Cell monolayers of the human hepatoma cell line Huh-7 were routinely grown in complete medium consisting of equal volumes of Dulbecco's modified minimal essential medium (Gibco) and RPMI 1640 (Gibco), supplemented with 1% l-glutamine (Gibco), 1% penicillin, 1% streptomycin, and 10% fetal bovine serum. Cell lines were passaged twice weekly after treatment with 0.05% trypsin-0.02% EDTA and seeding at a dilution of 1:10.
A rabbit polyclonal antibody against green fluorescent protein (GFP) and an anti-rabbit secondary antibody were purchased from Molecular Probes. A monoclonal antibody against glutathione S-transferase (GST) was purchased from Cell Signaling Technology.
Standard recombinant DNA technology was used to construct and purify all plasmids. All regions that were amplified by PCR were analyzed by automated DNA sequencing. Plasmid DNAs were prepared from large-scale bacterial cultures and purified by a Maxiprep kit (Marligen Biosciences). Restriction enzymes were purchased from New England Biolabs.
The Bart79I plasmid was described previously. Briefly, it was made by PCR mutagenesis (9
) of HCVrep1bBartMan/AvaII (3
) such that nucleotide 5336 was changed from a G to T, resulting in a change in NS5A amino acid 1179 from serine to isoleucine. This mutation results in a dramatic increase in replication efficiency of the HCV subgenomic replicon (3
). The NS4B NBM mutations of Bart79I (numbers represent the amino acid positions relative to amino acid 1 of NS4B), G129V, I131N, K135S, and K135R, were generated by site-directed mutagenesis using a PCR-based method. Briefly, complementary primers (forward primer 1, 3, 5, or 7 with primer 10 or reverse primer 2, 4, 6, or 8 with primer 9; Table ) and the enzyme Platinum Pfx
(Invitrogen) were used to generate by PCR two DNA fragments with overlapping ends containing the mutation. These ends were annealed to allow 3′ extension of the complementary strand with the 3′ overlap of each strand as a primer. The product was then further amplified by PCR using primers 9 and 10 (Table ). The PCR products and the Bart79I vector were cut with SspI and MluI, followed by ligation with T4 DNA ligase (Invitrogen) and transformation into chemically competent Escherichia coli
(One Shot Top10 competent cells; Invitrogen).
Sequences of the oligonucleotides used in this study
The plasmid PEF-NS4B-GFP was constructed in a two-step cloning procedure as follows. A PCR fragment of the NS4B gene amplified from the Bart79I plasmid with forward and reverse primers containing NcoI restriction sites (primers 11 and 12; Table ) was digested with NcoI and ligated with the NcoI-digested T7GFP plasmid (6
) to generate the plasmid T7NS4BGFP. The plasmid T7NS4BGFP was digested with BglII-KpnI, and the fragment corresponding to NS4B-GFP was inserted into BamHI-KpnI-digested PEF6myc-HisA (Invitrogen) to yield PEF-NS4B-GFP. To obtain the plasmids encoding mutations in the NBM in NS4B, coding sequences for proteins with G129V, I131N, K135S, and K135R mutations in the NBM (see above) were digested with NdeI-HpaI and the fragment corresponding to the mutated NBM was inserted into NdeI-HpaI-digested PEF-NS4B-GFP.
The GST-NS4B plasmid and those encoding the corresponding NBM mutant proteins were generated by using the Gateway technology (Invitrogen) according to the manufacturer's protocol. In brief, a forward primer introducing a recombination site and a TEV protease cleavage site (primer 13; Table ) and a reverse primer introducing a second recombination site and a stop codon (primer 14; Table ) were used to generate a PCR product encoding wild-type or mutant NS4B flanked by the two recombination sites. This product was first introduced into a donor vector (pDonor 201), from which it was transferred to the destination vector, pDEST15, to yield the GST-NS4B plasmid by a two-step recombination procedure. The 5A-GFP plasmid was described previously (6
Infection and transfection.
A vaccinia virus that expresses the T7 RNA polymerase (T7RNAP) was used to infect Huh-7 cells. Following a 45-min incubation at 37°C the cells were washed twice with Optimem (Invitrogen) and subjected to transfection with the appropriate construct by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells were supplemented with growth medium and incubated for 5 h at 37°C.
GTP binding assay.
Photoaffinity labeling of NS4B-GFP in membrane preparations with 32
P-labeled GTP-γ-4-azidoanilide ([γ-32
P]GTPγAA; 38 Ci/mmol; Affinity Labeling Technologies, Inc.) was carried out essentially as described previously (13
). Cellular membrane preparations were prepared from vaccinia virus-infected and transfected Huh-7 cells. Following infection and transfection the cells were collected by trypsinization, washed once with phosphate-buffered saline (PBS), and resuspended in HME buffer (20 mM HEPES [pH 7.4], 1 mM EDTA, 2 mM MgCl2
), which was supplemented with phenylmethylsulfonyl fluoride to a final concentration of 1 mM and a protease inhibitor cocktail (Sigma). The cells were lysed by two cycles of freeze-thaw in dry ice-ethanol and then passaged through a 27.5-gauge needle 10 times. Nuclei were removed by centrifugation at 250 × g
for 10 min, and the postnuclear supernatant was subjected to ultracentrifugation at 100,000 × g
for 30 min to obtain the membrane preparation. All steps were done at 4°C. One hundred and fifty micrograms of total membrane protein was resuspended in 20 mM Na-HEPES, pH 7.4. The assay mixture containing a 30-μl membrane preparation, 30 μl of 3× binding buffer (30 mM Na-HEPES [pH 7.4], 100 mM NaCl, 0.1 mM EDTA, 10 mM MgCl2
), and 30 μl of [γ-32
P]GTPγAA (total of 15 μCi) was incubated for 1 h at 30°C in the dark. Samples were then irradiated with UV light at a 3-cm distance for 1 min (2,000 μW, 254 nm; UVS-28; UV Products) to allow covalent attachment of the bound radiolabeled guanine nucleotide. Unbound nucleotides were removed by ultracentrifugation for 10 min at 100,000 × g
, and the membranes were resuspended in 1× binding buffer containing 2 mM dithiothreitol (for inactivation of the unbound material) and irradiated on ice for an additional 3 min with UV light.
Immunoprecipitation of labeled NS4B-GFP.
To identify the [γ-32P]GTPγAA-labeled NS4B-GFP, membrane preparations were incubated in 1 ml of TDB buffer (2.5% Triton X-100, 25 mM triethanolamine-Cl [pH 8.6], 20 mM NaCl, 0.5 M EDTA, 0.2% NaN3), followed by ultracentrifugation at 100,000 × g for 10 min. The supernatants were incubated overnight with a rabbit polyclonal antibody directed against GFP (Molecular Probes) and protein A-Sepharose (Amersham Biosciences). Following three washes in NET buffer (150 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl [pH 8.0]) immunoprecipitates were solubilized in sample buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Nitrocellulose membranes were also subjected to Western analysis with mouse anti-GFP antibodies (Roche) and horseradish peroxidase-conjugated donkey anti-mouse immunoglobulin G, followed by chemiluminescence (Amersham) development.
DNA constructs were transfected into Huh-7 cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Cells expressing GFP fusion proteins were fixed in 4% formaldehyde 18 h posttransfection and mounted with polyvinyl alcohol (Mowiol) mounting medium. Fluorescence images were captured with a Nikon E600 fluorescence microscope equipped with a SPOT digital camera and the Openlab (Improvision) image acquisition software.
Expression and purification of wild-type and mutant GST-NS4B.
Proteins were expressed and purified as previously reported (30
). Overnight cultures of E. coli
transformed with parental or recombinant pDEST15 plasmids were diluted 1:100 in 400 ml of fresh medium and grown at 37°C to an optical density of 0.6. Isopropyl-β-d
-thiogalactopyranoside (IPTG; Invitrogen) was then added to a final concentration of 0.1 mM. After 2 h of growth at room temperature, cells were pelleted and resuspended in 25 ml of lysis buffer (PBS [pH 7.3], 1% Triton X-100 [J. T. Baker], 100 U of DNase [Sigma]/ml, 100 μg of Lysozyme [Sigma]/ml, protease inhibitor cocktail [Sigma], 1 mM phenylmethylsulfonyl fluoride [Sigma], 2 mM MgCl2
). After 15 min of incubation on ice, cells were lysed by one cycle in a French press at a pressure of 10,000 lb/in2
for 1 min, followed by centrifugation at 12,000 × g
for 5 min at 4°C. The supernatant was mixed at 4°C on a rotating platform with 200 μl of 50% glutathione-agarose beads (Sigma). Beads were then washed three times with PBS. GST-NS4B was eluted by a 10-min incubation at room temperature in 100 μl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM reduced glutathione, 0.1% Triton X-100). Elution was repeated twice. Glycerol was added to the pooled eluates at a final concentration of 20% and stored at −20°C until use as described below. Expression and purification were monitored by SDS-PAGE, followed by Coomassie staining or Western blot analysis with an anti-GST antibody. We estimate the maximum amount of non-GST-containing protein to be <5%. In addition to the expected full-length GST-NS4B band at 58 kDa, some faster-migrating GST-containing bands were detected. The latter appeared to be the result of premature termination, as their size correlated with the positions of codons poorly recognized by standard E. coli
strains, and they were found to significantly decrease following the addition of appropriate tRNAs in an in vitro expression system (Rapid Translation system; Roche) (T. Danieli, unpublished data). Typical final yields were 5 μg of total protein per 100-ml bacterial culture. Of note, there were no differences in yield or purity between the NBM mutant proteins and wild-type GST-NS4B.
The standard GTPase assay was performed as previously described (25
). One-half microgram of purified protein was incubated in a 30-μl reaction mixture containing 20 mM HEPES-KOH (pH 6.8), 10 mM MgCl2
, 2 mM dithiothreitol, 40 μM cold GTP (Promega), and 15 μCi of [γ-32
P]GTP/ml (5,000 Ci/mmol; Amersham Biosciences). Serial aliquots were collected at different incubation time intervals (5, 15, 30, 45, and 60 min), while the reaction was performed at 37°C. The reaction was terminated on ice by the addition of EDTA to a final concentration of 5 mM. Aliquots (0.5 μl) were then spotted onto polyethyleneimine cellulose-coated thin-layer chromatography (TLC) plates (Merck). Plates were developed in 0.15 M LiCl-0.15 M formic acid (pH 3.5) in a TLC chamber, dried, and subjected to autoradiography and quantitative phosphorimager analysis.
In vitro RNA transcription.
Plasmid DNA of the wild-type HCV (Bart79I) replicon and replicons encoding the various NS4B NBM mutations were linearized with ScaI and treated with proteinase K, followed by phenol-chloroform extraction and precipitation with ethanol. The DNA was resuspended in RNase-free water to a final concentration of 1 μg/μl. Four micrograms of DNA was used as a template for transcription with the Ribomax RNA production kit (Promega) according to the manufacturer's protocol. The template DNA was digested by the addition of 5 U of RQ1 DNase (Promega) and a 15-min incubation at 37°C. The unincorporated ribonucleotides were removed by size exclusion with a Micro Bio-Spin P-30 column (Bio-Rad), and the transcribed RNA was extracted with phenol-chloroform, followed by precipitation in ethanol. The RNA pellet was washed with 70% ethanol and resuspended in H2O. Determination of the RNA concentration was performed by measurement of the optical density at 260 nm. The integrity of the RNA and its concentration were confirmed by 1% agarose gel electrophoresis and ethidium bromide staining.
Colony formation assays.
The standard replicon colony formation assay was performed as previously described (3
). Briefly, subconfluent Huh-7 cells were trypsinized and collected by centrifugation at 700 × g
for 5 min. The cells were then washed three times in ice-cold RNase-free PBS (BioWhittaker) and resuspended at 107
cells/ml in PBS. Five micrograms of in vitro-transcribed RNA was mixed with 0.4 ml of washed Huh-7 cells in a 2-mm-gap cuvette (BTX) and immediately pulsed (0.68 kV, five 99-μs pulses) with a BTX-830 electroporator. After a 10-min recovery at room temperature, pulsed cells were diluted into 10 ml of prewarmed growth medium. Cells were plated in 10-cm3
tissue culture dishes at different densities (4 × 106
, 4 × 105
, 8 × 104
, and 4 × 104
cells per dish) to permit accurate colony counting. Twenty-four hours postelectroporation, the cells were supplemented with plain Huh-7 cells to a final density of 106
cells/plate. Following an additional 24 h, the selecting drug, G418 (Invitrogen), was added to the medium to a final concentration of 1 mg/ml. Growth medium supplemented with G418 was replaced every 4 days for 3 weeks. The plates were then washed twice with PBS and incubated in 1% crystal violet made in 20% ethanol for 5 min, followed by three washes with H2
O to facilitate colony counting. The G418 transduction efficiency was calculated based on the number of G418-resistant colonies relative to the number of Huh-7 cells plated after electroporation. Results were expressed as number of colonies per microgram of transfected RNA of each mutant relative to the wild-type replicon.
RNA extraction, RT-PCR amplification, and sequencing.
Several G418-resistant clones were isolated from colony formation assays performed with the replicon harboring the G129V mutation. Total cellular RNA of individual clones was extracted with TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. The reverse transcriptase reaction and PCR amplification were performed with the Superscript One-Step reverse transcriptase PCR (RT-PCR) kit (Invitrogen) according to the kit's protocol. Briefly, the amplification reaction mixture included 1 μg of total RNA as the template and 10 pmol of each primer. Two sets of primers (4Left-for with 4Right-rev and 3800sp-for with 4Right-rev; Table ) were used to amplify 1-kb and 650-bp segments, respectively, each containing the NBM region coding sequence. Performing two independent amplification reactions for each clone provided further confirmation of the sequencing results. The RT reaction was performed at 50°C for 30 min and was followed by incubation at 95°C for 2 min. The DNA was amplified by 28 cycles of 95°C for 15 s, 60°C for 30 s, and 68°C for 1 min. A final elongation step was performed at 68°C for 10 min. The PCR products were purified from agarose gels with the Ultra Clean 15 DNA purification kit (MoBio) and sent for automatic sequencing on an ABI Prism 377 DNA sequencer (Sequetech).
Concentrations of purified protein and protein content in membrane preparations were determined by the Bradford dye binding procedure using a Bio-Rad (Richmond, Calif.) protein assay kit.