Cells and Viruses
Madin-Darby bovine kidney (MDBK) cells were grown in DMEM, supplemented with 10% heat-inactivated horse serum (HS), sodium pyruvate (1 mM), nonessential amino acids (0.1 mM), penicillin (100 units/ml) and streptomycin (100 μg/ml). Baby hamster kidney (BHK-21) cells were grown in DMEM, supplemented with 10% heat-inactivated calf serum (or Advanced DMEM supplemented with 1.5% FBS), nonessential amino acids (0.1 mM), penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells were maintained at 37°C in a 5% CO2
incubator. The cytopathic (cp) strain of bovine viral diarrhea virus (BVDV), NADL, was generated through the use of a cDNA clone, pNADLp15A [40
], supplied graciously by Ruben Donis, Center for Disease Control (CDC, Atlanta, GA).
BVDV NS4B and NS3 polyclonal antibodies were kindly supplied by Rubin Donis (CDC, Atlanta) and Charles Rice (Rockefeller University), respectively. Alkaline phosphatase (AP)-conjugated anti-rabbit and anti-mouse secondary antibodies were from Vector Laboratories (Burlingame, CA). TGN38 and GFP polyclonal antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Golgin-97 polyclonal antibody was from Abcam Inc, (Cambridge, MA) and Alexa Fluor 488- or 594-conjugated secondary antibodies were from Invitrogen (Carlsbad, CA). Penta-His monoclonal antibody was from Qiagen (Valencia, CA), whereas HA polyclonal antibody was from Affinity Bioreagents (Golden, CO). For immuno-EM studies, the secondary antibody used was conjugated to electron-dense quantum dots (Q-dots) 605 (Molecular Probes, Invitrogen, Carlsbad, CA).
To construct plasmids containing BVDV genes of interest, the desired gene was amplified from pNADLp15A. For recombinant vector containing NS4B-GFP, primers were designed to introduce a BglII site at the 5' end of the gene, a BamHI site at the 3' end, and an AUG start codon immediately upstream of the BVDV NS4B coding region. The resulting PCR product was cloned into pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA) and the sequence was confirmed. Recombinant vector containing NS4B was cleaved with EcoRI and BamHI and the purified fragment was subcloned into EcoRI- and BamHI-cleaved pEGFP-N1 vector (Clonetech, Palo Alto, CA). The resulting vector was cleaved with XhoI and NotI and the purified NS4B-GFP fragment was subcloned into SalI- and NotI-cleaved pIRES vector (Clonetech, Palo Alto, CA). For subsequent plasmid construction requiring DNA amplification, the genes of interest were cloned into pCR2.1 TOPO vector and sequences were confirmed. To construct a plasmid containing BVDV NS5A, NS5A was amplified with primers that introduced an XhoI site at the 5' end, a NotI site and 6xHis epitope tag at the 3' end, and an AUG start codon immediately upstream of the NS5A coding region. Recombinant pCR2.1 plasmid with NS5A-His was cut with XhoI and NotI and the purified NS5A-His fragment was subcloned into an XhoI- and NotI-cleaved pIRES vector. To construct the plasmid containing BVDV NS5B, NS5B was amplified with primers that introduced an EcoRI site at the 5' end, a NotI site, an epitope HA tag at the 3' end, and an AUG start codon immediately upstream of the NS5B coding region. Recombinant pCR2.1 plasmid with NS5B-HA was cut with EcoRI and NotI and the purified NS5B-HA fragment was subcloned into an EcoRI- and NotI-cleaved pIRES vector.
For each experiment, BHK-21 cells were trypsinized and grown overnight in 10 cm dishes 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. Cells were transfected according to the LipoD293 protocol from SignaGen (Ljamsville, MD). Ten micrograms of DNA to were added to 400 μl of OptiMEM, while 30 μl of LipoD293 were added to 400 μl OptiMEM. The LipoD293 mixture was then added directly to the diluted DNA and incubated for 15 min at room temperature. The DNA mixture was then added to each dish and incubated at 37°C for 24 h to 48 h.
In vitro transcription, electroporation and generation of infectious BVDV
To linearize BVDV genome, pNADLp15A was digested with SacII (New England Bio Labs, Ipswich, MA) at 37°C for 1 h, followed by incubation at 70°C for 15 min to inactivate SacII. pNADLp15A 3' overhangs were eliminated following incubation with 5 mM dNTPs and T4 DNA polymerase at 16°C for 30 min. The linearized DNA was then extracted using Phenol/Chloroform, followed by ethanol precipitation at -20°C for 2 h., The samples were resuspended in 10 μl RNase-free water. SacII-linearized pNADLp15A was used as template for in vitro transcription reaction with the T7 RiboMAX™ Kit (Promega, Madison, WI). The RNA was then isolated using the RNeasy miniprep kit (QIAGEN, Valencia, CA) and its integrity was assessed on a 0.8% agarose gel.
Before electroporation, MDBK cells were trypsinized and washed twice with PBS and resuspended to a final concentration of 1 × 107 cells/ml in RNase-free PBS. Three micrograms of in vitro-transcribed BVDV genomic RNA were mixed with 0.4 ml (4 × 106) of the cell suspension in a 2 mm-gap electroporation cuvette and pulsed with a Bio-Rad Gene Pulser (1 pulse; 125 μF; 0.28 kV). One milliliter of complete DMEM, antibiotic-free, was added to the cuvette and the resuspended sample was transferred to a 15 ml conical tube. Two milliliters of complete DMEM, antibiotic-free, was used to wash the cuvette to recover the remaining sample and was added to the same 15 ml conical tube. The resuspended sample was then divided into 3 wells in a 6-well plate. Complete medium (without antibiotic) was added to each well to bring the final volume to ca. 2 ml. Cells were incubated at 37°C in a 5% CO2 incubator. At 12 h p.t., the floating, dead cells were removed. Attached cells were washed twice with PBS and fresh complete DMEM was added to each well. Cells were observed at 24 h, 48 h, 72 h, and 96 h for cytopathic effects.
MDBK cells were seeded in 6-well plates at 3 × 105 cells per well. At the time of infection, cells were typically 70-80% confluent. On the day of infection, medium was removed and the monolayers were washed twice with PBS. The viral stock was diluted in serum-free DMEM. Cells were infected with 0.2 ml of the serially diluted virus (10-fold dilutions). Following adsorption, the monolayers were washed with 1 ml of complete DMEM and overlaid with DMEM-5% horse serum/0.5% agarose plugs. Plates were incubated for 15-30 min at RT to let the agarose solidify. Plates were then incubated at 37°C for 72 h. At 72 h post-infection, cells were fixed with 4% formaldehyde (in PBS) for 2 h. The agarose plugs were removed and the fixed monolayers were rinsed once with PBS. The monolayers were stained with 1% crystal violet (in 50% ethanol) for 10 min. The plates were rinsed with distilled water and plaques were counted. The viral titer was determined as follows: number of plaques × 5 × dilution factor. The resulting titer was expressed in plaque forming units per ml (pfu/ml).
BVDV Growth Kinetics
MDBK cells were seeded in 60 mm dishes at 4 × 105
cells per dish and grown overnight. The cell monolayers were then washed twice with PBS and infected at an MOI of 0.1 [5
]. After adsorption, the monolayer was washed with PBS, and 5 ml fresh complete media was added to each plate. For each time point (0 h, 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h post-infection), the medium was harvested from the plate and frozen. Fresh serum-free DMEM (5 ml) was added to the monolayer and the cells were lysed via two cycles of freeze/thaw. To determine the amount of infectious virus particles in the medium and lysate at each time point, plaque assays (as described above) were performed in duplicate. Each plaque assay was repeated three times.
Quantitative Real-time PCR
To examine the kinetics of viral BVDV synthesis at various times (0 h, 6 h, 12 h, 18 h, and 24 h) post infection, Real-Time PCR was performed. First, MDBK cells were infected with BVDV at MOI of 0.1. Total cellular RNA was collected at each time point using the RNeasy Mini Kit (Qiagen, Valencia, CA).
Total cellular RNA was prepared from virus-infected cells by using the RNeasy Mini Kit (Qiagen) and was treated with RNase-free DNase (Qiagen, Valencia, CA). 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 BVDV NS4B or GAPDH and the mixture was subjected to real-time quantitative PCR using the ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA).
Immunoblot analysis of BVDV Proteins
Infected MDBK cells were lysed using RIPA buffer containing 150 mM NaCl, 50 mM Tris pH 8, 1 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM PMSF and protein concentrations were measured via Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). One hundred micrograms of total protein were 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 10 min, and centrifuged at 12000 × g for 10 min. Samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and transferred onto Immobilon-P transfer membrane (Millipore, Billerica, MA). Antibody-bound proteins were detected by chemifluorescence (ECF, Amersham/GE Healthcare, Piscataway, NJ) and visualized on a phosphorimager (Typhoon 8600 Molecular Dynamics, Sunnyvale, CA).
Membrane Floatation Assay
For membrane floatation assay, BHK-21 cells were grown overnight and transfected with BVDV NS4B-GFP or control GFP construct according to the conditions described above. At 48 h p.t., the cells were resuspended in homogenization buffer (150 mM NaCl, 50 mM Tris pH 7.4, 2 mM EDTA) containing protease inhibitors (1 mM 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 × g/10 min at 4°C to pellet cellular debris and nuclei. A discontinuous iodixanol gradient (5%, 25% and 30%) [30
] was layered on the top of the homogenate and the samples were spun at 120,000 × g for 4 h 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 with equal volume of 20% TCA, 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.
Subcellular fractionation of BVDV NS4B protein
Subcellular fractionation of BVDV NS4B protein was performed as described by Hugle et al. [34
]. BHK-21 cells expressing BVDV NS4B were trypsinized at 48 h p.t. (p.t.) and resuspended in complete medium on ice. The cells were then spun at approximately 200 × g/5 min at 4°C, followed by two washes in PBS. The cells were finally resuspended in ice cold hypotonic buffer (10 mM Tris-Cl, pH 7.5, 2 mM MgCl2
) and lysed by 20 strokes of a dounce homogenizer to ensure approximately 90-95% lysis. Next, the lysate was spun at 1000 × g/5 min to pellet the nuclear fraction. Sixty micrograms of the supernatant were resuspended in RIPA buffer and labeled "lysate". The remainder of this supernatant was adjusted to 0.25 m sucrose and spun at 9000 × g/10 min to pellet the mitochondrial fraction. The supernatant from the mitochondrial centrifugation was then spun at 105,000 × g/40 min to obtain the microsomal pellet. Sixty micrograms of the remaining supernatant was saved for immunoblot analysis and labeled as "cytoplasmic".
MDBK cells were grown on coverslips and infected with BVDV. The coverslips were washed with PBS and fixed for 10 min in 4% formaldehyde/PBS. Fixed cells were permeabilized for 6 min at room temperature (RT) in 0.05% Triton-X 100/PBS, followed by staining with the primary polyclonal antibody (or antibodies in double labeling experiments) and Alexa fluor 594- (or 488)-conjugated secondary antibody. After three washes in PBS, the cells were stained with 0.36 mM DAPI in PBS for 10 min at RT, followed by three more washes in PBS. The coverslips were mounted on slides using Vectashield (Vector Co., Burlingame, CA) and nail polish. The samples were analyzed by fluorescence microscopy (Zeiss Axiovert 200M) at × 63 magnification and digital images were taken with a CCD camera Axiocam MRm. 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. Colocalization of green (FITC) and red (Cy3) signals results in yellow fluorescence.
For analysis of BVDV NS4B-expressing cells, BHK-21 cells were grown on coverslips and transfected in 10 cm dishes as described above. At 48 h p.t., the coverslips were washed with PBS and the cells stained for 30 min with 100 nm ER-Tracker, LysoTracker, or 1 μM MitoTracker (Invitrogen, Molecular Probes) in complete medium at 37°C in a 5% CO2 incubator. The cells were then washed in PBS and fixed for 10 min in 4% formaldehyde/PBS. For immunostaining of BHK-21 cells, fixed cells were permeabilized for 10 min at room temperature in 0.1% Triton-X 100/PBS, washed three times in PBS, and stained with the appropriate antibody for 1 h at room temperature, followed by three more washes in PBS. The cells were then immunostained with AlexaFluor 594-conjugated secondary antibody for 1 h followed by washing three times with PBS. The cells were mounted on glass slides and processed for fluorescence microscopy as described above.
MDBK cells were seeded at 6.8 × 105 cells per 100 mm dish. Cells were infected at MOI of 10 and collected at various times (0 h, 12 h, 18 h, 24 h, 48 h, and 72 h) post-infection. Briefly, at various times post-infection, the cells were resuspended in 2% glutaraldehyde/0.1 m sodium cacodylate buffer and incubated on ice for 30 min. After a brief spin, fresh 2% glutaraldehde/0.1 m 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 grids were double stained with uranyl acetate and lead citrate and the sections were examined with a JEOL 1200 EXII transmission electron microscope (Peabody, MA) at 80 kV.
For Immuno-EM analysis of infected cells, MDBK cells were plated in 8-chamber slides at 5.4 × 104 cells per chamber. Cells were harvested at 18 h.p.i. and fixed to the bottom of the chamber with 4% paraformaldehyde/0.1% glutaraldehyde for 10 min. Cells were permeabilized with 0.05% Triton-X for 6 min at RT. After permeabilization, cells were washed three times with PBS. Permeabilized cells were then blocked with 3% BSA in PBS for 30 min at RT. Immediately following blocking, anti-NS4B antibody, diluted 1:50 in 3% BSA in PBS, was applied to the fixed cells for 1 h at RT. The cells were washed three times in PBS (15 min each). The secondary anti-Rabbit 605-Quantum dots (Molecular Probes, Invitrogen, Carlsbad CA), diluted 1:125 in 3% BSA in PBS, were incubated with the cells for 2 h at 4°C, swirling gently. After incubation, cells were washed three times in PBS, 15 min each. Finally, nuclei were stained using 0.36 mM DAPI in PBS for 10 min at RT. Quantum dot labeling was observed via fluorescence microscopy. Labeled cells were fixed with 2.5% glutaraldehyde prior to sectioning and electron microscopy (see above).