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Wild-Type and Mutant SV40 1
1
Propagation of Wild-Type and Mutant SV40
James D. Tremblay, Kris F. Sachsenmeier, and James M. Pipas
1. Introduction
Simian virus 40 (SV40) is routinely propagated in established kidney cell
lines derived from the African green monkey (1). Generally, one of two cell
lines, BSC-1 or CV1, are used for this purpose (2,3). Both of these cell lines
are easy to maintain in culture and can be readily frozen and recovered from
frozen storage. Wild-type SV40 grows and plaques equally well in both cell
lines.
Because SV40 uses cellular machinery to carry out many steps in viral
infection, it has proved to be a powerful probe of molecular mechanisms of
transcription, DNA replication, and growth control. SV40 subverts cellular
systems toward virus production by acting on key regulatory cellular targets
and pathways. The analysis of SV40 mutants that are unable to act upon spe-
cific cellular targets has proved extremely useful in identifying key targets and
understanding how the virus alters their function (4,5). In this chapter, we describe
methods for propagating and assaying infectious SV40. We also briefly outline
the routine assays used in our laboratory to assess the effects of specific muta-
tions on viral productive infection, DNA replication, and transformation.
2. Materials
1. BS-C-1 or CV-1 cells: ATCC (Rockville, MD) cat. no. CCL-26 or CCL-70,
respectively.
2. Minimum essential medium (Gibco-BRL, Grand Island, NY) supplemented
with 10% or 2% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin, and
100µg/mL streptomycin (Gibco-BRL).
3. 1.8% Bacto-Agar (Difco) in distilled water. Autoclave and cool to 45°C.
From: Methods in Molecular Biology, vol. 165: SV40 Protocols
Edited by: L. Raptis © Humana Press Inc., Totowa, NJ
1
2 Tremblay, Sachsenmeier, and Pipas
4. 2X Modified Eagle’s medium (MEM) (without phenol red) (Gibco-BRL) supple-
mented with 10% FBS and 200 U/mL penicillin and 200 µg/mL streptomycin
(Gibco-BRL).
5. Neutral Red solution, 3.33 g/L (Gibco-BRL).
6. Diethylaminoethyl (DEAE) Dextran (Sigma) solution: 1 mg/mL in 2X MEM.
7. Qiagen plasmid purification columns (Qiagen Inc., Valencia, CA).
8. Transfectamine (Gibco-BRL).
3. Methods
3.1. Cell Culture
1. Culture BSC-1 or CV-1 cells as monolayers in MEM-10% FBS at 37°C in a
humidified incubator with 5% carbon dioxide and 95% air.
2. Change medium every 3–4 d and split cells prior to confluence.
3.2. SV40 Virus Stock Production
1. Prepare 80% to 100% confluent T75 flasks of BS-C-1 or CV-1 cells (seeNote 1).
2. Remove medium from dishes and replace with 2 mL of MEM-2% FBS contain-
ing the appropriate dilution of SV40 plaque forming units (PFU’s) to achieve a
multiplicity of infection (MOI) between 0.1 and 0.01 (seeNote 2).
3. Allow infection to proceed for 2 h in an incubator while rocking plates at 15-min
intervals to assure complete coverage and even distribution over the monolayer.
4. After this period, MEM-2% FBS should be added to each plate to obtain a final
volume of 10 mL of medium per 10-cm plate.
5. After 4 d of incubation, the medium should be replaced with fresh MEM-2%
FBS. After this point, cells should be checked daily for signs of cytopathic
effects (CPE) by comparing infected monolayers to the uninfected control (see
Note 3 and Fig. 1).
6. When CPE develops to the point of complete destruction of the monolayer with
floating clumps of cells, place flasks at –20°C overnight or until completely fro-
zen and then thaw at room temperature. Repeat for a total of three freeze/thaw
cycles.
7. This is the viral stock. It will contain cellular debris, which can be removed by
centrifugation at 200 RCF for 5 min if desired.
3.3. Titering SV40 by Plaque Assay
1. Prepare serial dilutions of the SV40 virus stock in MEM-2% FBS by putting
20 mL of stock solution into 2 mL of MEM-2 and vortex vigorously (10–2 dilution).
Repeat this procedure using the immediately preceding dilution in place of the
stock to make 10–4and 10–6dilutions. 10–7and 10–8dilutions should be made by
using 200 mL of the preceding dilutions in 2 mL of MEM-2% FBS (seeNote 4).
2. Use 1 mL of each dilution to infect 6-cm dishes of freshly confluent dishes of
BS-C-1 or CV-1 after removing the old medium. Be sure to include a dish with
1 mL of MEM-2% FBS without virus as mock control.
Wild-Type and Mutant SV40 3
Fig. 1. Cytopathic effect of BS-C-40 cells infected with SV40. (A) Confluent mono-
layer of BS-C-40 cells 17 h after infection. After 7 d, a CPE is clearly discernible by
comparing mock (B) with SV40 (C) infections.
4 Tremblay, Sachsenmeier, and Pipas
3. As in virus stock production, dishes should be incubated at 37°C for 2 h with
rocking at 15-min intervals to assure even coverage of the monolayer.
4. After this 2-h period, remove the 1 mL of MEM-2 and overlay the monolayers
with 4 mL of a fresh mixture containing 1 part melted 1.8% Bacto-Agar cooled to
45°C and 1 part 2X modified Eagle’s medium (without phenol red) with 10%
FBS at 37°C. Allow the overlay to harden at room temperature for approx 15 min
before returning dishes to the incubator (seeNote 5).
5. Add an additional 3 mL of this mixture every 3 d with the exception of the sixth
day, when neutral red should be added to the overlay mixture at a final concentra-
tion of 50 µg/mL, in order to visualize the plaques (seeNote 6).
6. Plaques should be visible and can be counted on the eighth day and beyond. The
number of plaques times the inverse of the dilution factor gives the titer of the
virus stock in pfu/mL.
3.4. Strategies for Analyzing SV40 Mutants
SV40 mutants continue to be generated for a variety of purposes. We will
not review the various methods used for selecting for SV40 mutants in vivo or
constructing them in vitro. Rather, we briefly describe the routine assays our
laboratory uses to assess the effects of mutations on virus growth and transfor-
mation. It is important to realize that the assays described here represent a
preliminary analysis of mutant activity. More thorough studies are needed to
understand the basis for each mutant phenotype.
Following sequencing to confirm the nature of the mutation, we perform
three tests to assess mutant activity. First, a viral plaque assay is performed as
aforementioned. In this test, the starting material is viral DNA introduced into
cells by transfection, rather than virus particles. In cases where the mutant
virus is defective for plaque formation, we attempt to rescue the mutant pheno-
type by complementation with a characterized defective SV40 mutant. Thus, if
the mutation of interest maps to T antigen, it should be rescued by any defec-
tive SV40 mutant that expresses a wild-type T antigen. For example, deletion
mutant dl 1007, lacking late regions coding for viral coat proteins can comple-
ment deletions in early regions, such as mutant 3213, which fails to bind to the
tumor suppressor pRb(4). Second, we assess the ability of the mutant to repli-
cate viral DNA. Finally, we perform a focus formation assay to test the trans-
forming ability of the mutant.
3.5. Analysis of Mutants
3.5.1. Transfection and Plaque Assays
1. Transfect freshly confluent 6-cm plates of BS-C-1 cells using 100 mL of 1 µg/mL
DEAE Dextran in 2X MEM mixed with equal parts of water containing 10 to
30 ng of purified DNA. Carefully add this mixture to the center of each plate
after removal of the old medium.
Wild-Type and Mutant SV40 5
2. After 15 min, overlay cell monolayers as described in steps 4–6ofSubheading 3.3.
3. Plaques should be scored by number, size, and time of appearance as compared
to wild-type SV40.
3.5.2. Complementation Assays
1. Complementation assays are carried out as above (see Subheading 3.5.1.)
except that equal quantities of the two virus mutants to be tested are mixed and
added to cells at once.
3.5.3. In Vivo DNA Replication Assay
1. Transfect freshly confluent 6-cm dishes of BS-C-1 cells with 45 ng of wild-type
SV40 DNA or 45 ng of each mutant DNA using the DEAE Dextran method afore-
mentioned with an 800 µL total volume.
2. After 15 min, feed dishes with 10 mL of MEM-2% FBS and return to the incuba-
tor for 2 or 3 d or until the desired time-point.
3. Trypsinize cells from the plates and extract DNA using Qiagen miniprep col-
umns, according to the manufacturer’s instructions.
4. Quantitate extracted DNA and then digest it with BclI to linearize newly replicated
DNA, and with DpnI to remove any residual nonreplicated input DNA (seeNote 7).
5. Separate DNA extracts by gel electrophoresis on a 0.8% agarose gel.
6. Transfer to a nylon membrane and hybridize with a 32P-labeled probe synthe-
sized using random primers on an SV40 genomic or cloned DNA template (6,see
Chapter 12).
3.5.4. Dense Focus Assay for Transformation
1. Transfect subconfluent plates of REF 52 cells with 2 µg of each DNA using
Transfectamine according to the manufacturer’s instructions (seeNote 8).
2. On the following day, rinse the Transfectamine and DNA from the cells and
replace the culture medium.
3. After 24 to 48 h, split cultures 1(cid:1)3 and feed twice weekly with MEM-10% FBS.
4. After 4 to 6 wk, stain plates with crystal violet and count foci using cells sub-
jected to mock transfections (e.g., vector alone) for comparison (seeFig. 2B).
For more details on transformation assays, see Chapter 11.
4. Notes
1. SV40 virus stock production procedures can easily be scaled up or down to any
size or number of dishes.
2. The MOI is the ratio of plaque-forming units (PFUs, i.e., number of infectious
particles) to the number of cells being infected. The most important parameter
for production of virus stock is keeping the MOI low. This is important to pre-
vent the replication of spontaneously occurring defective mutant viruses in the
stock. High MOI infections allow replication of defective viruses because they
are complemented in transby wild-type viruses infecting the same cell. Another
6 Tremblay, Sachsenmeier, and Pipas
Fig. 2. SV40 viral plaques and dense foci. (A) Plaques visible on a 60-mm
diameter agar plate after more than 2 wk of growth. The arrow indicates an individual
plaque.(B) A 75-cm2 flask stained for foci after 6 wk of culture. The arrow indicates
an individual dense focus.
important practice to reduce the occurrence of defective viruses in the stock is to
avoid serial passage of the virus when preparing new stocks. Because a relatively
small amount of virus is needed to produce a stock, it is best to have one master
virus stock which is used exclusively to produce working virus stocks for use in
experimental infections.
The next consideration after the MOI is the volume of medium used in the
infection. Ideally, the volume should be kept as low as possible while still com-
pletely covering the monolayer of cells being infected (approx 0.5 mL for a 3-cm
plate, 1 mL for a 6-cm plate, 2 mL for a 10-cm plate). This keeps the virions in
close proximity to the monolayer, promoting rapid absorption of the virions to
the cell surface and into the cells. Under these conditions most of the virus particles
will have entered the cells of the monolayer within the 1.5 h of incubation at 37°C.
3. The cytopathic effect associated with lytic SV40 infections in BS-C-1 cells can
be separated into four stages (Fig. 1). Stage 1: some rounded, refractile cells
appearing on the monolayer. Stage 2: many rounded, refractile cells on the mono-
layer with some forming groups. Stage 3: clumping of cells and the beginning of
deterioration of the monolayer. Stage 4: complete destruction of the monolayer
with floating clumps of cells. Some cells, such as CV1 show large vacuoles late
after infection.
4. Precise serial dilution is a key step in SV40 stock titering. It is critical that each
dilution be thoroughly mixed before it is used to make the subsequent dilutions.
It is also important to make the serial dilutions immediately before they are used
because the virus in very dilute solutions tends to be less stable.
Wild-Type and Mutant SV40 7
5. Another critical step in titering plaque assays is the preparation of the overlay
mixture. The key is to work quickly. The agar/medium mixture must not be too
hot when placed on the cells, so that it will start solidifying very soon after being
mixed together. If the mixture begins to harden before it is placed on the cells, the
overlay will have a rough texture which will make the plaques harder to see. Also
it is important to avoid bubbles in the agar/medium overlay.
6. Although one 3-mL agar/medium overlay with 50 mg/mL of neutral red is suffi-
cient to visualize plaques, the amount of neutral red can be increased in order to
improve the contrast between living cells that take up the red color and plaques
of dead cells that do not (seeFig. 2A). It is also important to note that a full 24 h
is often required for the neutral red to migrate through the agar/medium overlay
and evenly stain the monolayer.
7. There is a single BclI site in the SV40 genome. Thus, digestion with this enzyme
linearizes the circular genome DNA molecule, allowing for a single-band signal
during Southern blotting using a genomic SV40 probe. DpnI digests only methy-
lated DNA, The nonreplicated DNA (i.e., input DNA) is of bacterial origin and
will therefore be methylated, unlike genomic SV40 DNA replicated in the host
cell. DpnI will therefore selectively digest only the DNA added experimentally
as original SV40 template (6).
8. In our hands, transfectamine is at least as sensitive as DEAE-dextran or calcium
phosphate and allows the use of smaller amounts of DNA than is possible with
these methods.
References
1. Rapp, F., Kitahara, T., Butel, J. S., and Melnick, J. L. (1964) Synthesis of SV40
tumor antigen during replication of Simian Papovavirus (SV40). Proc. Nat. Acad.
Sci. USA53, 1138.
2. Hopps, H. E., Bernheim, B. C., Nisalak, A., Tjio, J. H., and Smadel, J. E. J. (1963)
Biologic characteristics of a continuous Kidney cell line derived from the African
green monkey. J. Immunol.91, 416.
3. Kit, S., Dubbs, D. R., DeTorres, R. A., and Melnick., J. L. (1965) Enhanced Thy-
midine kinase activity following infection of green monkey kidney cells by Sim-
ian Adenoviruses, Simian Papovavirus SV40, and an Adenovirus-SV40 hybrid.
Virology27, 453.
4. Pipas, J. M., Peden, K. W., and Nathans, D. (1983) Mutational analysis of Simian
Virus 40 T Antigen: isolation and characterization of mutants with deletions in
the T-Antigen gene. Mol. Cell. Biol.3, 203.
5. Peden, K. W., Srinivasan, A., Farber. J. M., and Pipas, J. M. (1989) Mutants with
changes within or near a hydrophobic region of Simian Virus 40 Large Tumor
antigen are defective for binding cellular protein p53. Virology168, 13.
6. Peden, K. W., Pipas, J. M., Pearson-White, S., and Nathans, D. (1980) Isolation of
mutants of an animal virus in bacteria. Science209, 1392–1396.
Propagation and Assay of the JC Virus 9
2
Propagation and Assay of the JC Virus
Christine K. Liu and Walter J. Atwood
1. Introduction
The human polyomavirus, JCV, is the etiological agent of a fatal central
nervous system (CNS) demyelinating disease known as progressive multifocal
leukoencephalopathy, or PML. Seroepidemiological studies have indicated that
greater than 70% of the human population worldwide is infected with JCV.
Like other polyomaviruses, JCV establishes a lifelong latent or persistent
infection in its natural host (1–5). Reactivation of JCV in the setting of an
underlying immunosuppressive illness, such as AIDS, leads to virus dissemi-
nation to the CNS, infection of oligodendrocytes, and the development of PML
(1,2,6,7). Not surprisingly, the incidence of PML has increased dramatically as
a result of the AIDS pandemic. A recent epidemiological study has found that
PML increased twentyfold from 0.2 cases per million people in 1979 to 3.3
cases per million people in 1994 (8). The increase in PML has renewed an
interest in studying the biology of this common human polyomavirus.
JCV was initially isolated from the brain of a patient with progressive mul-
tifocal leukoencephalopathy whose initials were J.C. (9). Isolation was
achieved by incubation of primary human fetal glial cells (PHFG) with extracts
prepared from PML brain taken at necropsy. PHFG cells and a few cell lines
derived from these cultures are the only cell types that are fully permissive to
the lytic growth of JCV. This chapter will focus on the methods and techniques
currently used to propagate and assay this virus in tissue culture.
2. Materials
2.1. Cell Culture
1. Cells: The human glial cell line, SVG is available from the American Type Cul-
ture Collection (ATCC) (Rockville, MD), cat. no. CRL-8621 (seeNote 1).
From:Methods in Molecular Biology, vol. 165: SV40 Protocols
Edited by: L. Raptis © Humana Press Inc., Totowa, NJ
9
PB Liu and Atwood
2. Eagle’s Minimal Essential Medium (EMEM, Mediatech, Gaithersburg, MD)
supplemented with 10% heat inactivated fetal calf serum (FCS) (Mediatech) and
100 U of penicillin/streptomycin (Gibco-BRL, Gaithersburg, MD). Store the
medium at 4°C, and let the solution come to room temperature before adding to
the cells. The complete medium is stable for about 60 d, as after this period the
serum proteins will begin to degrade and the effectiveness of the antibiotics will
diminish (seeNote 2).
3. JCV stock: ATCC, cat. no. VR-1397 (Mad-4 strain) or cat. no. VR-819 (Mad-1
strain). Store at –80°C or in liquid nitrogen (seeNote 3).
4. Trypsin-ethylenediaminetetraacetic acid (EDTA) (0.05% trypsin and 0.53 mM
EDTA-4Na) (Gibco-BRL).
5. Sterile phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na HPO , 1.4 mM KH PO , pH 7.2; sterilize by autoclaving.
2 4 2 4
6. 2.5% deoxycholic acid (DOC). Filter sterilize.
7. Receptor destroying enzyme: Crude neuraminidase from Vibrio cholerae, 0.2 U/mL
(Calbiochem, La Jolla, CA).
2.2. Hemagglutination Assay
1. 96-well U-bottom plate.
2. PBS (seeitem 5 in Subheading 2.1.).
3. Type O human blood collected in a Vacutainer tube containing anticoagulant
(seeNote 4).
4. Alsever’s solution: 0.1 M D-glucose (dextrose), 0.027 M sodium citrate, 0.07 M
sodium chloride. Adjust pH to 6.5 with citric acid and incubate for 30 min at
room temperature. Filter sterilize.
5. RBC Fix (ISOLAB, Inc. Akron, OH, cat. no. RS-3200), and RBC Wash
(ISOLAB, Inc. cat. no. RS-3100).
2.3. Indirect Immunofluorescence Assay
1. Six-well tissue-culture dishes.
2. Sterile 18-mm glass coverslips.
3. Monoclonal antibody directed against JCV V antigen (Novocastra Laboratories,
Newcastle upon Tyne, UK, cat. no. NCL-JC). A rabbit polyclonal anti-SV40 V
antigen antiserum (Lee Biomolecular Research, San Diego, CA, cat. no. 6201),
or a monoclonal antibody against SV40 T antigen (PAB416-AB2, cat. no. DP02)
which crossreacts with JCV T antigen, can also be used (seeNote 5).
4. Goat antimouse secondary antibody conjugated to a fluorogenic dye such as fluo-
rescein isothiocyanate (FITC). If the polyclonal serum was used as a primary
antibody, then the secondary must be antirabbit. Both can be obtained from a
number of sources.
5. PBS (seeitem 5 in Subheading 2.1.).
6. Copplin jars or other suitable staining dish.
7. Fixative: There are many fixatives that can be used. The two we use routinely are
acetone and 70% ethanol/30% 1X PBS (v/v): Mix 100% ethanol with the appro-
