Cells expressing full-length replicon, C5B, have been described by Ikeda et al. (Ikeda et al., 2002
). Parental (Huh7) and replicon cells (C5B) were grown as monolayers in advanced Dulbecco's modified Eagle's medium (DMEM) supplemented with 1.5% fetal bovine serum (FBS), non-essential amino acids (NEAA), 100 units/ml penicillin and 100 mg/ml of streptomycin at 37 °C in a 5% CO2
incubator. For replicon cells, the media also contained 0.3-0.5 mg/ml G418 (Geneticin). Huh7 and C5B cells were generously provided by Stanley Lemon (University of Texas Medical Branch, Galveston, TX). Described reagents were purchased from Invitrogen (Rockville, MD).
Rabbit polyclonal antibody to HCV NS4B was obtained from Covance (Denver, PA). Rabbit polyclonal antibodies to HCV NS3, NS5A and NS5B were kindly provided by Craig Cameron (Penn State, University Park, PA). Antibodies to Rab 1A, 2, 4A, 5A & B, 6 (Santa Cruz Biotechnology, Santa Cruz, CA), and Rab7 (Cell Signaling, Danvers, MA) were used in these studies. Horseradish peroxidase-conjugated secondary antibodies (used in chemiluminescence) were obtained from Vector laboratories (Burlingame, CA). Alexa Fluor 594-conjugated secondary antibodies (used in immunofluorescence) were from Invotrogen.
CFP-fused Wt Rab constructs (1, 2, 6, 7 and 11), their dominant negative versions (Rab1A-S25N, Rab2-S20N, Rab6A-T27N & Rab7-T22N), and GFP-fused Rab5A-S34N were provided by Won Do Heo (Korea Advanced Institute of Science and Technology, Korea). Wt GFP-Rab5 was kindly provided by Brian Knoll, Baylor College of Medicine (Houston, TX). To construct the YFP-Rab5 vector, pEYFP-C1 (Clontech, Mountain View, CA) was digested with NheI and NotI. The purified YFP fragment was subcloned into NheI- and NotI-cleaved GFP-Rab5 vector, thus replacing GFP.
For each experiment, Huh7 or C5B replicon cells were trypsinized and grown overnight in 6-well plates to obtain 60% confluent monolayer cells. Prior to transfection, the cells were washed with phosphate-buffered saline (PBS) and fed with 2 ml of complete medium. 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%.
Immunoisolation of a subcellular fraction containing HCV replicase proteins
The immunoisolation was done according to Stone et al. (Stone et al., 2007
) with a slight modification. Briefly, 4-6 × 100 mm dishes (1 × 106
cells/dish), each of parental (Huh7) and replicon cells (C5B), were grown for 48h. The cells were trypsinized, washed, and resuspended in phosphate-buffered saline (PBS) solution containing 0.25M sucrose (PBS/sucrose) and protease inhibitors (1mM PMSF and 1 tablet of Complete, Mini, EDTA-free; Roche, Nutley, NJ). The cells were then lysed with 7 passages in a ball-bearing homogenizer (Balch and Rothman, 1985
) to ensure approximately 90% lysis. Cell lysates were spun at 2500 xg/10 min at 4°C to pellet cellular debris and nuclei. The resulting supernatant will be referred to as the crude lysate or lysate throughout this paper. Purified NS4B antibody (1:200 dilution; Covance, Denver, PA) was added to equal amount (4 mg) of protein from either parental or replicon cells, and the 1.6 ml mixture (adjusted with PBS/sucrose and 3% final BSA) was incubated overnight at 4°C with constant rotation. The resulting homogenate was overlaid on a discontinuous iodixanol (Sigma-Aldrich, St. Louis, MO) gradient (4%, 8%, 12%, 20%, 30%, and 35%) in PBS/sucrose and the gradient was spun at 120,000 xg/1h 45min at 4°C in an Ti 80 rotor. A total of 12 fractions (630 l each) were collected from top to bottom. Typically, fractions 7 and 8 are endoplasmic reticulum (ER)-enriched, as determined by Calnexin-positive bands; in the replicon lysates, these fractions also contain viral replicase proteins including NS3, NS4B, NS5A and NS5B.
For membrane floatation assay, fractions 7 and 8 were combined and mixed with 60% iodixanol. A discontinuous iodixanol gradient (5%, 25% and 30%) (Elazar et al., 2004
) was layered on the top of the combined fractions and the samples were spun at 120,000 xg for 4h 25min at 4°C in a Ti80 Rotor. A total of 8 fractions (867 μl each) were collected from top to bottom. Typically, the floatation gradient fraction 2 contained most of the HCV replicase proteins.
To immunoisolate a subcellular fraction containing NS4B and associated host factors, secondary anti rabbit antibody-coated magnetic Dynabeads M-280 (4.3 × 107/ml; Invitrogen, Carlsbad, CA) were added to the floated membrane fraction 2 with 3% final BSA. After overnight incubation at 4°C with constant rotation, the mixture was placed in a magnetic rack (Invitrogen) for 2-5 min. The resulting supernatant was collected and labeled as “Unbound”. NS4B positive membrane-bound beads were labeled as “Bound”. The bound fraction was washed four times to get rid off loosely bound proteins or membranes, whereas the unbound supernatant microsomes were spun twice at 100,000 xg, each for 1 h at 4°C. Bound and unbound fractions were resuspended in 4x sample buffer (240 mM Tris pH 6.8, 4% SDS, 40% glycerol, 4% β-mercaptoethanol, 0.01% bromophenol blue) for immunoblot. For HCV RNA-dependent RNA polymerase activity, Bound and unbound fractions were resuspended in diethylpyrocarbonate (DEPC)-treated PBS.
Sypro Ruby staining of replicase fraction
To examine the protein profile in the different fractions, an equal amount of protein (1 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was fixed with 50% methanol/ 7% acetic acid and stained overnight with 75% Sypro Ruby Protein Gel Stain (Invitrogen, Carlsbad, California). After two washes in 10% methanol/7% acetic acid, the protein bands were visualized on a phosphorImager (Typhoon 8600, Molecular Dynamics, Sunnyvale, CA).
Protein identification by capillary LC-ESI-MS/MS
Protein bands mostly enriched in the bound fraction, but not the unbound fraction, were excised, divided into pieces no larger than 1 mm and subjected to in-gel digestion overnight with sequencing grade trypsin (Promega Corp., Madison, WI). Samples were analyzed by a capillary liquid chromatography–tandem mass spectrometry system with a nanospray ionization source (capLC-nanoESI-MS/MS) that consisted of a Surveyor Micro AS autosampler, a Surveyor MS Pump, and an LTQ linear ion trap mass spectrometer (Thermo Electron Corporation, Waltham, MA). The digests were desalted on-line and eluted by using a 30-min linear acetonitrile gradient (2% to 40 % acetonitrile in 0.1% formic acid) at a flow rate of ~0.3 μL/min obtained by pre-column splitting of a flow of 200 μL/min. The analytical column was a PicoFrit column (New Objective) packed in-house with 5 μm wide pore C18 particles (Supelco Co, Bellefonte, PA). From a survey scan performed over m/z 550 -1500, tandem mass spectrometry (e.g. collision induced dissociation or CID) was triggered for precursor ions above an intensity threshold determined based on the baseline. Raw tandem mass spectra were processed and searched against the protein sequence database using a locally licensed and maintained Mascot 2.2 search engine to identify proteins. Mass tolerance was 3 amu and 2 amu for precursor and product ions, respectively. Up to 2 missed cleavages were allowed for digestion by trypsin and methionine oxidation was considered as a variable modification. At least two unique peptides with a Mascot peptide score higher than 40 were required to confidently identity a protein.
Quantitative Real-time PCR
Total cellular RNA was prepared from bound and unbound samples by using the TRIZOL method (Invitrogen, Carlsbad, CA), followed by precipitation with isopropanol and resuspension in DEPC-treated water. First strand cDNA was synthesized from the DNA-free RNA using random primers and the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Triplicate samples of cDNA were mixed with a Taqman probe and a set of forward and reverse primers specific for either HCV NS4B (1b, strain N) or GAPDH and the mixture was subjected to real-time quantitative PCR using the ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA).
In-vitro HCV RNA synthesis
Typically, 25 μl of crude lysate, bound or unbound fraction was resuspended in a 50 μl reaction containing 1 mM ATP, GTP and UTP (CTP at 40 μM), 1 mCi of (α32P) CTP per ml, 1 × buffer (50 mM Tris-Cl pH 7.8, 50 mM KCl, 5 mM mgCl2, 10 mM DTT, 5 mM creatine phosphate and 25 μg/ml creatine kinase), 10 μg/ml actinomycin D and 800 U/ml RNasin. The reaction was incubated at 37°C for 1h, with gentle mixing (every 30 min), followed by RNA extraction using the TRIZOL method (Invitrogen, Carlsbad, CA). The RNA was precipitated with isopropanol, washed in ethanol, dried and resuspended in DEPC-treated water. The RNA was separated on 1% formaldehyde denaturing gel. After drying, the gel was exposed to a phosphor screen and the RNA was visualized with a phosphorImager.
Immunoblot of HCV and host proteins
To visualize proteins from the bound and unbound fractions, 50 μg of protein were resuspended in 4x sample buffer, while 50 μg of crude lysates were used as controls. The samples were separated on 10% SDS-PAGE, followed by transfer onto an Immobilon-P membrane (PVDF; Millipore, Billerica, MA). Following binding with the respective primary antibody and HRP-conjugated secondary antibody, proteins were visualized by enhanced chemiluminescence detection method (ECL western blotting substrate, Pierce, Rockford, Il).
Cells on coverslips were washed in PBS and fixe d in 4% formaldehyde in PBS for 15 min and permeabilized with 0.05% Triton X-100 for 5 min at room temperature (RT). The cells were washed three times with PBS and incubated in 3% bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted in 3% BSA and incubated with the cells for 1 h at room temperature. After three washes, Alexa fluor 594 (1:1000)-conjugated secondary antibody (Molecular Probes, Eugene, OR) was added to the cells for 1 h at RT. After three more washes in PBS, the cells were stained with DAPI/PBS for 10 min at room temperature, followed by three additional washes in PBS. The cells were mounted on glass slides in Vectashield (Vector Laboratories, Inc., Burlingame, CA). The samples were then examined with a confocal laser scanning microscope (Olympus Fluoroview FV1000). Image analysis was performed using Olympus Fluoroview version 1.7c software. Pearson's co-efficient (PC) for colocalization is calculated on a pixel by pixel basis; it is from a scale of −1 to 1 and is based on two variables (x and y) and the linear relationship between these variables. Numbers below 0.5 suggest little to no colocalization, whereas those above 0.5 indicate moderate to strong association. All the images were taken using similar conditions. Co-localization of green (FITC) and red (cy3) signals produces yellow, whereas co-localization of blue, green and red results in white. All images were saved as TIFF files, imported to and processed in Adobe Photoshop.
The day before transfection, C5B replicon cells were trypsinized and grown overnight in 6-well plates to yield a roughly 60% confluent monolayer. Prior to transfection, the cells were washed with phosphate-buffered saline (PBS) and fed with 1 ml of complete medium. Cells were transfected with 50nM siRNA according to the TransIT-TKO protocol from Mirus (Madison, WI). siGENOME SMARTpool siRNA was obtained from Dharmacon (Lafayette, CO), and was resuspended as a 20 uM stock in 1x siRNA buffer (Dharmacon). 50 nM of siCONTROL nontargeting siRNA pool was used as a control. Transfected cells were incubated for 48 h in complete media, and processed for RT-PCR analysis or western blotting.