This is the original protocol developed by Lucas Pelkmans for the growing of SV40 virus stock, and for purifying full and empty capsids from such a stock. This protocol can be adapted to other non-enveloped viruses, although specific buffers may be required to ensure capsid stability.
1. Grow the virus.
- Incubate 24 T175 flasks with confluent CV-1 cells at 37ºC and 5%CO2 with SV40 at a MOI of 0.01 (can be obtained from ATCC) for 2h in 5 ml of R-medium (RPMI 1640, 10mM Hepes pH 6.8, 0.2% BSA) per flask, using a rocker in the incubator.
- After that, add 50 ml of complete medium (DMEM, 10% FCS, 1x Glutamax) to each flask and incubate cells further at 37ºC and 5%CO2.
2. Harvest the virus stock.
- After 10 days, freeze-thaw cells by placing flasks in a -20ºC freezer and back in a 37ºC incubator 3 times.
- Collect all medium and cellular debris (using a cell scraper to maximize yield), and spin down debris at 10,000 x g for 10 min at 4ºC (using a Sorvall centrifuge that can handle large volumes).
- Save the supernatant and resuspend the pellet in 1/50 volume of supernatant.
- Freeze/thaw this suspension another 3 times and repellet debris.
- Combine both supernatants, which now represents the virus stock.
3. Purify empty capsids and capsids containing DNA.
- Load 20 ml of virus stock on a 10 ml cushion of CsCl (ρ = 1.4 g/ml) buffered with 10 mM Hepes in ultraclear centrifuge tubes.
- Band virus in the CsCl cushion by centrifugation at 24,000 rpm for 3 hours at 4ºC in a SW28 rotor (Beckman).
- Isolate the banded virus after first removing all medium from the top and some of the CsCl until just above the band, which is visible as a whitish narrow zone (careful inspection reveals that this zone actually consists of two bands on top of each other). Use a pasteur pipette or needle to collect this whole zone, but as little as possible of the CsCl above and below this zone.
- Check the density of the collected suspension (must be app. 1.34 g/ml) using a refractometer and dilute it 5 times into new Hepes buffered CsCl (ρ = 1.34 g/ml).
- Transfer this suspension to sealable ultraclear centrifuge tubes, and centrifuge to equilibrium at 40,000 rpm for 16 hours at 4ºC in a 70.1 Ti rotor (Beckman). Now, two bands are clearly visible. The upper virus band represents empty capsids, while the lower band represents capsids with DNA.
- Isolate both bands separately and dialyse extensively (use at least 20x more volume than the virus suspension) at 4ºC against virion buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM CaCl2) if no other modifications of the virus particles are required, or against 0.1 M carbonate buffer pH 8.3 for subsequent labeling with fluorophores (or other groups) via a succinimidyl ester linker. Exchange with fresh buffer after 2-3 hours of dialysis and continue dialysis overnight at 4ºC.
- The dialysed virus can be further concentrated, if necessary, by using a centricon with a large cut-off (100,000 NMWL).
- The purified virus is highly stable and can be stored at 4ºC, or if wished, at -20ºC by adding some glycerol (10%) to prevent freezing. It can also be frozen at -80ºC without the need to add glycerol, but it is then recommended to make aliquots to avoid multiple freeze-thawings.
- The yield of this protocol should be at least 2-3 mg of pure infectious SV40, but sometimes yields of up to 9 mgs have been achieved.
- Check the purity of your virus by loading it on a reducing SDS-PAGE gel. A Coomassie stain should nicely reveal the structural proteins VP1, VP2, and VP3, as well as the 4 histones from the host cell.