Huh-Lunet and Lunet-CD81 cells were maintained in Dulbecco's modified Eagle medium (DMEM-Glutamax) supplemented with nonessential amino acids and 10% fetal bovine serum (FBS) (DMEM complete medium). Huh-Lunet cells (20
) were obtained from ReBLikon GmbH (Mainz, Germany). Media and supplements were purchased from Gibco-BRL, Life Technologies Ltd. (Madison, WI). All cell lines were maintained in humidified incubators at 37°C and 5% CO2
Generation of stable Lunet-CD81 cell line.
pOTBR-human CD81, a plasmid carrying the human CD81 gene, was obtained from ATCC (Manassas, VA). A lentivirus encoding human CD81 was generated by using the ViraPower lentivirus expression system (Invitrogen, Carlsbad, CA) per the manufacturer's instructions. Huh-Lunet cells were transduced with the human-CD81 lentivirus and subsequently selected in complete medium supplemented with blasticidin (5 μg/ml). Stably transduced cells were stained with a CD81-specific monoclonal antibody (JS-81; Becton Dickinson Biosciences, San Diego, CA) and sorted for high CD81 expression at the Stanford University fluorescence-activated cell sorter (FACS) facility (Palo Alto, CA). Subsequently, cells were transduced and sorted a second time to generate a homogenous cell line that stably expresses high levels of CD81. This cell line was designated Lunet-CD81.
All constructs were verified by DNA sequencing (Tacgen, Richmond, CA). Restriction enzymes were purchased from New England BioLabs (Ipswich, MA). A chimeric HCV genome consisting of the J6 sequence (genotype 2a) from core through NS2 (genotype 2a) followed by the JFH-1 sequence (genotype 2a) of the 5′ untranscribed region (UTR) and then from NS3 through the 3′ UTR with a FLAG tag inserted in the NS5A coding sequence was called J6/JFH-1 Rluc and was previously described (30
). To create a nonreporter version of this genome, the Rluc gene was removed by using the flanking MluI restriction sites. The religated product did not contain the Rluc gene, and the resulting plasmid is referred to as pJ6/JFH-1. The individual mutations K78E (core), S401N (E2), W879R (NS2), Y904H (NS2), V1761L (NS4B), D2173G (NS5A), and V2417A (NS5A) were introduced into the pJ6/JFH-1 plasmid by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA.). Mutated regions were then exchanged into wild-type pUC19-J6/JFH-1 plasmid by using the restriction enzyme sets EcoRI/NsiI (for the region containing K78E), NsiI/MluI (for the region containing S401), MluI/SpeI (for the region containing W879R and Y904H), SpeI/BamHI (for the region containing V1761L), BamHI/RsrII (for the region containing D2173G), or RsrII/BsrGI (for the region containing V2417A). Plasmids containing a combination of mutations were created by using a QuikChange Multi kit (Stratagene) and up to three mutagenesis primers at once. A region containing both NS2 mutations (W879R and Y904H) was synthesized (GeneOracle, Mountain View, CA) with incorporated 5′ MluI and 3′ SpeI restriction sites and then subcloned into the virus cDNA by using these enzymes. The pJ6/JFH-All 7 construct containing all 7 mutations was synthesized through a combination of ligation and mutagenesis techniques. Single reversion mutants were created by reverting individual mutations in pJ6/JFH-All 7 back to the wild type using the primer sets described.
RNA transcription and electroporation.
In vitro transcripts were generated as previously described (30
). Briefly, plasmids were linearized with XbaI and purified by using a MinElute column (Qiagen, Valencia, CA). RNA was transcribed from 1 to 5 μg of purified template by using a T7 Megascript kit (Ambion, La Jolla, CA). Reaction mixtures were incubated at 37°C for 2 h and then digested with 3 U of DNase I (Ambion) for 15 min. RNA was purified by using an RNeasy kit (Qiagen) and quantified by absorbance at 260 nm. RNA integrity was determined by agarose gel electrophoresis and SYBR staining (Invitrogen). Each transcribed RNA (5 μg) was mixed with a suspension of trypsinized Huh-Lunet cells (7.5 × 106
cells/ml in a volume of 400 μl). Cells were then electroporated at 260 V and 950 μF in a Gene Pulser II apparatus (Bio-Rad, Hercules, CA). Electroporated cells were allowed to recover for 10 min at room temperature prior to addition of cell culture medium and plating in T75 flasks or 12-well plates.
Adaptation of wild-type J6/JFH-1 to Lunet-CD81 cells.
Five 96-well plates of Lunet-CD81 cells were infected by limiting dilution (one to four dilutions, from a multiplicity of infection [MOI] of 0.8 to 0.0008) of wild-type-J6/JFH-1-containing supernatant. Following the initial infection, supernatants containing virus were passed onto naive Lunet-CD81 cells. Supernatant passages were performed by collecting supernatant 72 h postinfection and transferring 100 μl virus-containing supernatant onto naive Lunet-CD81 cells, seeded at 3,500 cells per well in 100 μl of complete DMEM medium, so that cells in each well were maintained in a total volume of 200 μl. Overall, eight supernatant passages were performed during the initial adaptation. During the adaptation process, the percentage of cells infected at each passage was quantified by indirect immunofluorescence (see below). Following the 96-well supernatant passages, supernatants from wells consistently exhibiting >50% infected cells were passed into 24-well plates for two passages and then 6-well plates for four passages, T25 and T75 flasks for two passages, and finally a T225 flask. During the process, the percentage of cells infected was continuously monitored by indirect immunofluorescence, and virus supernatants producing greater than 80% infected cells were passed into larger vessels. Cell culture supernatants were assessed for infectious virus via indirect NS5A immunofluorescence or TCID50 assay, and cells were used for preparation of total RNA (see below).
Virus-containing supernatants were collected and filtered through 0.22-μm filters (Corning, Union City, CA). Following filtration, HEPES buffer, pH 7.5 (Gibco), was added to a final concentration of 10 mM. Viral supernatants were aliquoted and stored immediately at −80°C. Large stocks of virus were prepared by seeding 20 T225 flasks of Lunet-CD81 cells at 2.5 × 106 cells per flask. After cell attachment (>6 h postseeding), medium was removed and 5 to 7 ml of virus-containing supernatants were added at an MOI of >0.3. The next morning, 45 ml of complete DMEM was added to each flask. Three days later, supernatants were collected from subconfluent cells and fresh medium was added. Thirty hours later, supernatants were collected again. For high-titer virus stock collection, virus infection was the same as above. However, virus-containing supernatant was collected at 4-h intervals three times per day 2 to 5 days postinfection.
Preparation of total RNA, amplification of viral RNA by reverse transcription-PCR (RT-PCR), and cloning of amplified DNA fragments.
Total RNA was isolated from T75 flasks of confluent Lunet-CD81 cells infected with the adapted virus by using the QiaShredder (Qiagen) and RNeasy (Qiagen) kits or from infected cell supernatants by using the virus RNA QIAamp kit (Qiagen) as recommended by the manufacturer. One microgram total RNA and 50 pmol primer were used for cDNA synthesis by using the SuperScript III RT-PCR system (Invitrogen) as recommended by the manufacturer. Sequence analysis was performed with a set of primers covering the complete HCV genome (Tacgen).
NS3-4A protease assay.
The presence of viral NS3-4A protease was indirectly measured by using a europium-labeled NS3-4A protease substrate as described previously with slight modifications (43
). Briefly, virus-infected cells were grown in 96-well plates and lysed in 1× lysis buffer (Promega) containing 150 mM NaCl and 150 nM europium-labeled NS3 protease substrate (AnaSpec, Fremont, CA). Time-resolved fluorescence was measured by using a BioTek Synergy 2 instrument (Winooski, VT).
Infected cells were grown in 96-well plates and fixed with 50 μl/well glacial methanol-acetone (1:1) at room temperature for 20 min. Cells were then washed three times with phosphate-buffered saline (PBS). Immunostaining of NS5A was performed by using a mouse monoclonal antibody (9E10; Apath, Brooklyn, NY) at a dilution of 1:10,000 in PBS with 3% bovine serum albumin (BSA) for 1 h at room temperature. After two washes with PBS, bound primary antibodies were detected by using a mouse antibody conjugated to Alexa-Fluor 555 at a dilution of 1:3,000 in PBS containing 3% BSA for 20 min in the dark at 4°C. DNA was stained with 4′,6′-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Madison, WI) for 10 min at 4°C in the dark. Finally, cells were washed three times with PBS and imaged by using a Zeiss microscope with fluorescence capabilities (Thornwood, NY). The percentage of infected cells was quantified by visual inspection and counting. To further quantify the percentage of cells infected, images were instead acquired using the ImageXpress Micro system (Molecular Devices, Sunnyvale, CA). Following immunofluorescence with anti-NS5A antibody, Hoechst-stained cells were imaged in the Cy3 and 4′,6-diamidino-2-phenylindole (DAPI) channels, respectively, at magnification ×4 with a minimum of 4 images per well. Infected cells were distinguished from noninfected cells using the Cell Scoring module in the MetaXpress 2.0 software program (Molecular Devices).
Determination of virus titers in cell culture supernatants.
Virus titers were determined as described elsewhere, with slight modifications (21
). Lunet-CD81 target cells were seeded at a concentration of 5,000 cells per well in 96-well plates in a total volume of 100 μl complete DMEM/well. The next day, one to four serial dilutions of virus-containing supernatant were prepared and 100 μl of each dilution was added, with four wells per dilution. Three days later, cells were fixed according to the indirect immunofluorescence protocol described above. Virus titers (TCID50
/ml) were calculated based on the method of Reed and Muench (35
Determining specific infectivity.
Specific infectivity was calculated as extracellular TCID50
/ml at 72 h posttransfection/packaged extracellular RNA copies at 72 h posttransfection. Packaged extracellular RNA levels were measured by collecting 1 ml of extracellular medium from transfected Huh7-Lunet cell cultures growing on a 12-well plate 72 h posttransfection. In this cell line, viral spread is limited (20
). Aliquots of extracellular medium were separated by using equilibrium density gradient ultracentrifugation according to the method of Lindenbach et al. (22
). Briefly, 1 ml of extracellular medium was loaded at the top of 10 ml 10 to 40% iodixanol gradients. Iodixanol gradients were prepared by using 10% and 40% iodixanol solutions that each contained 10 mM HEPES, pH 7.5, and 0.02% BSA. In addition, the 10% iodixanol solution contained 125 mM NaCl and the 40% iodixanol solution contained 50 mM NaCl to maintain iso-osmolarity. Samples were centrifuged through the gradients at 40,000 rpm for 16 h at 4°C in an SW41 Ti swinging bucket rotor (Beckman, Brea, CA). Subsequently, each gradient was divided into 26 0.4-ml fractions, and each fraction was assayed for relative infectivity by using the NS3-4A protease assay described above. The packaged extracellular RNA was isolated from the peak infectious fraction (fraction 15) (data not shown) in each case using a QIAamp viral RNA isolation kit (Qiagen). Viral RNA was quantified by using quantitative RT-PCR (qRT-PCR).
Determining infectious HCV stability.
Infectious HCV stability was determined by incubating 1-ml aliquots of extracellular HCV in cell culture medium at 37°C and freezing 100-μl aliquots at −80°C at various times. After collecting samples for all of the time points, naive cells were infected with each sample for 72 h and the relative levels of infectivity were measured using the NS3-4A protease assay described above. The level of infectivity was normalized to 100% at time zero for each viral strain tested. The data were plotted as percent infectivity versus time (hours) and fit to a first-order decay equation by using the SigmaPlot software program (Systat, Chicago, IL) to determine the slope (−k
). The half-lives for each infectious HCV strain (t1/2
) were calculated in hours by using ln 2/k
Statistical analysis was performed using the Prism 4.0 software program (GraphPad, La Jolla, CA). All comparisons were made with two-tailed t tests. A P value of <0.05 is reported as significant.