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• W1997097856 abstract "Understanding the pathogenesis of hepatitis C requires the availability of tissue culture models that sustain viral replication and produce infectious particles. We report on the establishment of a culture system of nontransformed human fetal hepatocytes that supports hepatitis C virus (HCV) replication after transfection with full-length in vitro-transcribed genotype 1a HCV RNA without adaptive mutations and infection with patient sera of diverse HCV genotypes. Transfected and infected hepatocytes expressed HCV core protein and HCV negative-strand RNA. For at least 2 months, transfected or infected cultures released HCV into the medium at high levels and usually with a cyclical pattern. Viral replication had some cytotoxic effects on the cells, which produced interferon (IFN)-β as a component of the antiviral response. Medium from transfected cells was able to infect naïve cultures in a Transwell system, and the infection was blocked by IFN-α and IFN-λ. Viral particles analyzed by sucrose density centrifugation had a density of 1.17 g/ml. Immunogold labeling with antibody against HCV envelope protein E2 decorated the surface of the viral particles, as visualized by electron microscopy. This culture system may be used to study the responses of nontransformed human hepatocytes to HCV infection, to analyze serum infectivity, and to clone novel HCVs from infected patients. Understanding the pathogenesis of hepatitis C requires the availability of tissue culture models that sustain viral replication and produce infectious particles. We report on the establishment of a culture system of nontransformed human fetal hepatocytes that supports hepatitis C virus (HCV) replication after transfection with full-length in vitro-transcribed genotype 1a HCV RNA without adaptive mutations and infection with patient sera of diverse HCV genotypes. Transfected and infected hepatocytes expressed HCV core protein and HCV negative-strand RNA. For at least 2 months, transfected or infected cultures released HCV into the medium at high levels and usually with a cyclical pattern. Viral replication had some cytotoxic effects on the cells, which produced interferon (IFN)-β as a component of the antiviral response. Medium from transfected cells was able to infect naïve cultures in a Transwell system, and the infection was blocked by IFN-α and IFN-λ. Viral particles analyzed by sucrose density centrifugation had a density of 1.17 g/ml. Immunogold labeling with antibody against HCV envelope protein E2 decorated the surface of the viral particles, as visualized by electron microscopy. This culture system may be used to study the responses of nontransformed human hepatocytes to HCV infection, to analyze serum infectivity, and to clone novel HCVs from infected patients. An estimated 170 million people worldwide, including 1.5 to 2% of the U.S. population, are infected with hepatitis C virus (HCV).1Wasley A Alter MJ Epidemiology of hepatitis C: geographic differences and temporal trends.Semin Liver Dis. 2000; 20: 1-16Crossref PubMed Google Scholar Although some infected patients clear HCV by mounting a successful immune response, a chronic carrier state is established in the great majority of cases, resulting in liver injury that ranges from minimal to varying degrees of hepatic inflammation and fibrosis. After 20 to 30 years, 15 to 20% of patients develop liver cirrhosis that may lead to hepatocellular carcinoma. HCV-induced liver disease is the leading indication for liver transplantation in most U.S. medical centers.2Seeff LB Natural history of hepatitis C.Am J Med. 1999; 107: 10S-15SAbstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 3Williams I Epidemiology of hepatitis C in the United States.Am J Med. 1999; 107: 2S-9SAbstract Full Text Full Text PDF PubMed Google Scholar, 4Alter MJ Hepatitis C virus infection in the United States.J Hepatol. 1999; 31: 88-91Abstract Full Text PDF PubMed Google ScholarMany systems have been used in attempts to establish HCV replication in culture.5Kato N Shimotohno K Systems to culture hepatitis C virus.Curr Top Microbiol Immunol. 2000; 242: 261-278PubMed Google Scholar, 6Shimizu YK Iwamoto A Hijikata M Purcell RH Yoshikura H Evidence for in vitro replication of hepatitic C virus genome in a human T-cell line.Proc Natl Acad Sci USA. 1992; 89: 5477-5481Crossref PubMed Scopus (317) Google Scholar, 7Noguchi M Hirohashi S Cell lines from non-neoplastic liver and hepatocellular carcinoma tissue from a single patient.In Vitro Cell Dev Biol Anim. 1996; 32: 135-137Crossref PubMed Scopus (53) Google Scholar, 8Iacovacci S Manzin A Barca S Sargiacomo M Serafino A Valli MB Macioce G Hassan HJ Ponzetto A Clementi M Peschle C Carloni G Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.Hepatology. 1997; 26: 1328-1337PubMed Google Scholar, 9Loriot MA Permissiveness of human biliary epithelial cells to infection by hepatitis C virus.Hepatology. 1999; 29: 1587-1595Crossref PubMed Scopus (56) Google Scholar Most of theses systems are permissive for HCV infection but did not sustain efficient virus production. The lack of suitable HCV cell culture systems has been a serious impediment for progress in understanding the relationships between the virus and its natural host, nontransformed human hepatocytes. A major advance for culturing HCV was the development of a stable subgenomic replicon system,10Lohmann V Korner F Koch J Herian U Theilmann L Bartenschlager R Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2480) Google Scholar, 11Lohmann V Korner F Dobierzewska A Bartenschlager R Mutations in hepatitis C virus RNAs conferring cell culture adaptation.J Virol. 2001; 75: 1437-1449Crossref PubMed Scopus (398) Google Scholar, 12Pietschmann T Lohmann V Kaul A Krieger N Rinck G Rutter G Strand D Bartenschlager R Persistent and transient replication of full-length hepatitis C virus genomes in cell culture.J Virol. 2002; 76: 4008-4021Crossref PubMed Scopus (308) Google Scholar which was able to replicate autonomously and at a high level in the human hepatoma line Huh-7. A breakthrough occurred in 2005, following the isolation of the genotype 2a HCV JFH-1 virus from a patient with fulminant hepatitis. This virus replicates well in Huh-7 cells without adaptive mutations.13Kato T Date T Miyamoto M Furusaka A Tokushige K Mizokami M Wakita T Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.Gastroenterology. 2003; 125: 1808-1817Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 14Date T Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.J Biol Chem. 2004; 279: 22371-22376Crossref PubMed Scopus (97) Google Scholar, 15Kato T Non-hepatic cell lines HeLa and 293 cells support efficient replication of hepatitis C virus genotype 2a subgenomic replicon.J Virol. 2005; 79: 592-596Crossref PubMed Scopus (101) Google Scholar Wakita et al16Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2400) Google Scholar obtained virus production in cells transfected with the cloned JFH-1 genome, and Zhong et al17Zhong J Gastaminza P Cheng G Kapadia S Kato T Burton DR Wieland SF Uprichard SL Wakita T Chisari FV Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1507) Google Scholar established a highly efficient system for production of infectious virus in Huh-7.5.1 cells. Lindenbach et al18Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1943) Google Scholar have constructed full-length chimeric genomes J6/JFH and produced infectious particles in the Huh-7.5 cell line.Viral production in these systems relies on the transfection into transformed cells of a single virus, a genotype 2a HCV cloned from a rare case of fulminant hepatitis C. Very recently, the construction of intragenomic and intergenomic hepatitis C virus chimeras using JFH-1-derived sequences,19Pietschmann T Kaul A Koutsoudakis G Shavinskaya A Kallis S Steinmann E Abid K Negro F Dreux M Cosset FL Bartenschlager R Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.Proc Natl Acad Sci USA. 2006; 103: 7408-7413Crossref PubMed Scopus (614) Google Scholar the recovery of infectious JFH-1 virus from infected chimpanzee,20Lindenbach BD Meuleman P Ploss A Vanwolleghem T Syder AJ McKeating JA Lanford RE Feinstone SM Major ME Leroux-Roels G Rice CM Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.Proc Natl Acad Sci USA. 2006; 103: 3805-3809Crossref PubMed Scopus (378) Google Scholar and the transfection of Huh-7.5 cells with genotype 1a H77-S virus with five adaptive mutations have been reported.21Yi M Villanueva RA Thomas DL Wakita T Lemon SM Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells.Proc Natl Acad Sci USA. 2006; 103: 2310-2315Crossref PubMed Scopus (329) Google Scholar Nevertheless, the development of a system that can sustain the replication of HCV of various genotypes in nontransformed hepatocytes, after either transfection of nonchimeric virus or exposure to serum of patients infected with HCV virus, remains a challenging priority.22Berke JM Moradpour D Hepatitis C virus comes full circle: production of recombinant infectious virus in tissue culture.Hepatology. 2005; 42: 1264-1269Crossref PubMed Scopus (7) Google ScholarIacovacci et al8Iacovacci S Manzin A Barca S Sargiacomo M Serafino A Valli MB Macioce G Hassan HJ Ponzetto A Clementi M Peschle C Carloni G Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.Hepatology. 1997; 26: 1328-1337PubMed Google Scholar had reported the detection of replicative forms of HCV in human fetal hepatocytes (HFHs) exposed to serum of HCV-infected patients, indicating that HFHs are permissive for HCV replication. We have established and characterized long-term, serum-free primary and passaged cultures of nontransformed hepatocytes from human fetal liver, and recently isolated multipotent progenitor cells from these cultures.23Lázaro CA Croager EJ Mitchell C Campbell JS Yu C Foraker J Rhim JA Yeoh GCT Fausto N establishment, characterization and long-term maintenance of cultures of human fetal hepatocytes.Hepatology. 2003; 38: 1095-1106Crossref PubMed Scopus (161) Google Scholar, 24Dan YY Riehle KJ Lazaro C Teoh N Haque J Campbell JS Fausto N Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.Proc Natl Acad Sci USA. 2006; 103: 9912-9917Crossref PubMed Scopus (268) Google Scholar We have used the HFH culture system developed in our laboratory to determine whether HCV replication can be sustained after either transfection of these cells with cloned virus or infection with patients’ sera. We show that HFHs can sustain HCV replication after transfection with genotype 1a HCV or infection by patient sera of HCV genotypes 1, 2, and 3. After transfection or infection, high HCV titers were detected in the medium for at least 2 months, generally with a cyclical pattern, and viral-like particles were released into the medium. Viral infection could be transmitted to naive cells in a Transwell culture system, and the infection was abolished by exposure of the cells to interferon (IFN)-α or IFN-λ.Materials and MethodsCell Isolation and CultureLivers at 90 to 117 days of gestation were obtained from the Central Laboratory of Embryology at the University of Washington as approved by the University of Washington Institutional Review Board. Cells were cultured on collagen plates as previously described23Lázaro CA Croager EJ Mitchell C Campbell JS Yu C Foraker J Rhim JA Yeoh GCT Fausto N establishment, characterization and long-term maintenance of cultures of human fetal hepatocytes.Hepatology. 2003; 38: 1095-1106Crossref PubMed Scopus (161) Google Scholar for a minimum of 5 days and as long as 3 months before transfection with in vitro-transcribed RNA or infection with patient sera.Preparation of HCV RNAHCV genomic strand RNA (referred to as WT HCV RNA) was transcribed from the full-length HCV cDNA construct p90/HCVFLpU of genotype 1a, following the procedure of Kolykhalov et al.25Kolykhalov AA Agapov EV Blight KJ Mihalik K Feinstone SM Rice CM Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.Science. 1997; 277: 570-574Crossref PubMed Scopus (621) Google Scholar The ratio of RNA to DNA in the purified transcripts was 100,000:1 as determined by 10-fold serial dilution and amplified in the presence or absence of the reverse transcription step. The amount of the purified transcript was measured using RediPlate 96 RiboGreen RNA quantitation kit (Molecular Probes, Inc., Eugene, OR) and Packard Fusion Universal Microplate analyzer (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA). The 3′-UTR mutant RNA, in which the entire 3′-UTR and 52 amino acids of the C-terminal region of NS5B was deleted (428-bp deletion), was transcribed by digesting the full-length cDNA template with NotI that has a single recognition site near the 3′ end of HCV cDNA. The NS5B mutant RNA was transcribed from the full-length construct deleted of 12 amino acids (CTMLVCGDDLVV) in the NS5B polymerase active site.Transfection ProceduresFor transfection of wild-type (WT) or mutant HCV RNA, cells were rinsed with Opti-MEM medium (Invitrogen Corporation, Carlsbad, CA) and incubated with Lipofectin-RNA complex containing 1 μg of RNA per 35-mm dish. After incubation for a minimum of 5 hours at 37°C, the cells were rinsed 6 to 10 times with Hanks’ balanced salt solution (HBSS). The final wash was collected, and the medium was changed to feeding medium. The final wash contained negligible or no detectable amounts of HCV RNA. The medium was completely replenished at each feeding daily during the first 10 days, every 2 days from 12 to 20 days, and every 4 days thereafter.Infection of HFHs with Sera from HCV-Infected PatientsHCV-positive serum was obtained from patients with chronic or post-transplant HCV infection at the University of Washington, as approved by the Institutional Review Board of the University of Washington. Sera from individual patients and pooled sera from multiple HCV-infected donor of the same genotype were included.For serum infection, 50 μl of patient serum diluted in 1.5 ml of medium was added to cells plated in 35-mm dishes. After overnight incubation, the cells were rinsed 6 to 10 times with HBSS and 2 ml of fresh growth medium was added. The schedule of medium changes was the same as that for transfection experiments. The final wash was collected for HCV testing (shown as time 0 in the figures).Infection by Culture Medium Using a Transwell SystemFor these experiments, cultures transfected with WT HCV or the NS5B mutant RNA were cultured on collagen-coated (Vitrogen; Cohesive Technologies, Palo Alto, CA) transparent high-pore density polyethylene terephthalate track-etched membranes and in deep well dishes (Becton Dickinson Labware, Franklin Lakes, NJ). A 3.0-μm pore density was used to permit virus diffusion. At 16 and 37 days after transfection, the cell inserts were removed and placed in six-well plates that contained naïve HFHs from a different isolate. Transfected and naïve cells were cultured for 2 days in the same medium and then separated. Medium was collected from the infected cultures using the same collection schedule as described above.In some experiments, naïve cells were treated with 1 IU/ml recombinant human IFN-α (Biosource, Camarillo, CA) or 100 ng/ml recombinant human IFN-λ1 or IFN-λ2 (PreproTech Inc., Rocky Hill, NJ) beginning 1 day before co-culture. After the separation of the cultures, the infected cells were maintained in medium containing the appropriate IFN.RNA Extraction and Quantification of HCV RNAHCV RNA was isolated from culture medium using a QIAamp Virus BioRobot MDx kit (Qiagen, Valencia, CA). HCV-positive sera and unused culture medium were processed along with the samples to serve as the positive and negative controls, respectively. Extracted RNA was amplified using Taqman EZ RT-PCR Core reagents, and the amount of product was quantified by monitoring the increase in fluorescence of a FAM-labeled oligo probe using ABI PRISM 7700 or 7900HT real-time sequence detection system (Applied Biosystems, Foster City, CA). The amplification primers are located in HCV 5′-untranslated region and their sequences are 5′-CATGCCCCCGCAAGA-3′ (129F) and 5′-ACCCTATCAGGCAGTACCACAAG-3′ (199R). The sequence of the probe is 6-FAM-CATGCCGAGTAGCGTTGGGTTGCG-6-TAMRA. The thermal cyclic profile was 50°C for 2 minutes, 60°C for 30 minutes, 95°C for 2 minutes, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. Along with the sample RNAs, 10-fold serial dilutions of HCV WT RNA were amplified to serve as standards. The standard WT RNA was digested with DNase I to remove DNA and quantified by the RediPlate 96 RiboGreen RNA quantitation kit. The copy number of WT RNA was calculated using the concentration and the molecular weight of WT RNA.HCV RNA extraction by a QIAamp Virus BioRobot MDx kit and amplification using the Taqman method was calibrated in international units (IU)/ml. Serial dilutions of HCV standard serum (OptiQual HCV RNA High Positive Control, 2,000,000 IU/ml; AcroMetrix, Benicia, CA) were extracted and amplified. The linear range of this quantitative assay is from 40 to 200,000 IU/ml; 1 IU is approximately equivalent to 2.2 copies of WT RNA prepared by our laboratory. The detection limit of this assay is 10 IU/ml, which corresponds to approximately 25 copies per ml.Strand-Specific in Situ HybridizationTo detect HCV RNA in transfected or infected cells, HFHs were cultured on collagen-coated chamber slides (Nalgen Nunc International, Naperville, IL). The hybridization procedures as well as the controls have been previously described in detail.26Chang M In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google ScholarImmunohistochemistry (IHC) and ImmunofluorescenceTo detect HCV proteins in transfected or infected cells, HFHs were cultured on collagen-coated chamber and fixed with paraformaldehyde. HCV core protein was detected by IHC using the C7-50 monoclonal antibody (mAb) (subtype IgG1; Affinity Bioreagents, Golden, CO) and the ABC kit. Substitution of the primary antibodies with mouse IgG (Vector Laboratories, Burlingame, CA) was used as a control for the staining.For immunofluorescence, NS3 antibody (subtype IgG2b; Austral Biologics, San Ramon, CA) and fluorescent Alexa Fluor 594 goat anti-mouse IgG2b (Molecular Probes) were used. Nuclei were stained with blue fluorescent 4,6-diamidino-2-phenylindole (DAPI). A Nikon Eclipse E600 microscope with a QImaging Retigia EX CCD camera was used to capture black and white images of fluorescent signals. Green and blue colors were assigned to the images of NS3-positive signals and nuclei of cells, respectively.Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) and Viability AssaysTUNEL assay (in situ cell death detection kit; Roche Diagnostics, Indianapolis, IN) was performed according to the manufacturer's directions. Cell viability assays were performed using the Live/Dead Viability Cytotoxicity kit (Molecular Probes). The kit contains fluorescent calcein AM and ethidium homodimer-1. In viable cells, intracellular esterases hydrolyze calcein AM to calcein (green fluorescence). Ethidium homodimer-1 penetrates the membrane of dying cells and binds to DNA (red fluorescence). Cells in sterile glass slides were stained for 10 minutes, and the slides were examined in a fluorescence microscope.Equilibrium Density Gradient CentrifugationSucrose solutions (60, 50, 40, 30, and 10% w/v) prepared in NTE buffer (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, and 1 mmol/L EDTA) were sequentially loaded into Beckman polyallomer centrifuges tubes. One milliliter of culture supernatant was layered on the sucrose solutions, and a density gradient was generated by centrifuging at 315,000 rpm for 16 hours in an Optima ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). HCV measurements were done in sequential collections of 500 μl. The sugar content of each fraction was measured using a Leica ABBE Mark II refractometer (Reichert Analytical Instruments, Depew, NY).Electron MicroscopyFor all experiments, 400-mesh Formvar carbon-coated electron microscope nickel or copper grids (Electron Microscopy Sciences, Ft. Washington, PA) were glow-discharged before use. HCV cultures, filtered through a 1.0-micron membrane, were deposited onto grids by ultracentrifugation using a Beckman Airfuge with an EM 90 rotor (Beckman, Palo Alto, CA) at 26 lb/in2 for 30 minutes. Goat antibody against HCV 1a envelope protein E2 (Biodesign International, Saco, MA), diluted 1:10, and 10 nm of colloidal gold conjugate anti-goat IgG at a 1:25 dilution (Aurion, Wageningen, The Netherlands) were used for immunogold labeling. The controls included samples treated with goat anti-mouse IgG (Vector Laboratories) and omission of the primary antibody. Viral particles were negatively stained with 1% uranyl acetate and examined in a JEOL JEM 1230 transmission electron microscope (JEOL Inc., Peabody, MA).ResultsViral Replication in HFHs Transfected with HCV RNAAfter transfection with WT RNA, HFHs shed HCV into the culture medium for 64 days in a cyclical pattern, with peaks at 6, 16, 24, 40, and 64 days after transfection (Figure 1A). Fluctuation on HCV levels have been observed both in infected chimpanzees and in Huh-7.5 line infected with a chimeric JFH1 genome18Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1943) Google Scholar, 20Lindenbach BD Meuleman P Ploss A Vanwolleghem T Syder AJ McKeating JA Lanford RE Feinstone SM Major ME Leroux-Roels G Rice CM Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.Proc Natl Acad Sci USA. 2006; 103: 3805-3809Crossref PubMed Scopus (378) Google Scholar and may reflect the effect of host responses to the virus, as discussed below. Although in our experiments the cyclical pattern of virus detection was most commonly observed after transfection of WT HCV RNA, virus persistence with a continuous pattern occurred occasionally (data not shown). In either the cyclic or the continuous pattern of virus detection, HCV levels in the medium reached high concentrations ranging from 105 to 107 copies/ml during the 2-month culture period. In marked contrast, in HFH cultures transfected with mutant HCV RNAs, either deleted of 3′-UTR or the NS5B catalytic motif (see Materials and Methods), HCV RNA levels progressively declined, and virus was no longer detectable in the medium 24 days after transfection (Figure 1B). The progressive decline of virus levels after transfection of HCV mutant viruses reported here is almost identical to the pattern described by Wakita et al16Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2400) Google Scholar for Huh-7 cells transfected with JFH1 mutants. Measurements of viral levels in cells and the culture medium revealed that nonreplicating viruses are slowly released from the cells into the medium for up to 30 days.16Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2400) Google ScholarWe used strand-specific in situ hybridization to detect the presence of negative-strand HCV RNA in cells transfected with WT and mutant HCV.26Chang M In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.J Virol. 2000; 74: 944-955Crossref PubMed Scopus (55) Google ScholarIn situ hybridization for HCV negative-strand RNA in cultures transfected with WT HCV RNA revealed clusters of cells with strong cytoplasmic staining (Figure 2, A and B). In contrast, staining was barely detectable in cells transfected with NS5B mutant RNA (Figure 2C), and only a few weak positive cells and cell debris were detected in the cultures transfected with 3′-UTR mutant RNA (Figure 2D). We speculated that truncated, functional RNA polymerase proteins might have been generated after 3′-UTR mutant RNAs were transfected,27Ferrari E Wright-Minogue J Fang JW Baroudy BM Lau JY Hong Z Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli.J Virol. 1999; 73: 1649-1654Crossref PubMed Google Scholar, 28Lohmann V Korner F Herian U Bartenschlager R Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.J Virol. 1997; 71: 8416-8428Crossref PubMed Google Scholar and produced a small amount of negative-strand RNAs in a nontemplated manner.29Ranjith-Kumar CT Gajewski J Gutshall L Maley D Sarisky RT Kao CC Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis.J Virol. 2001; 75: 8615-8623Crossref PubMed Scopus (95) Google Scholar, 30Ranjith-Kumar CT Sarisky RT Gutshall L Thomson M Kao CC De novo initiation pocket mutations have multiple effects on hepatitis C virus RNA-dependent RNA polymerase activities.J Virol. 2004; 78: 12207-12217Crossref PubMed Scopus (28) Google Scholar Core protein expression occurred in clusters of HFHs transfected with WT RNA surrounded by cells that exhibited little or no immunoreactivity and was localized to the cell cytoplasm in a punctated pattern (Figure 2E). However, no staining of core protein was detected in cultures that were transfected with the 3′-UTR mutant RNA (Figure 2F). Core staining in cells transfected with WT RNA was not detectable until 4 days after transfection, but high expression was present at days 8 and 16, demonstrating a fluctuating pattern (not shown). Cytoplasmic NS3 was detectable by immunofluorescence in clusters of cells transfected with WT HCV RNA (Figure 3).Figure 2Detection of HCV negative-strand RNA by strand-specific in situ hybridization and expression of core protein in transfected HFHs. HFHs transfected with WT RNA (A and B), NS5B mutant RNA (C), or 3′-UTR RNA (D) were fixed on slides 11 days after transfection for detection of HCV negative-strand RNA by strand-specific in situ hybridization. Digoxigenin-labeled riboprobes were detected using an antibody conjugated to alkaline phosphatase with Vector Red as the substrate. Cells were counterstained with methyl green. HCV negative-strand RNA (red staining) was detected in clusters of cells (A) and was localized to the cytoplasm (B). Little or no staining was detected in cultures transfected with 3′-UTR or NS5B mutant HCV RNAs (D and C, respectively). Core protein expression demonstrated by immunohistochemistry was localized to the cytoplasm with a punctated distribution in cultures transfected with WT RNA (E). Cells transfected with 3′-deleted mutant HCV showed very faint staining (F). Original magnifications, ×4 (A); ×100 (B–D); ×40 (E–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Staining HCV NS3 proteins in transfected HFHs. HFHs were transfected with WT HCV RNA and examined for the expression of NS3 proteins 12 days after transfection. A shows the superimposed images of DAPI nuclear staining and fluorescent staining of NS3 protein; B is a negative control without NS3 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of HCV Recovered from Cell Culture SupernatantsTo determine whether HCV virions could be recovered from the culture medium of HFHs transfected with WT HCV RNA, we examined the buoyant density distribution of HCV-RNA in two samples of medium collected at days 5 and 40 after transfection. At these times, viral levels in the medium were in the range of 104 HCV copies/ml (Figure 4A). Sucrose gradient density centrifugation showed that the day 5 sample contained HCV distributed at varying densities, with one of the main fractions having a density of 1.12 g/ml (Figure 4B). By contrast, in the medium collected 40 days after transfection, particles had a homogeneous distribution at a density of 1.17 to 1.18 g/ml (Figure 4C), similar to the value reported by Wakita et al16Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizoka" @default.
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• W1997097856 title "Hepatitis C Virus Replication in Transfected and Serum-Infected Cultured Human Fetal Hepatocytes" @default.
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• W1997097856 doi "https://doi.org/10.2353/ajpath.2007.060789" @default.
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