Cells expressing the NS5AGFP replicon (Moradpour et al., 2004
) were derived from Huh7.5 cells, a highly permissive cell line for HCV replication (Blight, McKeating, and Rice, 2002
), and were obtained from Dr. Charles Rice (The Rockefeller University, New York, NY). Replicon and parental cells were grown as monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (NEAA, Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2
incubator. Replicon cells were also cultured in the presence of 0.3–0.5 mg/ml G418 (Geneticin, Invitrogen, Carlsbad, CA).
To construct plasmids carrying NS4B (WT and deletion mutations) with a C-terminal GFP fusion, NS4B was amplified from genotype 1B, strain Con 1 or J4L6S (Lohmann et al., 1999
; Yanagi et al., 1998
). Specifically, truncations were introduced into J4L6S NS4B sequence, whereas missense mutations were engineered into Con 1 NS4B sequence. Primers () were designed to introduce a Sal
I site at the 5’end, a Bam
HI site at the 3’ end, and an AUG start codon immediately upstream of the NS4B coding region. The PCR products were digested with Sal
I and Bam
HI, and the purified fragments were subcloned into Sal
I- and Bam
HI-cleaved pEGFP-N2 vector (Clontech, Mountain View, CA).
List of primers used in these studies
For site-directed mutagenesis, the QuikChange PCR method (Stratagene, La Jolla, CA) was used. Briefly, two PCR primers, one with NS4B mutation and another with the Wt sequence, were used to amplify recombinant pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) containing a 1.2 kb fragment (including NS4A-NS4B-NS5A sequence) flanked by NsiI at the 5’end and MluI at the 3’ end. To subclone NS4B mutations into pEGFP-N2 vector, NS4B was amplified using two PCR primers, one flanked by SalI and the other by BamHI. The PCR product was digested with SalI (5’ end) and BamHI (3’ end) and cloned into SalI- and BamHI-cleaved pEGFP-N2 to generate the recombinant vector expressing Wt or mutant NS4B alone.
The NS4B mutant replicon constructs were engineered in two steps. First, pI/5A-GFP-6 (generously provided by Charles Rice, The Rockefeller University) (Moradpour et al., 2004
) was digested with Ssp
I (5’ end) and Mlu
I (3’ end) to remove an 868 bp fragment containing NS4B flanked by part of NS4A and NS5A sequences. Then, a 1.6 kb fragment [containing EMCV IRES and some pEGFP-N2 vector (Clontech) sequence] was digested with Ssp
I and Mlu
I, followed by ligation into Ssp
I- and Mlu
I-cleaved pI/5A-GFP-6 to create an intermediate vector. Finally, Wt or mutant NS4B DNA fragment (in pCR 2.1-TOPO vector) was cleaved with Ssp
I and Mlu
I. The purified DNA was ligated into the Ssp
I- and Mlu
I-cleaved intermediate vector to generate recombinant pI/5A-GFP-6 vector expressing Wt or mutant NS4B protein. DNA sequences from Wt and NS4B mutant replicons were confirmed by DNA sequencing.
We also engineered a recombinant pIRES vector (Clontech) containing NS5AGFP subgenomic replicon (pIR/I5AGFP) DNA sequence to express Wt and mutant HCV polyproteins under the transcriptional control of both CMV and T7 promoters. To this end, pI5AGFP-6 (Moradpour et al., 2004
)] vector was digested with Xba
I (5’ end) and Mlu
I (3’ end) and the resulting 5’ UTR-neo-NS3-NS4A-NS4B-NS5A fragment was ligated into Nhe
I- and Mlu
I-cleaved pIRES vector. Next, pI5AGFP-6 vector was digested with Mlu
I and EcoR
V and the resulting NS5A-NS5B-3’ UTR fragment was ligated into Mlu
I- and Sma
I-cleaved pIRES vector. Finally, the recombinant pIRES vector, with NS5A-NS5B-3UTR, was digested with Mlu
I and Not
I, and the purified Mlu
I fragment was ligated into the Mlu
I- and Not
I-cleaved pIRES vector, containing 5’ UTR-neo-NS3-NS4A-NS4B-NS5A fragment, to generate pIR/I5AGFP vector.
Rabbit polyclonal antibody to HCV NS4B was obtained from Covance (Denver, PA) whereas mouse monoclonal antibody to NS4B was from Abcam (Cambridge, MA). Rabbit polyclonal antibodies to HCV NS3, NS5A and NS5B were generous gifts from Craig Cameron’s laboratory (Penn State University, University Park). Rabbit polyclonal antibody to GFP and Rab5 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Calnexin and GAPDH antibodies were from Stressgen (Ann Arbor, MI) and Fitzgerald industries International, Inc. (Concord, MA), respectively. Horseradish peroxidase-conjugated secondary antibodies were obtained from Vector Laboratories (Burlingame, CA). Alexa fluor-conjugated secondary antibodies were obtained from Invitrogen (Carlsbad, CA).
For each experiment, parental cells (Huh7.5) or NS5AGP subgenomic replicon-expressing cells were trypsinized and grown overnight in 10 cm dishes or 6-well plates to obtain 70–80% confluent monolayer cells. Prior to transfection, the cells were washed with phosphate-buffered saline (PBS) and fed with 10 ml of fresh complete medium (for 10 cm dishes) or 2 ml of complete medium per well for 6-well plates. Cells were transfected according to the TransIT-LT1 protocol from Mirus(Madison, WI). The DNA mixture was added to each dish and incubated at 37°C for 24 or 48 h. With this procedure, DNA transfection efficiency was usually 70–90%.
Membrane Floatation Assay
For membrane floatation assay, 2–3 × 100 mm dishes (7 × 105
cells/dish) of parental cells (Huh7.5) were grown overnight and transfected as described above. Transfected cells were resuspended in homogenization buffer (150 mM NaCl, 50 mM Tris pH 7.4, 2 mM EDTA) containing protease inhibitors (1mM PMSF and 1 tablet of Complete Mini; Roche, Nutley, NJ). The cells were then lysed with 6–8 passages in a ball-bearing homogenizer to ensure approximately 90% lysis. Cell lysates were spun at 2500 xg/10 min at 4°C to pellet cellular debris and nuclei. A discontinuous iodixanol gradient (5%, 25% and 30%) (Elazar et al., 2004
) was layered on the top of the homogenate and the samples were spun at 120,000×g for 4h 25 min at 4°C in a Ti80 Rotor. A total of 8 fractions (867 µl each) were collected from top to bottom. Each fraction was precipitated as described above, separated on 10% SDS-PAGE and processed for western blotting as described above. Typically, membrane-bound proteins were associated with fractions 1 to 4 whereas soluble proteins were prominent in fractions 5 to 8.
Metabolic Labeling and Immunoprecipitation
Huh7.5 cells were seeded at 1.5 × 105 cells/10 cm dish approximately 24 h prior to transfection. Before transfection, the cells were washed once in PBS and fed with fresh DMEM/10%FBS. The cells were co-transfected with 10 µg of DNA encoding Wt or mutant NS5A-GFP replicon in pIRES vector (pIRES-I/5A-GFP) and 5 µg of plasmid DNA encoding T7 polymerase (pCAGGS-T7 kindly provided by Biao He, The Pennsylvania State University). Parental cells, co-transfected with pIRES and T7 polymerase-encoding vector, were used as negative controls. DNA transfection with TransIT-LT1 reagent was done as described above.
At 48 h post transfection, the cells were trypsinized and washed twice with PBS. Each sample was then resuspended in 1 ml of DMEM without cysteine and methionine (Invitrogen) and incubated at 37°C with gentle rotation for 1h. After starvation, the cells were labeled with 200 µl of 500 µCi/ml Express 35S protein labeling mix (Perkin-Elmer) for 10–15 min. The cells were evenly split into aliquots, and some were lysed immediately in 1 ml of ice-cold RIPA buffer [150 mM NaCl, 50 mM Tris, pH 8.0, 1 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor cocktail (Roche)]. Alternatively, the remaining label was removed after brief centrifugation (500 xg/1 min), replaced with complete DMEM containing 10 mM cysteine/methionine (pH 7.4), followed by a chase and lysis with 1 ml of ice-cold RIPA buffer. Cell lysates were precleared by incubation for 1 h at 4 °C with Protein A/G Plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). The supernatants were then incubated with HCV-specific antibodies at a dilution of 1:500 (anti-NS3, NS5A, or NS5B) or 1:250 (anti-NS4B) for 3–12 h at 4°C, mixed with protein A/G Plus agarose and incubated for 2h at 4°C. Protein A/G agarose Plus-bound complexes were collected by centrifugation at 500 xg/5 min, washed once with RIPA buffer, once with RIPA buffer/500 mM NaCl and once more with RIPA buffer. To examine the immuno-precipitates by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), the samples were resuspended in 30 µl of loading buffer (Laemmli, 1970), heated at 95 °C/5–10 min and centrifuged at 14,000 xg/2 min. The supernatants were separated on 10% SDS-PAGE, fixed in 20% methanol/7% acetic acid for 10 min at room temperature, and dried for 1 h at 80°C. Labeled proteins were visualized and quantitated on a PhosphorImager (Typhoon 8600, Amersham Pharmacia Biotechnology Inc./Molecular Dynamics, Piscataway, NJ).
In vitro transcription, electroporation and generation of G418-resistant cells
Plasmid DNA containing subgenomic NS5AGFP replicon (pI/5A-GFP-6)(Moradpour et al., 2004
) constructs were linearized with Sca
I and purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). RNA was synthesized using the T7 RiboMAX Express Large Scale RNA Production Systems Kit (Promega, Madison, WI) according to the manufacturer’s instructions. The RNA was then isolated using the RNeasy miniprep kit (QIAGEN, Valencia, CA). Prior to electroporation, subconfluent Huh7.5 cells were trypsinized and resuspended in complete DMEM. The cells were then washed three times and resuspended at a concentration of 6.25 × 106
cells/ml in OptiMEM. For each electroporation, 0.4 ml of the cell suspension (2.5 × 106
cells) was mixed with 5 µg of HCV replicon RNA and electroporated in a 0.2-mm gap cuvette in a BioRad Gene Pulser (1 pulse, 0.13 kV). The cell suspension was recovered in complete DMEM for 10 min on ice prior to plating in a 10 cm dish. Resuspended cells (1 × 104
, 1 × 105
, 2.5 × 105
and 5 × 105
) were seeded in 10 cm dishes together with cells electroporated with polymerase-deficient replicon RNA to obtain 5 × 105
cells per dish. After 24 h incubation, the medium was changed and replaced with complete medium supplemented with 0.5 mg/ml G418. The medium was changed every three to four days for three weeks. G418-resistant colonies were fixed with 4% formaldehyde/PBS and stained with 0.1% crystal violet in 70% ethanol. G418-resistant clones were counted and used to calculate the colony forming efficiency per microgram of input RNA (CFU/µg).
Huh7.5 cells were seeded onto coverslips and transfected in 10 cm dishes or 6-well plates as described above. At 48 h posttransfection, the coverslips were washed with PBS and fixed for 10 min in 4% formaldehyde/PBS. The cells were then permeabilized for 5 min at room temperature in 0.05% Triton-X 100/PBS, followed by staining with NS3 rabbit polyclonal antibody (or NS4B staining with a mouse monoclonal antibody) and Alexa fluor 594-conjugated secondary antibody. After three washes in PBS, the cells were stained with DAPI/PBS for 10 min at room temperature, followed by three more washes in PBS. The cells were mounted on glass slides in Vectashield (Vector Laboratories, Inc., Burlingame, CA) and the coverslips sealed with nail polish. The samples were then examined by fluorescence microscopy (Zeiss Axiovert 200 M) with a 63x lens. Digital images were taken with an Axiocam MRm CCD camera. An image stack was deconvolved using the iterative mode of the Axiovision software to exclude out-of-focus information. Images were saved as TIFF files, imported and processed in Adobe Photoshop.
Transfected cells or replicon cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin) and protein concentrations determined by Bio-Rad protein assay. Fifty to one hundred micrograms of total protein were typically resuspended in 4x SDS loading buffer (240 mM Tris pH 6.8, 4% SDS, 40% glycerol, 4% β-mercaptoethanol, 0.01% bromophenol blue) and boiled for 5 min at 95°C. The proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) followed by transfer onto an Immobilon-P membrane (PVDF; Millipore, Billerica, MA). Antibody-bound proteins were detected by enhanced chemiluminescence detection method (ECL, Pierce, Rockford, Il).
For ultrastructural analysis, cells expressing GFP, NS4BGFP or the various mutant constructs were trypsinized, resuspended in complete DMEM and processed as follows. Briefly, the cells were resuspended in 2% glutaraldehyde/0.1M sodium cacodylate buffer and incubated on ice for 30 min. After a brief spin, fresh 2% glutaraldehde/0.1M sodium cacodylate was added to the pellet and the pellet was incubated overnight at 4°C. The cell pellet was rinsed with 0.1 M sodium cacodylate prior to postfixation with 1% osmium tetroxide/0.1 M cacodylate for 1–2 h at 4°C. After rinsing and en bloc staining in aqueous uranyl acetate, samples were dehydrated with graded ethanol concentrations, infiltrated with eponate resin and embedded overnight in eponate at 65 °C. Ultrathin sections were cut on Leica Ultracut UCT microtome (Wetzlar, Germany), collected on copper grids and stained with 1% uranyl acetate-1% lead citrate. The sections were imaged at 80kV in a JEOL JEM 1200 EXII (Peabody, MA) electron microscope.