Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis

Authors

Errata

This article is corrected by:

  1. Errata: Erratum: Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis Volume 53, Issue 2, 720, Article first published online: 27 January 2011

  • Potential conflict of interest: Nothing to report.

Abstract

Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease. Hepatology 2010;51:1922–1932

Studies of normal human liver functions and host response to hepatic pathogens or hepatotoxins has been hampered by low yield of primary hepatocyte cultures, limited replication capacity of primary cells, and the inability to sustain differentiated liver cell function in vitro cultures. We developed procedures for cultivating functionally stable primary hepatocytes derived from human donor tissues and validated their physiological relevance based on the ability to support productive infection with hepatitis C virus (HCV). The culture system offers significant improvement for maintaining viable cultures of human primary hepatocytes, which will be suitable for hepatotoxicity studies, and should significantly improve studies of host response to hepatic pathogens.

HCV infection is a major cause of chronic hepatitis and hepatocellular carcinoma (HCC). Studies of the virus life cycle have been constrained by the fact that HCV infects only humans and chimpanzees, and until recently, cell culture systems for the replication of the full-length HCV genome was limited to JFH-1 isolate (genotype 2a). Virus replication in hepatoma cell lines required culture-adaptive mutations; thus, most of our understanding of HCV replication and virus-host interactions is based on subgenomic and genomic replicons.1-3 Of necessity, these studies relied on surrogate experimental systems that only approximate the virus-host interaction in natural infection.2

A significant advance in studying HCV replication in cell cultures came from the identification of JFH-1 isolated from a Japanese patient with fulminant hepatitis.4 The JFH-1 replicons do not require adaptive mutations for efficient viral RNA replication in cell cultures.5 Further advances in HCV replication in vitro came from successful replication of the full-length genomic clone of JFH-1, or chimeric constructs derived from the JFH-1, J6-JFH (genotype 2a).6-9 Buck10 described efficient replication of HCV isolates from patients' sera in human primary hepatocytes. A better understanding of host-pathogen interactions, however, would require normally differentiating long-term cultures of primary human hepatocytes that support productive infection with HCV strains.

We developed long-term cultures of primary human hepatocytes that express markers of normal hepatic differentiation and support efficient replication of infectious HCV clones. Virus recovered from the culture media of infected cells established an entry factor, CD81-dependent infection of naïve primary hepatocytes. These properties, along with the sensitivity to interferon (IFN), suggest that HCV replication in primary hepatocyte cultures could serve as physiologically relevant in vitro model for studies of HCV pathogenesis and development of potent antiviral measures.

Abbreviations:

Alb, albumin; Alpha-FP, alpha-fetoprotein; BrdU, Bromodeoxyuridine, Br-UTP, 5-Bromo Uridine 5'-Triphashate; CFSC-8B, fat-storing stellate cell line-8B; CK-18, cytokeratin-18; DMEM, Dulbecco's Modified Eagle Medium; ECM, extracellular matrix; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HCV, hepatitis C virus; HDM, hepatocyte-defined medium; HSA, hepatocyte-specific antigen; HSC, hepatic stellate cell; IFN-α R1, 2, interferon alpha-receptors 1 and 2; IgG, immunoglobulin G; ITS, insulin-transferrin sodium selenite; MEM, minimal essential medium; MMP-2, MMP-13, matrix metalloproteinases 2 and 13; PPH, postattachment primary hepatocyte; RT-PCR, reverse-transcriptase polymerase chain reaction; TGF-β 1, transforming growth factor-beta.

Materials and Methods

Primary Hepatocyte Coculture.

Hepatic stellate cell line (CFSC-8B), used as a feeder cell layer, was plated at 106 cells per 25 cm2 culture flasks in minimal essential medium (MEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), antibiotics (penicillin/streptomycin 100 IU/mL; Quality Biological), and 1% nonessential amino acids (Cellgro). After 2 hours the MEM was replaced and 5 × 106 freshly isolated human hepatocytes suspension (Cambrex, Walkersville, MD) was seeded over the feeder cell line in a hepatocyte-defined medium (HDM, human hepatocyte growth medium). Following 3 hours incubation, and monitoring cell attachment, the hepatocyte cocultures were grown in HDM comprised of: Dulbecco's modified Eagle medium (DMEM) with D-glucose 0.45%, L-glutamine 5 mM, without sodium pyruvate, and supplemented with albumin 0.2%, glucose 0.2%, galactose 0.2%, ornithine 0.01%, proline 0.003%, nicotinamide 0.061%, insulin-transferrin-sodium selinite (ITS, Sigma) 0.1%, ZnCl2 0.544 μg/mL, ZnSO4 0.75 μg/mL, CuSO4 0.2 μg/mL, MnSO4 0.025 μg/L, transforming growth factor-alpha (TGF-α) 20 ng/mL, and penicillin 100 units/mL, streptomycin 100 μg/mL, and dexamethasone 10−7 M. The HDM culture medium was replaced every 48 hours. Primary hepatocyte cultures form spherical masses after 30 days in coculture. The hepatocyte cultures containing spherical masses are harvested with 0.05% trypsin in HDM (supplemented with 1% FBS) and reseeded at 5 × 104 cells/six-well plates and propagated in HDM (the plating efficiency was 90-95%). The monolayer cultures in six-well plates (which were used for most experiments) were reseeded at 10-day intervals. The “maintenance cultures” when allowed to grow for more than 4 weeks in HDM reform the spherical masses (from which monolayer culture of primary hepatocyte was derived as outlined). The “passage” numbers of postattachment primary hepatocytes (PPH) cultures refer to reseeding from the reformed primary hepatocyte containing spheroids in maintenance cultures. Experiments described in these studies used various “passages” (passages denote reseeding from “maintenance” cultures). PPH cells denote the sustained phenotype of primary hepatocyte cultures.

HCV Permissive Properties of Primary Human Hepatocytes.

To assess whether the primary hepatocyte culture support HCV replication we transfected the cells with either the wildtype (pI/5A-GFP), or the polymerase-minus mutant (pPol /5A-GFP) of HCV-GFP constructs.17 The runoff transcripts (1 μg each) of the wildtype or the polymerase mutant were transfected (FuGENE-6, Roche) into primary hepatocyte; 24 hours after transfection the medium was replaced with defined growth media (HDM) and the cells were allowed to grow for the indicated period (HDM was replaced at 3-day intervals). To visualize nascent GFP-tagged viral proteins, cells were fixed with 4% paraformaldehyde and observed at 486 nm.

To determine the replication of nascent viral RNA, transfected cells were metabolically labeled with Br-UTP (25 mM) in the presence of actinomycin D (5 μg/mL, added 30 minutes prior to Br-UTP to inhibit cellular DNA-dependent RNA polymerases). Following Br-UTP incorporation for 45 minutes (at 37°C in 5% CO2 incubator), the unincorporated Br-UTP was “chased” with fresh HDM media supplemented with 5% FBS for 30 minutes. The cultures were then washed with phosphate-buffered saline (PBS), the cells were fixed with 4% paraformaldehyde, and free formaldehyde groups were blocked with 0.2% glycine for 10 minutes. The cells were then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes, treated with 5% normal donkey serum in PBS for 40 minutes at 4°C, and incubated with primary antibody (anti-bromodeoxyuridine mouse IgG monoclonal PRB-1, Molecular Probes, 1:500 dilution) and secondary Alexa Fluor 568 goat antimouse IgG (H+L) conjugated to Texas red (Molecular Probes, 1:80) for 1 hour at room temperature.

Replication of Genomic Clones of HCV Genotypes 1a, 1b, and 2a.

To validate the replication of full-length HCV genomic clones in human hepatocyte cultures (PPH), run-off transcripts of HCV genotype 1a (pCV-H77c), HCV genotype 1b (pCV-J46LS), and HCV genotype 2a (pJ6CF-2a)21 were introduced by transfection (1 μg viral genomic RNA/106 cells; FuGENE-6, Roche). Virus replication was followed by analysis of viral RNA within total cell RNA with nested reverse-transcription polymerase chain reaction (RT-PCR) reaction using primers as described22; viral RNA genomic equivalents (GE) were estimated on the basis of real-time quantitative RT-PCR (Applied Biosystems), using World Health Organization (WHO) HCV standards (Acrometrix, Benicia, CA).

Infection of Naïve Hepatocytes with the Virus from Cell Culture Media.

To evaluate whether infection competent HCV particles were released in culture media of cells transfected with full-length genomic RNAs of HCV genotypes 1a, 1b, and 2a, conditioned media from the transfected PPH cultures (filtered through 0.25 micron filters) were used to inoculate (with equal GE virus RNA) naïve PPH cultures (1 mL/106 cells) in the presence of 12 μL of lipofection solution (FuGENE-6, Roche). Viral RNA replication was evaluated at indicated times after infection, and the efficiency of virus released in the culture media was validated with WHO HCV standards (Acrometrix).

RNA Isolation, Reverse-Transcription, and Quantitative PCR.

Total cell RNA was extracted using Trizol LS Reagent (Invitrogen) according to the manufacturer's instructions. The purity of total cell RNA was determined by Agilent RNA 6000 Nano chip Bioanalyzer (Applied Biosystems). The purified RNA was used for the real-time quantitative PCR using the 7300 Real-Time PCR System (Applied Biosystem). The standardization of HCV particles (international units, IU) was carried out using WHO HCV standards with the Taq Man Kit (Acrometrix). Viral RNA amplification was performed in a total volume 50 μL containing 25 μL of 2× master Mix, 1.25 μL of 4× RNase inhibitor, forward primer (50.0 μM) 0.5 μL, reverse primer (100.0 μM) 0.5 μL, probe (25.0 μM) 0.5 μL, and water 12.25 μL. Each 50 μL reaction contained 40 μL master mix plus 10 μL of test sample (RNA) in PCV tubes. All test samples were run in duplicate along with the WHO HCV standard. Data were analyzed using the 7300 Real Time software program. The following primers and probe were used for amplification of HCV1a virus. Primers HCV- Con256F-AGYGTTGGGTYGCGAAAG; HCV-ConR-CAC TCG CAA GCR CCC T and probe HCV-278MGB-CCT TGT GGT ACT GCC TGA. Results are presented in GE and IU of HCV.

RNase-Protection Assay (RPA) for ± Sense Viral RNAs.

The RPA probes were prepared by cloning a 344-basepair Hind III / Eco R1 fragment of NS5B region from pH77c into a pSP72 vector as indicated in Fig. 6A. The total cell RNA from mock-infected or HCV1a-infected cells, or the RNA from virus particles recovered from the culture medium of HCV1a-infected cells, were purified as outlined above. The labeling of the probe, the hybridization (5 μg total cell RNA, hybridized with 2 × 104 CPM of either positive or negative strand probe), and the analysis of RNase protected fragments was carried out according to the Ambion RPA III kit.

Western Blot Analysis of HCV1a Core and NS5A Protein.

PPH culture was infected with HCV1a virus in triplicate. On day 6 the total cell protein was separated by 4-12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The blots were first blocked with 5% nonfat dry milk in Tris-Hcl buffer (pH 7.5) saline containing 0.1% Tween-20 (TBST) and then probed with the primary antibodies against HCV core protein (mouse monoclonal C7-50, ab 2740-100), and HCV NS5a (388) protein (mouse monoclonal C7-50, ab 20342-100) for 1 hour. After extensive washes the blots were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour and washed four times with TBST. Protein bands were developed by enhanced chemiluminescence reagent and were detected using x-ray film.

Statistical Analysis and Reproducibility.

The results are given as the mean ± standard error. Statistical analysis of the data was performed by using Student's t test, Fisher's Exact test, or otherwise as described.

Results

HCV Permissive Hepatocyte Culture.

A common theme in primary hepatocyte cultures has been the contribution of nonparenchymal cells that stabilize liver-specific functions and hepatic differentiation.11, 12 We pursued primary hepatocyte cultures by adherence to preattached stellate cells (CFSC-8B) that abundantly produce extracellular matrix.13 Freshly isolated primary human hepatocytes in suspension (supplied by a commercial source, Cambrex; Supporting Information Table S1) was seeded onto an ≈30% confluent culture of stellate cells (in MEM supplemented with 5% FBS). After 2 hours the MEM was replaced with HDM, lacking FBS. During this period of growth, primary hepatocytes form distinct spherical masses that can be dispersed with 0.05% trypsin and propagated (independently of the stellate cell layer) for up to 6 months as PPH. The spherical masses of primary hepatocytes reform in monolayer cultures that are maintained for over 4 weeks, termed “maintenance” cultures (maintenance cultures are grown either in 25 cm2 flasks or in six-well plates). For the experiments described here, monolayer cultures of primary hepatocytes were grown in six-well plates reseeded from the maintenance cultures; monolayer cultures were grown in HDM by repeated passages at 10-day intervals.

We first validated the depletion of stellate cell layer by monitoring GFP-positive stellate cells in cocultures with primary hepatocytes (Fig. 1A). Quantitative assessment of the GFP-tagged feeder cells showed progressive elimination of stellate cells within 4 weeks (Fig. 1B). A schematic representation of the primary hepatocytes cultures used in these studies is shown (Fig. 1C).

Figure 1.

(A-C) Stellate cells were transduced with lentiviral vector expressing GFP under PGK promoter for tracking the depletion of feeder cells in cocultures in HDM medium. GFP-positive CFSC 8B cells in MEM medium (MEM supplemented with 5% FBS) prior to coculture with primary human hepatocytes (A, panel a); 12 days in coculture (panel b), and 21 days in coculture (panel c). Panel (d) shows a phase-contrast image of the 21-day coculture. (B) Depletion of the GFP-positive feeder cell layer was quantitated (by cell count) during the 30-day coculture with primary hepatocytes in HDM medium. (C) Schematic representation of the primary hepatocyte culture system.

To ascertain whether the primary hepatocytes retain proliferative capacity, we tagged the replicating cells in vivo with CFSE (carboxyfluorescein diacetate, succinimidyl ester, CellTrace, Molecular Probes; Fig. 2). The succinimidyl ester group of CFSE reacts with intracellular amines, forming fluorescent conjugates that are retained through cell division. The dye-protein adducts formed in CFSE-labeled cells are diluted through cell replication. As a control, we analyzed 30-minute CFSE pulse-labeled cells that were synchronized (by Nocodazole treatment to arrest cells at G2/M phase of cell cycle), followed by 24-hour growth in HDM (Fig. 2A). In parallel, a 45-day-old unsynchronized culture of primary hepatocytes was similarly pulse-labeled with CFSE and the dye was “chased” by allowing cell proliferation in HDM medium for 6 days (Fig. 2B). Replication of the primary hepatocytes in PPH cultures was also monitored by bromodeoxyuridine (BrdU) incorporation in a 30-minute pulse followed by a 3-day chase (Supporting Information Fig. S1, Supporting Information Materials).

Figure 2.

Primary human hepatocytes (45-day-old PPH cultures) were stained with CellTrace CFSE (Invitrogen). (A) As a control, primary hepatocytes were synchronized at G2/M with Nocodazole (1 μg/mL for 30 minutes) and chased in HDM medium for 24 hours. The synchronized cells undergo one cell generation. (B) Unsynchronized cells were similarly pulse-labeled with CSFE for 30 minutes followed by chase in fresh HDM medium for 6 days. Dilution of CFSE in successive generations of cells is shown (based on a computer enhanced program, ModFit LT, Verity Software House, Topsham, ME).

Morphologic Features of PPH Cultures.

Figure 3 (upper panels), shown as phase contrast images of a 180-day-old maintenance culture, suggests vertical accumulation of primary hepatocytes in the maintenance cultures. The spherical masses of primary hepatocytes show morphologic properties of hepatocytes in normal liver tissues (Fig. 3, lower panels). Histological (hematoxylin and eosin [H&E]-stained) sections through the spheroid mass indicates clusters of binucleated cells (lower panel, A,B). Immunohistochemical staining for factor VIII indicated sinusoidal endothelial cell lining (lower panel, C).

Figure 3.

Morphologic features of primary human hepatocyte (PPH) cultures. (A, upper panel) Maintenance culture (180-day-old from the time they were harvested from the coculture) are shown as phase-contrast images at different magnifications, suggesting three-dimensional growth of primary hepatocytes “spherical mass” (4× magnification). (B) 10× magnification focused on the upper cell layer. (C) 20× magnification of primary hepatocytes showing large nuclei and binucleated cells. Lower panel (A,B) H&E staining of 5-μm sections through the spherical mass of primary hepatocytes showing hepatocytes organized in clusters with defined nucleus and binucleated cells (arrows). (C) Immunohistochemical staining for Factor VIII indicates sinusoidal endothelial cell lining (arrow).

Biochemical Characteristics of PPH Cultures.

To assess whether markers of normal liver cell differentiation are expressed in long-term PPH cultures (ranging in age up to 180 days), we performed RT-PCR assays utilizing primers specific for human transcripts and compared them with the respective rat primers (primer sequences are shown in Supporting Information Table S2). Among the markers of hepatic differentiation we examined expression of plasma proteins (albumin [Alb], alpha-fetoprotein [α-FP], epithelial markers cytokeratins [CK18], TGF-β [Supporting Information Fig. S2A], type-1 collagen, matrix metalloproteases [MMP] 2 and MMP13), IFN receptor (IFN-R1 and IFN-R2, Supporting Information Fig. S2C), and drug metabolism (cytochrome P450) (Supporting Information Fig. S2D).

Expression of these hepatic markers, along with the endothelial cell marker (Factor VIII, Fig. 3C), suggests an overall capacity of the primary hepatocytes to sustain normal liver function during their long-term cultures in HDM, arguing that the human primary hepatocytes maintained in in vitro cultures assumed morphologic features and functional characteristics of liver differentiation in vivo. The expression of hepatic markers in the primary hepatocytes was independent of measurable contributions from the feeder cell layer.

Ethanol-induced hepatocyte injury in great part is due to generation of reactive oxygen species (ROS) on exposure of hepatocytes to ethanol, as it is oxidized by enhanced cytochrome P450 2E1 (CYP2E1).14, 15 To determine whether the primary human hepatocytes propagated in vitro cultures will be suitable for alcohol toxicity studies, we evaluated CYP2E1 expression by western blotting. Supporting Information Fig. S2D shows an accumulation of CY2E1 in primary hepatocytes during a 3-week growth in HDM. For comparison we observed the accumulation of albumin in the same cells (Supporting Information Fig. S2D, lower panel).

We analyzed the expression CK18 in freshly plated primary hepatocytes and compared it with cells grown for 30 days in cocultures. As controls, we compared the expression of albumin and AFP in freshly plated and 30-day-old cocultures (Supporting Information Fig. S3). Low levels of CK18 synthesis were detectable in freshly cocultured primary hepatocytes; however, we observed an induction of CK18 expression in 30-day cocultures. Overall, the results define phenotypic restoration of long-term cultures of primary hepatocytes consistent with normal hepatic functions.

In addition to comparing the synthesis of CYP 2E I and CK-18 in freshly plated primary hepatocytes and 30-day-old cultures, we analyzed the expression of hepatic functions in normal and HCV1a-infected cells from long-term cultures (Supporting Information Tables S3, S4). In contrast to the results reported by Richert et al.,16 where hepatic functions are drastically reduced after reseeding, our PPH culture maintained hepatic functions through repeated reseeding in long-term culture.

Primary Hepatocyte Cultures Are Permissive to HCV-GFP Replication.

To ascertain whether they support HCV replication, we introduced run-off transcripts of full-length HCV-GFP replicons17 into PPH cultures. An increase in the GFP-positive foci is observed at 3-6 days posttransfection (Supporting Information Fig. S4). As control, cells transfected with the polymerase mutant virus showed background GFP fluorescence (Supporting Information Fig. S4).

Newly synthesized viral RNA was visualized by Br-UTP incorporation. Virus replication was carried out in the presence of 5 μg/mL actinomycin-D (to suppress Br-UTP incorporation into host cell transcripts); as a result, we observed background level of Br-UTP incorporation in uninfected cells (Fig. 4, control). Cells transfected with polymerase mutant showed no BrUTP incorporation (Fig. 4[I], pPol/5A), as compared with cells infected with wildtype HCV (Fig. 4[I], pI/5A). The Br-UTP-labeled viral RNA colocalized with GFP-positive foci in the cytoplasmic structures (Fig. 4[II]).

Figure 4.

(I, upper panel) Br-UTP labeled nascent HCV RNA detected (by immunostaining with anti-Br-UTP antibody): (A) Uninfected host cell control: primary hepatocyte cultures were incubated with 5 μM actinomycin D for 30 minutes prior to 45 minutes Br-UTP (25 μM) incubation at 37°C, followed by two 15-minute chases with fresh medium supplemented with 5% FBS. (B) Wildtype HCV transcript (3 days posttransfection) was detected (by immunostaining with anti-Br-UTP antibody) in either the “wildtype” HCV transfected cells (pI/5A) or (C) The polymerase mutant (pPoI/5A) HCV RNA transfected cells. (II, lower panel) The confocal image of a group of cells showing: (A,E) uninfected control cells; (B,F) Viral RNA (red) in cultures transfected with pI/5A-GFP (wildtype) HCV replicon; (C,G) nascent viral protein (green); (D,H) the viral RNA (red) and protein (green) are merged.

To validate the inhibition of viral RNA accumulation in cells transfected with the polymerase mutants, we compared viral RNA levels in primary hepatocytes 6 days after the introduction of either the wildtype or the polymerase mutant HCV genome. There was marked inhibition of viral RNA accumulation in cells transfected with polymerase mutant (Supporting Information Fig. S5). Overall, the results suggest that primary hepatocyte cultures are permissive for replication of HCV genome.

Replication of Full-Length HCV Genomic RNA in Primary Hepatocyte Cultures.

We next asked whether the primary hepatocytes support replication of genomic clones of HCV by transfecting the PPH cultures with full-length HCV genotype 1a RNA (Fig. 5A). Equal amounts of HCV1a genomic RNAs were introduced into PPH cultures using the FuGENE (Roche) transfection method. Transfection efficiency was monitored by cotransfection with a control GFP expression plasmid. We noted the accumulation of viral RNA in HCV transfected primary hepatocytes over a 6-day period. The input viral RNA was detected in cells immediately following transfection (shown as “Day 0” in Fig. 5A). The results indicate an accumulation HCV RNA in the primary hepatocytes over 6 days posttransfection. Control cells transfected with RNA polymerase mutants (Supporting Information Fig. S5) showed no viral RNA accumulation during a similar 6-day period.

Figure 5.

(A) Kinetics of HCV genotype 1a RNA replication: primary hepatocyte cultures were transfected with full-length run-off transcripts of HCV genotype 1a using FuGene 6. Total cell RNA was prepared from infected cells at indicated times. Results represent input viral RNA (0 hours), immediately after transfection. Viral RNA accumulation at 0, 24, 72, and 144 hours after the introduction of full-length genomic RNA was analyzed as indicated. The results are represented as GEs/μg total cell RNA. Estimates of the HCV RNA were normalized with WHO HCV standard. The data shown represent three independent assays. (B) Inhibition of HCV RNA replication by anti-CD-81 monoclonal antibody. (B) Transfection: primary hepatocyte cultures were either preincubated with CD-81 monoclonal antibody (10 μg/mL, Santa Cruz Biotechnology) for 30 minutes prior to transfection or with nonspecific antibody as negative control followed by transfection with full-length genomic RNA of HCV genotype 1a. (C) Infection: primary hepatocytes were preincubated with CD-81 antibody or a nonspecific control as in (B), followed by infection with HCV1a virus (recovered from filtered media of HCV1a-infected cells). Viral RNA was analyzed as in (A). Viral RNA levels are shown from 6 days postinfection. Data represent average of three experiments. P < 0.05. (D) Infection of primary hepatocytes with virus recovered from filtered culture media. Primary hepatocyte cultures were incubated with an equal amount of virus recovered from the culture media of cells transfected with full-length genomic clones of HCV genotype 1a (pCV-H77c) (lane 1), 1b (pCV-J4L6S) (lane 2), and 2a (pJ6CF) (lane 3). Viral RNA replication was determined by RT-PCR 6 days postinfection (as in A). The replication of JFH-1 genomic RNA in primary hepatocytes or Huh 7.5 cells (lanes 4 and 5, respectively) is shown for comparison. The error bars represent the average of three experiments.

CD81 is an essential HCV entry factor expressed on most cells.18 Next, we determined whether its binding to viral envelope glycoproteins, an essential step in HCV infection, is required for the propagation of HCV in PPH cultures. Pretreatment of cells with monoclonal antibody against CD81 prior to transfection with HCV1a genomic RNA inhibited HCV RNA accumulation (Fig. 5B).

In the FuGENE6 transfection protocol, HCV genomic RNA is “artificially” introduced into the PPH cultures. To determine whether CD81 entry factor is important in reinfection of naïve hepatocytes we preblocked the cells with CD81 antibody prior to infection with virus (recovered from the culture medium of cells transfected with HCV1a genomic RNA). Untreated and CD81 entry receptor blocked cells were infected with equal amounts (GE) of virus recovered from the spent medium. HCV infection and propagation in primary hepatocytes appears to be dependent on the availability of CD81 entry receptor (Fig. 5C).

Infection of Naive Cells with Virus from Filtered Culture Media.

Next we asked whether HCV infection could be transferred to naïve cells by inoculating primary hepatocytes with equal amounts (genomic RNA equivalents) of virus recovered from filtered culture media of cells 6 days posttransfection with full-length HCV genotypes 1a, 1b, and 2a. For comparison, we replicated equal amounts HCV JFH-1 full-length genomic RNAs introduced either into the primary hepatocytes or the Huh-7.5 hepatoma cells (Fig. 5D, columns 4 and 5, respectively).

Viral RNA replication in cells infected with three different HCV strains was compared by transfecting PPH cultures with equal amounts of full-length genomic RNAs of HCV genotypes 1a (pCV-H77c), 1b (pCV-J4L6S), and 2a (pJ6CF). Replication of HCV RNA was assessed by nested RT-PCR reactions of total cell RNA. Transfer of HCV infection to naïve hepatocytes was carried out with equal amounts of virus recovered from the culture media and was quantitated by qRT-PCR (TaqMan, Applied Biosystems), using WHO standards for HCV virus (Acrometrix). Comparable GEs of the three strains of HCV (recovered from 0.25-μm filtered culture media) were used to inoculate primary hepatocyte cultures (same passage as PPH cultures). Replication of viral RNA in cells infected with the three HCV genotypes (shown in Fig. 5D, columns 1-3) was determined by qRT-PCR 6 days postinfection. Replication of HCV1a appeared consistently higher as compared to HCV genotypes 1b or 2a. For comparison, we transfected genomic RNA from the JFH-1 strain into either the primary hepatocyte (PPH) cultures or the Huh-7.5 hepatoma cells and utilized the virus particles recovered from the culture media to inoculate either naïve primary hepatocytes (Fig. 5D, column 4), or the Huh-7.5 hepatoma cells (Fig. 5D, column 5). The accumulation of viral RNA (6 days postinfection) suggests that the replication of JFH-1 strain in primary hepatocytes is equally efficient (Fig. 5D, column 4) as compared to its replication in Huh-7.5 hepatoma cells (Fig. 5D, column 5). Overall, the results argue that infectious virus particles are recovered from culture media of primary hepatocytes transfected with full-length genomic clones of HCV genotypes 1a, 1b, and 2a.

Formation of the negative sense RNA replicative intermediate is an important indicator of virus replication in infected cells. We monitored the relative ratios of positive and negative sense viral RNAs in cells infected with HCV1a and, as a control, in the virus particles recovered from the culture medium. The viral RNA concentration was monitored by RPA using run-off probes of metabolically labeled transcripts from the NS5B region. Figure 6A outlines the probes used for detecting the positive and negative sense viral RNAs. Comparison of the positive sense and the negative sense viral RNAs 6 days postinfection showed higher levels of the replicative intermediate (negative sense RNA) in HCV1a-infected primary hepatocytes (Fig. 6B). Estimates of the copy number, based on titration of the probes, suggested that the negative sense RNA in HCV1a-infected hepatocytes was 2.5 times more abundant than the positive sense viral RNA (Fig. 6B, lanes 5 and 6). As controls, the RPA assay showed only the positive sense genomic RNA in mature virus particles, recovered from the culture media (Fig. 6B, lanes 7 and 8); no viral RNA was detected in the mock-transfected cells (Fig. 6B, lanes 3 and 4).

Figure 6.

RNase-protection assay for ± sense viral RNAs. (A) The RPA probes were prepared by cloning a 344 bp Hind III/Eco R1 fragment of NS5B region from pH77c into a pSP72 vector. (B) Lanes 1 and 2 show the positive and the negative probes, respectively. The RPA products using either positive and negative probes are shown, from total cell RNA of mock-infected (lanes 3 and 4), HCV1a-infected cells (lanes 5 and 6), or the RNA from virus particles recovered from the culture medium of HCV1a-infected cells (lanes 7 and 8).

Replication of HCV in Primary Hepatocytes Is Sensitive to IFN.

Patients with HCV genotype 2a infection respond to IFN therapy better than HCV1a infections. As shown in Fig. 7, HCV genotype 1a appears relatively resistant to IFN treatment as compared with the genotype 2a-infected primary hepatocytes cells (HCV 2a replication is undetectable at 400 IU of IFN-β).

Figure 7.

Sensitivity to IFN-α treatment of the HCV-infected primary hepatocytes. Primary hepatocytes infected with HCV1a, 1b, or 2a were either untreated or treated with increasing concentration (IU) of IFN-α, and the inhibition of virus RNA replication was evaluated at 6 days postinfection. The level of inhibition is presented as a fraction of the untreated controls.

Replication of Viral RNA and the Release of Infectious Virus in the Culture Medium.

We next asked whether the HCV particles released in the culture medium could passage infectivity to naïve hepatocytes. HCV genomic RNA units were estimated by qRT-PCR in the virus fraction recovered from the culture medium of infected primary hepatocytes. In the experiments shown in (Fig. 8A) we compared the viral RNA of HCV1a-infected primary hepatocytes (PPH) with that of Huh-7.5 hepatoma cells infected with HCV1a (in each case, infection was initiated with an equal amount of virus recovered from HCV1a-infected primary hepatocytes). We estimated the efficiency of virus released in the culture media of either the infected primary hepatocytes or Huh-7.5 hepatoma cells, based on qRT-PCR analysis of HCV genomic RNA. The results (Fig. 8B) represent virus released in the culture media for up to 20 days postinfection. As shown, efficient virus recovery was sustained from infected primary hepatocytes. By contrast, the virus released from Huh-7.5 hepatoma cells infected with HCV1a rapidly declines.

Figure 8.

(A) HCV RNA accumulation in infected PPH and Huh 7.5 cells: quantitative RT-PCR analysis of viral RNA from total cell RNA of PPH and Huh 7.5 cells 20 days postinfection is presented as relative amounts of HCV RNA/μg total cell RNA, lane 1, RNA from uninfected control PPH cells; lane 2, RNA from PPH infected with HCV1a; lane 3, RNA from cells transfected with run-off transcripts of HCV1a in PPH; lane 4, uninfected Huh 7.5 cells; lane 5, RNA from Huh-7.5 cells transfected with HCV1a genomic RNA; lane 6, RNA from Huh-7.5 cells infected with HCV1a virus recovered from infected primary hepatocytes. (B) Release of infectious virus particles: primary human hepatocytes were infected with equal amounts of HCV1a virus and the virus released in the culture media was collected at the indicated days postinfection. Quantitative RT-PCR analysis of released viral RNA was validated with OptiQuant (Applied Biosystem, AcroMetrix) using WHO standard HCV RNA. The efficiency of virus released from HCV1a-infected primary hepatocytes is maintained over 20 days postinfection (by contrast, the virus released from Huh-7.5 hepatoma cells declines rapidly).

Interestingly, although full-length HCV1a genomic RNA when introduced directly into Huh-7.5 hepatoma cells (by transfection) failed to replicate (Fig. 8A, lane 5), Huh-7.5 hepatoma cells infected with the HCV1a virus (recovered from infected primary hepatocytes) initiates virus replication, albeit at a lower efficiency (Fig. 8A, compare lanes 2 and 6). However, as noted above (Fig. 8B), the release of mature virus from Huh-7.5 hepatoma cells infected with HCV1a is restricted.

Discussion

Long-term cultures of primary hepatocytes are desirable in vitro as a model for studying effects of hepatotoxins on liver function and host response to hepatopathogens. In this study we examined the expression of normal human liver cell markers to determine whether primary human hepatocytes sustain hepatic functions through long-term cultures and whether the primary hepatocyte cultures can support productive HCV replication, a major hepatopathogen. Essential hepatic functions sustained in long-term cultures include synthesis of plasma proteins, drug metabolism, and signal transduction.

In the present study we established a culture system of primary human hepatocytes (derived from liver biopsy tissues of donors ranging in age from 13 months to 58 years; Supporting Information Table S1), and validated their physiological relevance as an in vitro cell culture model for productive replication of an important hepatic pathogen. The primary hepatocyte cultures described here are able to sustain replication through multiple passages as well, as they are able to retain liver-specific functions, making it possible to use the in vitro culture system for repeat applications in studies with hepatopathogens and hepatotoxicity.

We validated the physiological relevance of primary hepatocyte cultures by studying productive replication of infectious clones of the three prevalent HCV genotypes. Virus recovered from the culture media of HCV-infected primary cells was able to transfer infection to naïve hepatocytes. Recovery of infectious virus from Huh-7.5 is limited as compared with the infected primary hepatocytes. Supporting Information Fig. S8 provides a schematic presentation of replication-competent virus produced from primary cells. Although the reasons for restriction of native HCV replication in hepatoma cell line Huh-7.5 is yet unclear, our studies point to an important role of primary human hepatocytes as appropriate host cells for productive infection with a wider repertoire of native HCV genotypes and examination of host response to hepatopathogens.

We observed robust replication of infectious virus particles from HCV1a, 1b, and 2a-infected primary hepatocytes. The efficiency of HCV replication in its natural host, human hepatocytes, reflects the innate replicative properties of the HCV genotypes (Supporting Information Table S3). In natural infections, HCV genotype 1a is known to cause the most severe liver disease; as well, infection with HCV genotype 1a/b is less responsive to IFN therapy. We observed that the replication of HCV1a/1b is less sensitive to inhibition by IFN, suggesting that HCV infection in primary hepatocytes imitates natural HCV infections. The infectivity of recovered HCV1a virus, however, declines over a month following the infection. It is likely that the effect of HCV replication on cholesterol biosynthesis might interfere with sustained virus production, packaging, and release of stable virus particles.19, 20

The results described here provide a physiologically relevant in vitro system for efficient replication of full-length infectious strains of HCV genotypes 1a, 1b, and 2a. An in vitro system for the replication of infectious HCV clones in primary hepatocytes should facilitate analysis of host factors that modulate virus replication and will be useful in in vitro models for molecular analysis of virus-host interactions and studies with hepatotoxins.

Acknowledgements

We thank Drs, Charles M. Rice (Rockefeller University) for the HCV-GFP wildtype and polymerase mutant clones and the Huh-7.5 cell line; Jens Bukh and Suzanne Emerson (NIAID, NIH) for the HCV1a,1b, and 2a genomic clones; Robert Purcell (NIAID, NIH) and Michael Gale Jr. (Fred Hutchinson Cancer Research Center) for helpful comments; Marcos Rojkind (George Washington University) for the stellate cell line and valuable advice with the cocultures; Sita D. Gupta (USUHS) for valuable comments; and Sanjeev Gupta (Albert Einstein College of Medicine, NY) for the stably transduced GFP-CFSC-8B cells. We thank Dr. Songchan Liang for help with the RPA experiment.

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