative (LP) responses (35%) compared to coinfected persons who exhibited chronic HCV viremia (7%, p = 0.016), but less frequent compared to HCV controllers who were not HIV infected (86%, p = 0.003). Preservation of HCV-specific LP responses in coinfected individuals was associated with a higher nadir CD4 count (r2 = 0.45, p < 0.001) and the presence and magnitude of the HCV-specific CD8+ T cell interferon-γ response (p = 0.0014). During long-term follow-up, recurrence of HCV viremia occurred in six of 25 coinfected individuals with prior control of HCV, but in 0 of 16 HIV-1-negative HCV controllers (p = 0.03, log rank test). In these six individuals with recurrent HCV viremia, the magnitude of HCV viremia following recurrence inversely correlated with the CD4 count at time of breakthrough (r = −0.94, p = 0.017).
These results indicate that HIV infection impairs the immune response to HCV—including in persons who have cleared HCV infection—and that HIV-1-infected individuals with spontaneous control of HCV remain at significant risk for a second episode of HCV viremia. These findings highlight the need for repeat viral RNA testing of apparent controllers of HCV infection in the setting of HIV-1 coinfection and provide a possible explanation for the higher rate of HCV persistence observed in this population.
HIV infection impairs the immune response to HCV. Even individuals who have cleared HCV infection remain at significant risk for a second episode of HCV viremia.
Editors' Summary
Because of shared transmission routes (contaminated needles, contaminated blood products, and, to a lesser extent, unprotected sex), a large proportion of HIV-infected individuals (estimates range between 25% and 33%) are also infected with the hepatitis C virus (HCV). In most but not all individuals infected with HCV, the virus infection is chronic and causes liver disease that can eventually lead to liver failure. Disease progress is slow; it often takes decades until infected individuals develop serious liver disease. In people infected with both HCV and HIV, however, liver disease caused by HCV often appears sooner and progresses faster. As highly active antiretroviral therapy (HAART) and prophylaxis of opportunistic infections increase the life span of persons living with HIV, HCV-related liver disease has become a major cause of hospital admissions and deaths among HIV-infected persons.
Why Was This Study Done?
A sizable minority of people who are infected with HCV manage to control the virus and never get liver disease, and scientists have found that these people somehow mounted a strong immune response against the hepatitis C virus. CD4+ T cells, the very immune cells that are infected and destroyed by HIV, play an important role in this immune response. The goal of the present study was to better understand how infection with HIV compromises the specific immune response to HCV and thereby the control of HCV disease progression.
What Did the Researchers Do and Find?
The researchers recruited four groups of patients, 94 in total, all of whom were infected with HCV. Two groups comprised patients who were infected with HIV as well as HCV, with either high or undetectable levels of HCV (30 patients in each group). The two other groups included patients not infected with HIV, either with high or undetectable levels of HCV (17 patients in each group). The researchers focused on the individuals who, despite coinfection with HIV, were able to control their HCV infection. They found that those individuals managed to maintain relatively high levels of CD4+ T cells that specifically recognize HCV. However, a quarter of these patients (six out of 25) failed to keep HCV levels down for the entire observation period of up to 2.5 years; their blood levels of HCV rose substantially, most likely due to recurrence of the previously suppressed virus (the researchers could not be certain that none of the patients had become infected again after a new exposure to HCV-contaminated blood, but there was no evidence that they had engaged in risky behavior). The rise of HCV levels in the blood of the relapsed patients coincided with a drop in overall CD4+ T cell numbers. Following relapse in these individuals, HCV did not return to undetectable levels during the study. During the same period none of the 16 HIV-uninfected people with controlled HCV infection experienced a recurrence of detectable HCV.
What Do These Findings Mean?
Despite the relatively small numbers of patients, these results suggest that recurrence of HCV after initial control of the virus is more likely in people who are coinfected with HIV, and that HCV control is lost when CD4+ T cell counts fall. This is one more reason to test all HIV-positive patients for HCV coinfection. Coinfected patients, even those who seem to be controlling HCV and would not automatically receive HCV treatment, should be regularly tested for a rise of HCV levels. In addition, maintaining CD4+ T cells at a high level might be particularly important for those patients, which means that doctors might consider starting HAART therapy earlier than is generally recommended for HIV-infected individuals. Additional studies are needed to support these recommendations, however, especially as this study did not follow the patients long enough to determine the consequences of the observed loss of control of HCV.
Additional Information.
Please access these Web sites via the online version of this summary at
AIDS Treatment Data Network factsheet on HIV/HCV coinfection
US CDC factsheet on HIV/HCV coinfection
American Liver Foundation, information on HIV and HCV
MedlinePlus pages on HCV
PMCID: PMC1705826  PMID: 17194190

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