Propagation of Synthetic Papillomaviruses
Chris Buck and Cindy Thompson
(rev. May 2007)
Note: a more detailed version of this protocol is available from Current Protocols in Cell Biology.
When expressed in mammalian cells, the papillomavirus capsid proteins, L1 and L2, promiscuously package <8 kb segments of DNA, including transfected plasmids. This protocol outlines the use of a plasmid, p16L1L2, that expresses HPV16 L1 and L2, carries an SV40 origin of replication (Ori) and is small enough to become packaged within L1/L2 capsids. When the plasmid is transfected into 293TT (or 293-dTH cells), which express high levels of SV40 T antigen, the resulting self-packaged plasmid can be used as a seed stock to produce L1/L2 capsids by infecting a second or third round of cells. Using this system, it is possible to generate at least a milligram of purified virions from a 75 cm2flask of cells. Because of the promiscuity of packaging, it is possible to co-propagate reporter plasmids carrying the SV40 Ori as part of a viral swarm. This allows inexpensive, high-yield production of reporter pseudoviruses. Systems for propagation of synthetic HPV18 and BPV1 are available, but are not as well characterized and virion yields generally aren’t as high as for synthetic HPV16.
CAUTION: If the L1+L2 plasmid were to pick up SV40 T antigen (resident in 293TT cells), it could theoretically become an autonomous tumor virus. Synthetic papillomaviruses should be handled according to the recommendations of an institutional biosafety committee or other appropriate advisory body. Our laboratory has received authorization to handle pseudoviruses under biosafety level 2 conditions. See this CDC linkfor further information. It is also possible to use 293-dTH cells, which express a “safety-modified” SV40 T antigen mutant that is deficient for pRb and p53 binding. Although the virion yields from 293-dTH cells is not quite as good, the improved safety of the virion stock may be desirable. Laboratory personnel may also wish to consider receiving the new HPV vaccine, Gardasil (Merck), as an added safety precaution.
Step 1: producing a seed stock:
An initial crude seed stock is produced according to a slightly modified versionof the standard pseudovirus production protocol. The modified “ripcord” protocol selectively removes capsids associated with cellular DNA. This results in a substantial improvement in the stock’s particle to infectivity ratio.
Day 0: Late afternoon, pre-plate 7.5 million 293TT cells in 20 ml of DMEM-10 in a 75 cm2flask.
Day 1: Morning, transfect the cells with p16L1L2 using Lipofectamine 2000 (Invitrogen 11668-019), basically following the package insert. It is critically important that the cells be less 50% confluent for efficient transfection. Optional: co-transfect with a reporter plasmid, such as pCIneo-GFP, to monitor the infectivity of the stock. Cells should be about 50% confluent at the time of transfection.
•Mix 80 µl of Lipofectamine 2000 reagent with 2 ml of OptiMEM-I (Invitrogen 31985-062). Incubate 10 minutes.
•Mix 38 µg of p16L1L2 plasmid (or 19 µg each of p16L1L2 and reporter plasmid of interest) with 2ml of OptiMEM-I.
•Combine the two mixtures and incubate 20-30 minutes.
•Add the lipid/DNA complexes directly to the flask (no need to change medium).
•Incubate cells with lipid complexes for 4-6 hours, then change cells into fresh DMEM-10 pre-warmed to 37º C.
Day 3: Harvest and lyse cells.
•Remove and discard medium. Rinse away residual FCS by gently cascading 2 ml of trypsin along the top edge of the cell monolayer.
•Remove first trypsin rinse, then gently add 2 ml of fresh trypsin. Incubate at 37º C for 5 minutes, rocking flask occasionally. Cells should be completely detached from the flask by gentle rocking.
•Rinse cells to the bottom of the flask with 7.5 ml of DMEM-10. Triturate several times and transfer cells to a 15 ml conical tube. Collect residual cells by rinsing flask with an additional 4 ml of DMEM-10.
•Pellet cells 10 min at 100 x g. Remove supernatant. Resuspend cells in 0.5 ml of DPBS-Mg (Invitrogen 14040-141 supplemented with 9.5 mM MgCl2and 1x pen-strep-fungizone (Invitrogen 15240-112)). Transfer suspension to a 1.5 ml sterile screw-cap siliconized tube (e.g., VWR 60828-818). Collect residual cells from the 15 ml conical tube by rinsing it with an additional 0.5 ml of DPBS-Mg.
•Pellet cells. Carefully remove supernatant. Estimate the cell pellet volume by side-by-side comparison to fluid in a dummy tube. Pellet volume is typically 60-80 µl.
•Add one cell pellet volume of DPBS-Mg. For example, add 80 µl of DPBS-Mg to a cell pellet of 80µl. Resuspend cells by gently flicking or vortexing the tube. It is critical that the cells be suspended at very high density (at least 100 million per ml).
•Add 1/12thof a cell pellet volume of 10% Brij-58 stock. This gives a final Brij concentration of about 0.4% in the final cell suspension.
•Add 1 µl of RNase cocktail (Ambion 2286).
•Allow virions to mature by incubating the cell lysate at 37º C for 24 hours. It may be helpful to resuspend the lysate by gently flicking the tube at ~1 hr and ~18 hrs (the resuspension is not essential).
Day 4: Clarify and aliquot seed stock
•Chill the matured lysate briefly on ice. Centrifuge at 5,000 x g for 10 min.
•Transfer virus-containing supernatant to a fresh siliconized tube. Mix then divide into 20 µl aliquots and freeze at –80º C.
Step 2: Virion production
Day 0: Pre-plate a T-225 flask with 12 million 293TT cells.
Day 1: Add a 20µl aliquot of seed stock directly to the pre-plated flask of cells.
Day 2: Change cells into fresh DMEM-10 medium. This media change is not essential.
Day 4: Harvest cells. Their morphology should look fairly bad – rounded cells growing in a webby-looking monolayer. Under hemacytometer, cells should look swollen. For bulk production of capsid, prepare cell lysates exactly as outlined in the standard pseudovirus production protocol, using Benzonase and Plasmid Safe nucleases. Note that the use of DNases results in the liberation of capsids containing 8 kb linear fragments of cellular DNA. Capsids associated with cellular DNA outnumber plasmid-containing capsids by about 20:1. Thus the use of DNases substantially increases to total yield of capsids. If the goal is simply to make an infectious pseudovirus stock, use Rnase (no DNases) and clarify without additional salt, as described above for seed stock production.
Day 5: Clarify lysate (with or without salt, depending on whether DNases have been added). Purify virions using Optiprep gradients (see standard protocol) or by agarose gel filtration.
Alternative procedure: Production of L1-only virus-like particles.
Produce a seed stock by co-transfecting cells with p16sheLL and p16L1-GFP. Since p16sheLL is too large to be taken up into virions, only the L1 plasmid will be incorporated into infectious particles. p16L1-GFP carries a GFP expression cassette and can be titered on 293TT cells, as outlined in the standard pseudovirus production protocol. If the initial seed stock has been titered, use it at an moi of 3 to infect fresh 293TT cells, as described above.