Preparation of virus antigens
The 4 prototype dengue viruses were used in this study. All viruses were propagated in Aedes albopictus C6/36 cells grown in Leibovitz 15 media supplemented with 5% heat inactivated foetal calf serum, antibiotics and 10% tryptose phosphate broth. Antigens were prepared by inoculating C6/36 cell monolayers with the different DENV serotypes as described previously and harvested when syncytium formation was extensive. Cell culture fluids were clarified by centrifugation before use. Fluids similarly prepared from mock infected C6/36 cells were used as negative antigen controls.
Preparation of cell and membrane extracts
Just confluent flasks of the porcine kidney cell line PS Clone D were used in the preparation of detergent extracts for separation by 2D gel electrophoresis. Monolayers were washed twice with phosphate buffered saline (PBS) before subjecting to treatment with 1% β octyl-glucopyranoside (βOG) in a hypotonic buffer containing 10 mM HEPES, 1.5 mM MgCl2, 5 mM KCl and a protease inhibitor cocktail (Boehringer Mannheim GmbH, Germany), pH 7.5 rocking at 4°C for 1 hour. The solution was removed and designated βOG-extract.
The remaining membranes, cytoskeleton and nuclei were then washed for 1 hour at 4°C with a solution containing 2% CHAPS in the same buffer as described above. The resulting solution was discarded and the residual material solubilized by rocking at 4°C for 1 hour in the above buffer containing 1% sodium deoxycholate (NaDOC). The resulting solution was removed and designated βOG-insoluble NaDOC-extract.
All extracts were spun in a microfuge at 14,000 rpm for 10 minutes and the supernatants stored at -20°C until use.
Sample preparation for 2D gel electrophoresis
All samples were prepared for 2D gel electrophoresis using the Ready Prep 2-D Cleanup Kit (BioRad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions.
2D gel electrophoresis
Protein pellets were resolubilized in IPG strip rehydration solution (8 M urea, 2% CHAPS, 40 mM DTT, 0.5% IPG buffer pH3-10, bromophenol blue) at room temperature for 30 minutes, then spun in a microfuge at 14,000 rpm for 10 min. 125 ul of the resulting supernatant was used for each IPG strip (ReadyStrips pH 3-10, 7 cm, BioRad Laboratories, Hercules, CA, USA) and rehydration was achieved at 50 uA for 15 hours at 20°C using the IPGphor IEF system (Amersham Pharmacia Biotech, Uppsala, Sweden). Subsequently IEF was carried out for 30 minutes at 500 V, 30 minutes at 1000 V and 2 to 2.5 hours at 8000 V with a step-and-hold gradient until a total of 8500 volt-hours had been achieved.
IPG strips were then washed with distilled water and then equilibrated by rocking for 20 minutes at room temperature in SDS equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 10 mg/ml DTT, allowing for at least 5 ml of buffer per strip.
Strips were then washed with distilled water and placed on the top surface of the second dimension gel which was a 10% SDS polyacrylamide gel polymerized overnight. Molecular weight markers were applied onto small pieces of chromatography paper and inserted next to each strip on the top of each gel, after which the strips and markers were sealed with 0.7% agarose in 0.125 M Tris-HCl pH 6.8. The second dimension separation of proteins by molecular mass was achieved at a constant 140 V (Mini Protean 3, BioRad Laboratories, Hercules, CA, USA).
Electrotransfer of 2D gels to nitrocellulose
The Hoefer TE series Transfor Electrophoresis Unit (Hoefer Scientific Instruments, San Francisco, CA, USA) was used to electrotransfer proteins from 2D gels to nitrocellulose membranes at 200 mA for 1 hour in ice cold Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol). Nitrocellulose blots were then stained using Ponceau S. A record of the positions of the visible protein spots on each blot was made by scanning the Ponceau S probed blot using ImageScanner (Amersham Pharmacia Biotech, Uppsala, Sweden) and the software ImageMaster Labscan v3.00 (Amersham Biosciences, UK). After scanning, the Ponceau S was stripped by washing in water and the blots were then blocked by rocking for 1 hour in PBS containing 5% skimmed milk.
Virus overlay protein binding assay (VOPBA)
The 2D blots were incubated overnight with rocking at room temperature with clarified antigen preparations and mock-infected controls. The blots were then washed with PBS and incubated with the anti-flavivirus monoclonal antibody 4G2 in another overnight incubation at room temperature. After washing with PBS, the blots were incubated with rabbit-anti-mouse Ig HRP (DAKO, Glostrup, Denmark) at 1:1000 dilution in 5% skimmed milk in PBS for 2 hrs at room temperature. The blots were then washed with PBS and reactive protein spots were visualized by developing with the chromogenic substrate, 4-chloro-1-naphthol/hydrogen peroxide. Reaction was stopped after 1 hr by washing with water. The membranes were scanned and compared with the Ponceau S images scanned previously using Adobe Photoshop version 5.0 LE (Adobe Systems Inc., San Jose, CA, USA).
In-gel trypsin digestion and analysis by MALDI-TOF MS
Reactive spots seen on the blots were identified in the Ponceau S scans which had been recorded previously and the corresponding spots in the coomassie blue-stained gel were picked and stored in UHQ water in 0.5 ml microfuge tubes at 4°C. During all steps in the digestion process the buffer used was 5 mM NH4HCO3. Gel spots were first destained with 50% HPLC grade methanol. Destained spots were dehydrated with acetonitrile for 10 minutes before incubation with 10 mM DTT for 50 minutes at 55°C. This was followed by incubation with 55 mM iodoacetamide (IAA) for 30 minutes at room temperature in the dark. The spots were then washed twice with buffer for 20 minutes each time, dehydrated with acetonitrile for 10 minutes and rehydrated with buffer. Finally the gel spots were dehydrated twice with acetonitrile for 10 minutes each time and dried completely by centrifugation under vacuum (DNA Speed-Vac DNA110, Savant Instruments Inc, Farmingdale, NY, USA) for 10 minutes. Each gel spot was then reswelled in 5 ul of 12.5 ng/ul of sequencing grade trypsin (Promega, Madison, WI, USA) in 5 mM NH4HCO3 for 45 minutes on ice. Excess trypsin solution was then removed, the spots were covered in 5 ul buffer and digestion was allowed to proceed at 37°C overnight. Digests were stored at -20°C until analysed.
Analysis by MALDI-TOF MS
For MALDI analysis, digests were thawed, spun in a microfuge at 14,000 rpm for 10 minutes. One ul of the supernatant was mixed in a 1:1 ratio with a 1:10 dilution of saturated α-cyano-4-hydroxycinnamic acid (ACCA) matrix in 0.25% trifluoroacetic acid, 50% acetonitrile, 50% water. This mixture was spotted onto MALDI target plates and spectra were acquired using Voyager-DE STR Biospectrometry workstation (Applied Biosystems, Foster City, CA, USA). Peptide mass lists were submitted for search against the NCBI database (National Institutes of Health, Bethesda, MD, USA) using the MASCOT search engine (Matrix Science, London, UK). No constraints were set for species but carbamiodomethylation of cysteine residues and possible missed-cleavages were included.
Immunostaining of 2D gel blots
The 2D blots were incubated with polyclonal rabbit antisera against LAMR1 and Lamin B1 diluted 1:200 in PBS with 5% skimmed milk at room temperature, overnight with rocking. After extensive washing with PBS, the bound antibodies were detected with anti rabbit Ig conjugated with horseradish peroxidase, and visualized using the chromogenic substrate 4-chloro-1-naphthol/hydrogen peroxide as described above. Antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Surface staining of cells and photomicrography
Cells were resuspended at 1 × 105 cells per ml in Leibovitz 15 media containing 3% heat inactivated foetal calf serum, antibiotics and tryptose phosphate broth. Resuspended cells were delivered in 25 ul volumes to individual wells of multitest slides (Erie Scientific Co., Portsmouth, NH, USA) and allowed to adhere overnight in a moist box at 37°C. Cells were then washed in PBS and fixed with 3.7% paraformaldehye in PBS at pH 7.4 for 15 minutes followed by a shift to 2% paraformaldehyde in PBS at pH 8.5 for a further 15 minutes. After washing in PBS slides were air dried and stored at -20°C until use.
Prior to staining, slides were incubated in 50 mM ammonium chloride in PBS for 5 minutes, washed thoroughly and blocked in 1% foetal calf serum in PBS for 30 minutes. Immunofluorescence staining of the surface of cells was achieved by incubation for 1 hour with polyclonal rabbit antisera against LAMR1 at 1:25 dilution in PBS containing 1% foetal calf serum. After washing with PBS the cells were incubated with anti rabbit Ig conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) at 1:1000 dilution for 30 minutes following by washing with PBS. DAPI was used to counterstain nuclei. Slides were viewed using an Axiovert 200 (Zeiss, Germany) with filter sets appropriate for FITC and DAPI.
Photomicrography was achieved using a cooled CCD monochrome 12 bit camera Evolution QEi and Image-Pro 5.0 software (Media Cybernetics Inc., Canada) was used for preparing fluorescence composite images with pseudocolour. Adobe Photoshop version 5.0 LE was used to compose and present the figure collage.