Propagation of Synthetic
Papillomaviruses
Chris Buck and Cindy Thompson
Laboratory of Cellular Oncology,NCI
(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 cm2
flask 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 arenÕt
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 link for further
information. It is also possible
to use 293-dTH cells, which express a Ņsafety-modifiedÓ 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 version of the
standard pseudovirus
production protocol. The
modified ŅripcordÓ protocol selectively removes capsids associated with
cellular DNA. This results in a
substantial improvement in the stockÕs particle to infectivity ratio.
Day 0: Late afternoon, pre-plate 7.5 million
293TT cells in 20 ml of DMEM-10 in a 75 cm2 flask.
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 37¼
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 37¼ 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 MgCl2 and 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 1/12th of 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 37¼ 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 –80¼
C.
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.