Cells and viruses
A cell culture-adapted BTV serotype 10isolate (BTV-10; Colorado isolate) was used. The virus was propagated in Vero (African Green Monkey Kidney) cells and its titre was determined by plaque assay in BSR (Baby Hamster Kidney) cells. Vero and BSR cells were were incubated at 37°C in Dulbecco's modified Eagle's medium (Gibco BRL) containing 10% foetal calf serum (FCS), 100U penicillin/ml, and 100 μg streptomycin/ml (Sigma-Aldrich Chemical Co., St. Louis, Mo.). Recombinant baculovirus expressing bluetongue virus VP2 (AcMNPV-VP2) was propagated in Spodoptera frugiperda (Sf21) cells grown in suspension cultures at 28°C in TC100 medium (Sigma-Aldrich) supplemented with 10% foetal calf serum. Purification of recombinant VP2 was completed as described [4,40].
Antibodies and pharmacological reagents
Monoclonal antibody against BTV-10 VP2 was a gift from N J. MacLachlan (School of Vet. Med., Davis, CA). Polyclonal antibodies against vimentin (goat), actin (goat) and GFP (rabbit) were purchased from Santa Cruz Biotech, CA. Polyclonal antibodies against ubiquitinin, tubulin and nuclear lamin were purchased from Abcam (Cambridge UK). Tetramethyl rhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were purchased from Sigma-Aldrich. For pharmacological experiments, molecular grade acrylamide and cholchicine were purchased from Sigma-Aldrich.
Construction of fusion proteins and VP2 mutants
Plasmids expressing VP2 were constructed by PCR amplifying the full-length L2 gene from BTV-10 and ligating it downstream of the Pol II or T7 RNA polymerase promoters in the pCAG-GS (The CABRI Consortium) or pCITE-2a vectors (Novagen), respectively.
To express the N-terminal GFP tagged VP2 fusion protein (GFP-VP2), GFP was amplified by PCR without its stop codon and inserted in-frame upstream of the VP2 protein. To express C-terminal GFP tagged VP2 (VP2-GFP) the normal VP2 stop codon was mutated to a restriction site using the QuickChange™ mutagenesis system (Stratagene, La Jolla, CA) and the GFP gene was excised from the pEGFP plasmid and ligated in-frame to the modified VP2 gene. VP21–118GFP was produced by cloning EGFP as a blunt-end fragment from pEGFP (Clontech, Mountain View, CA) into a plasmid containing the full-length BTV-10 VP2 gene cut with AflII. For deletion analyses, site-directed mutagenesis was used to introduce unique restriction sites in VP21–118GFP plasmid and internal deletions were introduced by cutting at the inserted restriction sites and religating the plasmid. Mutations were designed to introduce minimal changes to the amino acid sequence of the resulting deletion mutants. The QuickChange™ system (Stratagene) was used to generate amino acid substitution mutants in the VP21–118GFP plasmid according to the manufacturer's protocol.
To express full-length VP2 with the DVD mutation at amino acids 70–72 the transfer vector pAcYM10.2 [40] expressing wild-type VP2 was mutated using the same primers and protocol as to introduce the mutation into the VP21–118GFP plasmid. The orientation of constructs in all the plasmids was examined by restriction enzyme analysis, and the authenticity of each construct was confirmed by DNA sequencing using the ABI Prism® Big Dye™Terminator cycle sequencing ready reaction kits (Applied Biosystems, Foster City, CA).
Isolation of recombinant baculoviruses expressing mutant VP2 protein
Recombinant baculovirus expressing the DVD mutant for VP2 was produced using standard baculovirus recombination procedures as described [41]. Recombinant baculoviruses were plaque purified and propagated in Sf21 cells as described elsewhere [41].
Expression of proteins from transfected plasmids and virus infection
Vero cells were plated in six well plates and plasmid DNA was transfected with Lipofectamine Plus™ transfection reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Expression was analysed at 12–72 hours post-transfection as indicated in the text.
Confocal microscopy
BTV infected or plasmid transfected cells were fixed on coverslips by incubation for 30 min at room temperature with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS). Cover slips were then incubated for 15 min at room temperature with 1% Triton X in PBS (pH 7.5) and blocked for an hour with PBS containing 1% bovine serum albumin. Cells were incubated with primary antibodies diluted (1:100) in blocking buffer for 1 h, washed 5 times with PBS then incubated with appropriate secondary antibodies conjugated to TRITC (1:64) or FITC (1:128) prior to washing with PBS and mounting in Vectashield mounting media (Vector Laboratories, Burlingham, CA). Coverslips were examined with a Zeiss Axiovert 200 M laser-scanning microscope fitted with a helium-argon laser. Images were acquired and analysed using LSM 510 confocal software (Zeiss). For GFP imaging, cells grown on cover slips were transfected with the different GFP chimeras, washed and fixed at 12 hour intervals, from 0 to 48 hours post transfection as described previously.
Isolation of Detergent Soluble (DS) and Insoluble (DI) fractions
Fractions were essentially prepared as described [42], with some minor modifications. In brief, 72 hours post transfection, cells were rinsed twice with ice-cold PBS, re-suspended and centrifuged at 1000 g for 5 minutes (Sanyo Micro Centaur, Jepson Bolton & Co Ltd, Watford, UK). The cell pellet was lysed for 30 min at 4°C in 0.2 ml of a 25 mM Tris-HCl (pH 7.5) buffer, containing 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100, followed by 30 strokes in a dounce homogeniser. detergent soluble and insoluble fractions were separated by centrifugation for 15 minutes at 12,000 g (4°C) and resolved by SDS/10% Polyacrylamide gel electrophoresis (PAGE) [43]. In some experiments, cell lysis was carried out at 37°C instead of 4°C, and 1% Nonidet P-40 (NP40) was used instead of 1% Triton X-100 in the lysis buffer, as indicated in the text. SDS PAGE gels were stained with Coomassie brilliant blue as standard.
Analysis of the oligomeric nature of VP2
Sf21 cell monolayers were infected with recombinant baculoviruses (pAcNPV.VP2 or pAcNPV.VP2M1) at a MOI of 3. Cells were harvested at 72 hours post infection, washed with PBS and lysed in 20 mM sodium phosphate buffer containing 150 mM NaCl [pH 7.5], 1% [v/v] Triton X and protease inhibitors (Sigma-Aldrich) at 4°C for 10 minutes. The lysate was clarified by centrifugation for 10 min at 10,000 rpm (Sanyo Micro Centaur, Jepson Bolton & Co Ltd, Watford, United Kingdom) and sample buffer added to a final concentration of (1% SDS, 15% glycerol, 10 mM Tris-HCl [pH 6.8], 0.02% [wt/vol] bromophenol blue) without 1% β-mercaptoethanol, without heating, and resolved by SDS-7% PAGE followed by western blotting.
Western blot analysis
SDS-PAGE separated proteins were transferred onto a Hybond enhanced chemiluminescence nitrocellulose membrane (GE Healthcare, Uppsala Sweden) as described previously [44] and were probed with appropriate antibodies to GFP, vimentin, tubulin, lamin and actin. Subsequently, the blots were incubated with alkaline phosphatase conjugated secondary antibodies and developed with BCIP-NBT substrate (Sigma-Aldrich).
Treatment of the cells with cytoskeleton inhibitors
Cells were washed 48 hours post transfection and incubated in medium with 50 μM of acrylamide (24 hours) or 5 μg/ml of colchicine (2 hours). They were then processed for immunofluorescence staining as described above. To examine the effect of the inhibitors on viral yield at different time points, the cells were first infected as described previously and then pharmacologically treated. Cell supernatant and cell pellets were collected and were subjected to three freeze/thaw cycles to disrupt cells and to release virus. All samples were collected, the viral titer of each sample was determined in triplicate by virus plaque assay in BSR cell monolayer cultures. Infectivity titers were calculated as log10 pfu/ml, and the mean and standard error of the reduction mediated by each inhibitor was calculated (Sigma-AldrichPlot 2000, SYSTAT Software Inc.). All the pharmacological treatments were performed in duplicate and the plaque assay in triplicate.
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