1
Preparation and Titration
of CsCl-Banded Adenovirus Stock
Ann E. Tollefson, Terry W. Hermiston, and William S. M. Wold
1. Introduction
An important step m the development of modern experimental virology was
the development of the plaque assay, first with bacteriophage, then with
eukaryotic viruses. In order to obtain quantitative, interpretable, and reproduc-
ible results, it is necessary to know how much virus is bemg used in the experr-
ment. With adenovuuses, several approaches have generally been used to
quantitate vrrus stocks. First, virus particles are counted, e.g., in an electron
microscope (1,2). Another approach is to quantitate virion DNA by optical
absorbance (2). The problem with these approaches is that many adenovirus
particles are not infectious, perhaps because they have a defective complete
genome or they lack fiber or some other protein. The second approach is to
determine the number of plaque-forming units (PFU) per mL. Here, the analy-
sis quantitates the number of vnions capable of a full infectious cycle. This
approach will be described in detail in this article.
Experimental reproducibility also requires that adenovirus stocks be pre-
pared in a consistent manner. Such stocks are stable for years when stored
at -7OOC. One simple approach is to prepare a cytopathic effect (CPE) stock.
Here, an isolated plaque is picked and a small dish of permissive cells (e.g.,
A549) is infected. After approx 4-5 d, the cells in the monolayer will show
typical adenovirus CPE, i.e., their nuclei will become enlarged and they will
round up and detach from the dishes into individual floating cells as well as
grape-like clusters. These floating cells remain alive for some time (cell death
and the release of adenovnus from the cells begins at approx 3 d postinfection
for subgroup C adenoviruses). These cells are collected, adenovirus is released
by repeatedly freezing and thawing, and then it is used to infect a larger mono-
From Methods m Molecular Medrcme, Vol 21 Adenowrus Methods and Protocols
Edited by W S M Wold @ Humana Press Inc , Totowa, NJ

1
2 Tollefson, Hermiston, and Wold
layer, such as a T-150 flask. After CPE appears, cells are collected and aden-
ovirus is released by three rounds of freeze-thawing followed by sonication;
then the adenovirus is titered by plaque assay. These CPE stocks, which are
typically 1 Os-1O’o PFU/mL, are adequate for exploratory studies. Large-scale
vnus stocks are usually prepared by banding the virus in CsCl equilibrium-
density gradients, and this procedure will be described. CsCl-banding yields
large quantities of high-titer (10’ t PFU/mL) adenovirus stocks.
2. Materials
2.1. Cell-Culture Media and Stock Solutions
1 Dulbecco’s modified Eagle’s medium (DMEM) from powdered medmm (20 L):
DMEM (with high glucose, with L-glutamine, with phenol red, without sodmm
pyruvate, without sodium bicarbonate; Gtbco-BRL, Gaithersburg, MD; JRH Bto-
sciences [Lenexa, KS]) (2X 10 L), 74 g (3.7 g/L) of sodmm bicarbonate (tissue-
culture grade, Gibco-BRL or Sigma [St. Louis, MO]). Adjust the pH of the
solution to approx 6.9 with 1 N HCl or 1 N NaOH (pH will increase 0.3 to 0.4 U
with filtration). Add commercral penicillin-streptomycm stock (1 mL/L; Gibco-
BRL). Medium is membrane-sterilized by posrtive or negative pressure (postttve
pressure IS preferable in mamtaming pH) through a 0 22-p filter (Mrllipore,
Bedford, MA; Coming [Coming, NY], Nalge [Rochester, NY])
2. MEM (Joklik-modified) for suspension cultures (20 L) (see Note 1) mmimum
essential medium (S-MEM) (Jokbk-modified) (with L-glutamine, with 10X phos-
phate, without sodmm bicarbonate; Gibco-BRL or JRH Biosciences) (2X 10 L),
40 g sodium bicarbonate (2 g/L) Adjust pH to 6.9 with 1 N HCI or 1 N NaOH,
then add streptomycin-penicillin stock (1 mL/L). Sterilize medmm by membrane
filtration (0.22~pm filter).
3. 2X DME for plaque assay overlays (5 L) (see Note 2): Combine 4.5 L ddHzO
(tissue-culture grade), 0.6 g penicillin G (sodium salt, 1670 U/mg), 1 .O g strepto-
mycm sulfate (787 U/mg), DME powder (1X 10 L) (with htgh glucose, with

L-glutamine, with phenol red, without sodium bicarbonate, without sodium pyru-
vate; Gibco-BRL). AdJust to 5 L. pH is not adjusted at the time of preparation
(sodium bicarbonate stock is added at the time of overlay preparation). Sterilize
by membrane filtration through a 0.22-u filter
4. Penicillin and streptomycin: penicillin/streptomycin stock (1000X) contains
10,000 U/mL penicillin G sodium, and 10,000 U/mL streptomycin sulfate in
0.85% saline (Gtbco-BRL). Store frozen until use in preparation of DMEM and
Joklik-modified MEM.
5. Phosphate-buffered saline (PBS): prepare Dulbecco’s phosphate-buffered saline
(PBS) (without calcium chloride, without magnesium chloride; Sigma) with trs-
sue-culture grade water and sterilize by filtration through a 0.22~~.un filter.
6. Ttypsin-EDTA stock solution (4 L): dissolve 4 g trypsin (1:250, DIFCO Labora-
tories, Detroit,
MI), 2 g EDTA (disodium salt), 4 g dextrose, 20 mg phenol red
(sodium salt, Fisher, Pittsburgh, PA), 0.25 g penicillin G (sodium salt, 1670 U/mg,
&Cl-Banded Adenovirus Stock
3
Sigma), 0.45 g streptomycm sulfate (787 U/mg, Sigma) m a final volume of 4 L
Dulbecco’s PBS (calcium- and magnesium-free). Adjust pH to 7.2. Sterilize by mem-
brane filtration. Store aliquoted stocks frozen until they are needed as workmg stocks.
7. Horse serum and fetal bovine serum (see Notes 3 and 4): Sera are needed for
supplementation of the tissue culture media. Heat horse serum at 56°C for 30 min
to inactivate complement prior to use in growth or during infection of KB cells.
8. Tris-salme-glycerol (TSG) (for dilution of cesium-chloride-banded vnus): Solu-
tton A: 900 mL ddHz0, 8.0 g NaCl, 0.1 g NaHPO, (dibasic), 0.3 g KCl, 3.0 g
Trisma base (Tris); adjust pH to 7.4 by addition of approx l-2 mL concentrated
HCl. Solution B: 2.0 g MgCl,, 2 0 g CaCl,, 100 mL ddH20. Combine 700 mL
solution A with 3.5 mL solution B. Add 300 mL ultrapure glycerol (Gibco-BRL).
Heat solution m microwave and filter sterilize through 0.22~pm filter (unheated
solution is too viscous to filter).

9. 1.8% DIFCO Noble agar stock: Add 1.8 g Noble agar per 100 mL of tissue-culture
grade water (9 g of Noble agar in 500 mL water is convenient). Autoclave for
25-30 min to sterilize. Store at 4°C until approx 1 h prior to preparation of overlay.
10. Agar overlay medium for plaque assays: Final volumes of components for 100 mL
of overlay are as follows: 50 mL of 2X DME (see item 3), 5
mL 7.5% (w/v)
sodmm-bicarbonate stock solution (Gtbco-BRL), 2 mL fetal bovme serum (FBS),
43 mL 1 8% agar Noble stock (see item 9). Microwave 1.8% agar Noble stock to
melt; then reduce temperature to 56’C in a 56°C water bath prior to addition to
the other overlay components. Mix the other components of the overlay and keep
at 37°C in a water bath. These temperatures are used to keep the agar from solidt-
fying and to ensure that the overlay will not be hot enough to result in cell killmg
when it is layered onto the monolayer. If stock of 2X DME was prepared 1 mo or
more prior to the date of plaque assays, add 1 mL of glutamine stock (200 mM;
Gibco-BRL) per 100 mL of overlay. Neutral red is added to the second overlay.
Add 0.45 mL of neutral red stock (3.333 g/L neutral red sodium salt m distilled
water, membrane-filtered; Gibco-BRL) per 100 mL overlay. Addition of neutral
red to the first overlay may inhibit plaque formation and expansion. Do not reheat
or reuse the overlay mixture.
3. Method
3.1. Growth of Monolayer and Suspension Cultures
3.1.1. KB Cells
US-suspension cultures are a clonal line derived in the laboratory of Maurice
Green (from a KB-suspension culture received originally from Harry Eagle) and
grown in the laboratories of Maurice Green and William Wold. This clonal line is
reported to produce higher yields of virus than the parental KB cell line (3,#).
1 Grow KB cells in suspension in Joklik-modified MEM (5% heat-inactivated horse
serum) in spinner
flasks (Bellco Glass [Vineland, NJ]). Maintain cells m culture
with daily dilution of cells to maintain cultures in the 1.5-4.0 x lo5 cells/ml range.

4
Tollefson, Hermiston, and Weld
2. Split the culture each day to a cell density of 1.5-2.0 x lo5 cells per mL. Cells
typically double in 24 h. Add new medium to the cell suspension and discard
excess cells. Replace only a portion of the medium because conditioned medium
appears to have some beneficial effect on the growth of the cells (perhaps from
autocrine effects).
3.1.2. A549 Cells
A549 cells (CCL 185; American Type Culture Collection, Rockville, MD)
are grown in DMEM supplemented with glutamine and 10% FBS (HyClone,
Logan, UT; BioWhittaker [Walkersville, MD]; or Gibco-BRL).
1, For routine passage, remove medium from the plates and add trypsin-EDTA (2 mL
for 100~mm dish or 5 mL for a 1 75-cm2 flask).
2. When cells have rounded up (usually in 3-5 min), remove cells from the dish
by adding DMEM (10% FBS) with gentle pipettmg. Use of 10% FBS/DMEM
inhibits continued trypsm action and cells remain more intact with higher
viability.
3. Centrifuge cells at 600-1000 rpm (lOO-25Og) in a table-top centrifuge (e.g.,
Beckman GS-6) to pellet cells
4. Remove trypsin/medium solution and resuspend cells in DMEM (10% FBS), and
plate at 1:5 to 1:20 dilutions relative to the original cell density.
5. Cells are usually passaged at 2- to 3-d intervals. Grow cells at 37’C with 6% CO*
in humidified incubators in 100~nun dishes or 175-cm* flasks. Cells should not
be allowed to become very heavy in routine culture or they will not have good
survival in plaque assays.
3.1.3. Large-Scale Adenovirus Preparation
Spinner KB cells are used for large scale production of adenovnuses.
Grow cells m mmimal essential medium, Joklik-modified (Gibco-BRL or
JRH Biosciences), a suspension medtum with reduced calcium and
increased levels of phosphate, with 5% horse serum (heat-inactivated at

56OC for 30 min to inactivate complement). For infection, it 1s typical to
use a 3-L volume of cells that have reached a density of 3-3.5 x lo5 cells/ml.
Reduce volume for infection to 1 L by centrifuging 2400 mL of suspension
culture to pellet cells, resuspend these cells in approx 400 mL of serum-
free Joklik-modified MEM, and return cells to spinner. Infect cells with 5-
20 PFU/cell with stock viruses (lower MO1 will result in fewer defective
particles). Virus is adsorbed with spinning at 37OC for 1 h; at the end of the
adsorption period, add 2 L of medium (with 5% horse serum) to the infec-
tion. Maintain infected cells in spinner flasks at 37°C for 40-46 h; then
harvest. Given viruses may be incubated for more extended times if cell
lysis is not occurring (especially if the E3 gene for the adenovirus death
protein, previously named E3-11.6K, is absent).
CsCI-Banded Adenovirus Stock
5
Day 1:
1. Prepare 3 L KB spmner cells in Joklik-modified MEM/S% horse serum at approx
2 x lo5 cells per mL.
Day 2:
2. Do cell count on hemacytometer to determine the cell number. This cell number
will be used to determine the volume of virus to use for infection.
3. Reduce the total cell volume to 1 L by centrifugation. Cells (2400 mL) are
pelleted in a table-top centritige (Beckman GS-6 centrifuge) in 750-mL Beckman
centrifuge tubes at 1000 rpm (250g) for 10 min. Because the centrifuge bottle
bottoms are flat, the rotor is not braked at the end of the spin.
4 Resuspend cells in 400 mL Joklik-modified medium (serum-free) and return to
the spinner flask.
5. Add virus (5-20 PFWcell or use a portion of a CPE stock from a flask). If using
small volumes of banded vnus, it is best to dilute the virus in serum-free Joklik-
modified MEM in a 50-mL centrifuge tube (Falcon, Coming) prior to addition to
the spinner. Adsorb 1 h at 37°C with spinning.

Day 4:
6. Pellet infected cells in 750-mL Beckman centrifuge tubes in table-top centrifuge
(1000 rpm [25Og], 10 min, 4’C). Do not use brake at end of centrifugation.
7. Remove medium. Resuspend cell pellets m a total volume of 150-200 mL of
cold PBS (4°C) and transfer to a 250-mL conical centrifuge tube (Corning). Cen-
trifuge at 1000 rpm (250g) at 4°C for 10 min.
8. Repeat PBS wash and pelleting of cells twice.
9. Resuspend cell pellet in enough cold 10 mM Tris-HCl, pH 8.0 (4°C) to give a
final volume of 24 mL.
10. Ahquot 8 mL into each of three sterile polypropylene snap-cap tubes (15 mL
size), wrap caps with parafilm, and freeze at -7O’C or m ethanol/dry ice bath for
at least 1 h (processing can be left at this point for one or more d before complet-
ing the remainder of the protocol).
11. Thaw tubes m 37°C water bath. Repeat these freeze-thaw steps two more times
and then place tubes on ice.
12. Disrupt cells by sonication on me in the cup of a Branson sonifier 250. Settings
are as follows: duty cycle on “constant,” output control on 9 (scale of l-lo), and
3-mm cycles. Repeat three times for each sample.
13. Transfer somcated material to sterile 50-mL flip-cap centrifuge tubes and centnmge
at 10,000 rpm (12,000g) for 10 min at 4°C in a Beckman J2-HC centrifuge. Remove
supematant (which will contain released virions) and discard cell-debris pellet.
14. Determine the volume of supernatant and multiply by 0.5 1. The resulting number
will be the grams of CsCl to be added to the preparation. For example: 20 mL
of supernatant x 0.5 1 g of CsCl per mL equals 10.2 g of CsCl for addition to
the supernatant.
17. Stop the ultracentrifnge without using the brake.
18 The virus will appear as a white band that will be at approximately the middle of
the tube. Collect by syringe puncture at the bottom (puncture top of tube as well).
Band will visibly move down the tube. Collect the band region in a 15- or 50-mL
sterile centrifuge tube (Coming, Falcon) as it drips from the bottom Altema-

tively, the virus band can be removed by side puncture of the tube at the level of
the virus band with a syringe and withdrawing the band wtth the syringe
19. Dilute virus 5- to lo-fold in Trts-saline-glycerol (TSG) (see Subheading 2.1.,
item 8); this will usually result m a stock which is 101o-lO” PFU/mL when using
wild-type viruses.
20 Aliquot in l- to 3-mL volumes in sterile 6-mL snap-cap polypropylene tubes or
m cryovials and store at -70°C unttl needed.
21. Determine titer of the vtrus by plaque assay on A549 cells (see Note 5).
3.2. Plaque Assays for Determination of Adenovirus Titers
1.
2
3.
One day prior to plaque assay, plate A549 cells at 2.0 x lo6 tells/60-mm dish
(Coming, Falcon).
On the day of the plaque assay, wash dishes of confluent A549 cells with 5 mL
serum-free DMEM for 30-60 mm prtor to addition of the diluted vn-us; remove
this wash medium immediately before the addition of the VIIUS dilutions.
Make serial dtlutions of vtrus in serum-free DMEM; perform dilutions within a
laminar flow hood. Dilute virus in sterile-dtsposable snap-cap polypropylene
tubes and vortex well after each dilution (5-10 s at an 8-9 setting on a I-10
scale). Typically for cesium-chloride-banded stocks, the initial two dilutions are
1: 1000 (10 pL mto 10 mL), followed by dilutions of 1:lO. Be sure to change
micropipet tips after each dilution; avoid contamination of the mrcropipet barrel
(use of barrier tips will help avoid contammatton). Care should also be taken to
avoid transfer of virus stock on the outside of the micropipet tip by avoiding
dipping the tip into the solutton, especially in expellmg the volume. For cesium-
banded stocks, the range of dilutions that are usually countable are 1 W8-1 O-lo.
Place a volume of 0.5 mL of the appropriate dilution on confluent A549 cells
(each relevant dilution 1s assayed in triplicate).
Rock dishes to distribute medium over the monolayer at lo- to 15-mm intervals.

Incubate cells at 37°C with 6% CO,.
Immediately prior to addition of overlay to the cell monolayers, mix the agar
stock with the remaining ingredients
At the end of the 1 -h adsorption period, add 6 mL overlay (see Subheading 2.1., item
9) to the edge of the dish and rotate the dish to blend overlay wtth the medium used
for infection (the 0.5-n& volume of medium used for infection IS not removed).
6
Tollefson, Hermiston, and Weld
15. After mixing wtth CsCI, divide sample into two Ti50 quick-seal tubes.
16. Centrrfuge in Ti50 rotor at 35,000 (110,OOOg) rpm m Beckman ultracentrifuge at
4°C for 16-20 h to band the virus
Day 5:
CsCI-Banded Adenovirus Stock
7
8. Leave dishes at room temperature on a level surface for 5-15 mm in order to
allow the overlay to solidify.
9 Transfer dishes to 37°C 6% CO, and mcubate for 4-5 d. At that time add a
second overlay (5 r&/60-mm dish) containing neutral red (see Subheading 2.1.,
item 9). It is important to have a humidified atmosphere in the incubator, but
avoid very high humidity because it may cause excess moisture on and around
the overlay, resulting in plaques that diffuse excessively and inconsistently.
10. Begin counting plaques 1 d after the neutral-red overlay is added. On A549 cells
it is not necessary to add more than the first and second overlays
11. Count plaques at 2- to 3-d intervals until new plaques are no longer becommg
apparent. For Ad2 and Ad5 wild-type viruses, this may be 12-l 5 d postinfectton,
with other serotypes ($6) and with group C adenoviruses that have mutations or
deletions in the adenovuus death protein, this may be approx 30 d postinfection
(7,s). Plaques are most apparent when holding the dish up toward a light source
and observing an unstained circular area that has altered light diffractron. Cells
initially may not be rounded up and may simply appear unstained, but plaques

will typically become more apparent with time.
12 Use dishes with 20-100 plaques for the calculation of titer (plaque assays are
done m triplicate for each of the serial dilutions for more accurate numbers).
4. Notes
1. Incubate test bottles of medium at 37°C for 5 d to ensure sterility for each batch.
Sterility can also be tested on blood agar plates or in nutrient broth. Care should
be taken to cover the medium during preparation and to prepare medium in an
area not normally used for handling of virus (adenovirus vtrions can pass
through a 0.22-p filter). Bottles, 20-L container, and stir bar used for media
preparation should be dedicated for tissue-culture use and not mixed with chemi-
cal glassware.
2. 2X DME is usually ahquoted in 500~mL volumes and is stored at 4’C; care should
be taken to avoid increases in pH (cell survival of monolayers m plaque assays is
significantly decreased m medium that has become “basic” or if the pH of the
overlay is too high initially).
3. Serum testing: sera (HyClone, BioWhittaker, Gibco-BRL) are purchased in large
lots to reduce experimental variation caused by differences m serum lots. Sera
are tested with relevant cell lines in two different assays.
a. To determine cloning efficiency of cells at low-cell density, plate 100-500
cells per tissue culture plate. After 10-12 d, fix clones in methanol (10 min at
-20°C) and stain wtth Giemsa staining solution or with crystal violet. The
number, stze, and morphology of clones can then be compared for dtfferent
lots. Determination of cloning efficiency is of importance for experiments in
which small numbers of cells will be present on a tissue-culture dish (as m
production of stable transfectants or in limited dilution for selectton of clonal-
cell populations). One can also check the appearance of clones and make sub-
jective judgments about the growth of the cells (flatness or overgrowth of the
8
Tollefson,
Hermisron,

and Wold
clones, size of the cells, cell uniforrmty, vacuoles, mitotic index, and relative
“health” of the cells).
b Cell-growth rates are calculated by plating lo5 cells per 60-mm tissue-culture
dish; cell counts are done at daily intervals to determine the kinetics of growth
for the different serum lots.
4. The FBS is not heat-inactivated (heat-inactivation will substantially decrease the
survival of A549 cells in plaque assays).
5. Verification of virus stocks. Virus preparations are tested periodically by Hirt
assay (see Chapter 2) or other assays (such as immunofluorescence or PCR) to
confirm the “fidelity” of the vnus preparations. It may be necessary to plaque-
purify a stock periodically (see Chapter 2) to eliminate possible contaminants
(this is especially important for viruses that grow less efficiently than wild-type
virus or that are released from cells less efficiently during infection).
6. Plaque-assay consistency: Careful and consistent plaque assays will typically
result in less than twofold differences in determined titer of the same virus prepa-
ration in separate experiments. It is often preferable to plaque assay a large panel
of mutants that will be used in the same experiments simultaneously so that the
relative titers will be quite accurate.
7. Adjusting plaque assays for small-plaque morphologies (viruses that lack the
subgroup C adenovnus death protein gene and serotypes that produce small
plaques): It is important to have consistent PFU informatton in order to perform
infections with viruses m which comparisons are made between the phenotypes
of various virus mutants. The most direct method of generating infectrous titers 1s
by doing plaque assays for PFU. In the study of E3 mutants, we have determined
that given mutants have a small plaque morphology and therefore a number of
modifications have been made in previous plaque-assay methodologies to
accommodate the requirements for these mutants. It is necessary for the cell
monolayers to remain viable for an extended time (approx 28-30 d) to see the
full extent of the plaque development. Plaque assays are done on A549 cells

(ATCC) that have a very good survival time under the overlay in plaque assays.
Cell survrval of A549 cells is also somewhat dependent on the type of tissue-
culture dish used
References
1. Pinteric, L. and Taylor, J. (1962) The lowered drop method for the preparation of
specimens of parttally purified virus lysates for quantitative electron mtcrographtc
analysis. YzroIogy l&359-371.
2. Mittereder, N., March, K. L., and Trapnell, B. C (1996) Evaluatton of the con-
centration and bioactivity of adenovirus vectors for gene therapy. J Vlrol. 70,
7498-7509.
3. Green, M. and Pina, M. (1963) Biochemical studies on adenovnus multiplication. IV.
Isolation, purification, and chemical analysis of adenovuus. Virology 20, 199-207.
4. Green, M. and Wold, W. S. M. (1979) Human adenoviruses: growth, purificatton,
and transfection assay. Methods Enzymol. 58,425-435.
CsCI-Banded Adenovirus Stock
9
5. Hashimoto, S., Sakakibara, N., Kumai, H., Nakai, M., Sakuma, S., Chiba, S., and
Fujinaga, K. (1991) Fastidious human adenovirus type 40 can propagate effi-
ciently and produce plaques on a human cell line, A549, derived from lung carci-
noma. J Vwol. 65,2429-2435.
6. Green, M., Pma, M., and Kimes, R. C. (1967) Biochemical studies on adenovirus
multiplication. XII. Plaquing efficiencies of purified human adenoviruses. Dis-
cussion and preliminary reports. Vlrohgy 31,562-565.
7. Tollefson, A. E., Scaria, A., Hermiston, T. W., Ryerse, J. S., Wold, L. J., and
Wold, W. S. M. (1996) The adenovirus death protein (E3-11.6K) is required at
very late stages of infection for efficient cell lysis and release of adenovirus from
infected cells. J. Viral 70,2296-2308.
8 Tollefson, A. E., Ryerse, J. S., Scaria, A., Hermiston, T. W., and Wold, W. S. M.
(1996) The E3-11.6kDa adenovirus death protein (ADP) is required for effi-
cient cell death: characterization of cells Infected with adp mutants. Virology

220, 152-162.
Construction of Mutations in the Adenovirus
Early Region 3 (E3) Transcription Units
Terry W. Hermiston, Ann E. Tollefson, and William S. M. Wold
1. Introduction
The E3 transcription umt of the well-studied subgroup C adenovtruses (Ad)
(prototypic serotypes 2 [Ad21 and 5 [Ad5]) is located between map units 76-
86 (see
Fig. 1).
The E3 region 1s surrounded by the genes for virion protein
VIII and fiber, and is transcribed off the r-strand. The E3-region transcription
unit is complex Extensive sphcmg controls expression of seven identified E3
proteins, four of which have functions that modify the host immune response
to the viral mfection (reviewed m
ref. I, Table 1)
Human adenovirus has served as a model system for studying persistent
infections and has enjoyed widespread interest as a gene therapy vector. In
both cases, understanding the immune response to the virus is paramount for
further advancements m these fields. This will require mutational analysis of
the immune-response modifying E3 region and its proteins, their reincorpora-
tion mto the viral genome, and study of the viral infection in vwo A series of
protocols will be presented that have been used for the creation of adenoviruses
with mutations in the E3 region A method detailing the tsolation of adenovirus
DNA containing the terminal protein (TP-DNA) will be described (8-10). This
will serve as the startmg material into which the mutated E3 region will be
inserted, either by overlap recombmation (11) or by ligation insertion (12). In
either method, transfection of the DNA into the cell is required for infectious
virus to be generated, and a protocol will be given. Lastly, a procedure will be
provided that can be used to screen for the desired E3-mutated adenovnus.

Mutagenesis techniques will not be discussed here but excellent reviews are
available (13,14).
From Methods m Molecular Medune, Vol 21 Adenovws Methods and Protocols
Edited by W S M Wold 0 Humana Press Inc , Totowa, NJ
11
12 Hermiston, Tollefson, and Wold
A
Ela,
Elb
E3 )
4 E2a
IE4
B
EC0 RI
76 l?coRl 83
ECORI 100
t
A c I a
20 40 60 ’ so ’
I I I I
Ad5 TP-DNA
r%
Mutoled Ad2
83
w
Mulated Ad2
Em RI m.w,c,ion en? me
digeuion 0r ~d5 w- L NA
Fro RI cleaved Ad5
TP- DNA lnlo human A549 &lr

Ad5
EcoRl A
’ 76 Ad2 83 Ad 5
oR1 D I IlkoR B
Ad5. Ad 2- Ad5
v
E3 mutated adenovnn plaques
Fig 1 Methods used to insert a mutated E3 region mto the viral genome (A) Sche-
matic tllustratmg some of the adenovirus transcription units expressed during early
stages of infection (B) Construction of E3 mutations Ligation method. Cleave cloned
Ad2 EcoRI-D fragment contammg a mutation with EcoRI, then ligate to EcoRI-
cleaved Ad5 TP-DNA complex. Transfect legation mixture mto A549 cells and allow
plaques to form. Overlap recombmatton method. Cotransfect cloned Ad2 KpnI-A frag-
ment containing a mutation m the E3 region with EcoRI-cleaved Ad5 TP-DNA com-
plex into A549 cells and plaques were allowed to form
2. Materials
1 CsCl-purified adenovtrus from 6 L of KB cells (preparation described m
Chapter 1)
2. 8 M GuHCI: aqueous solution (Stgma, St. LOUIS, MO, cat. no. G9284). 8 M
GuHCl (guamdme HCl) can be made up from a powder (Sigma cat no G4505)
m ddH20/2 WPefabloc (Boehrmger Mannhetm, Indtanapolls, IN) Microwave
400 mL ddH,O to near botlmg, add 764 2 g GuHCl, and stir Adjust volume to 1 L
by the addition of Pefabloc to 2 mM and ddH20. Use this stock for lys~s of aden-
ovuus virions and to generate 4 M GuHCl.
3. Pefabloc: Boehringer Mannheim cat no 1 429 876
Table 1
Location of the E3 Genes Within the Ad5 and Ad2 Genomic Sequence
Coding sequence
E3 protein
Ad5 (2)

ACQ (3)
start stop start stop
Function
12.5 K 27,858 28,179 27,919 28,220 Unknown
67K 28,547 28,736 28,630 28,8 19 Unknown
gp19 K 28,735 29,215 28,812 29,289 Inhibits Ad-specific cytotoxic T-lymphocyte recognition
and killing of the virally infected cell by binding
MHC class I molecules and inhibtting their transport
z
to the cell surface
11.6 K (ADP) 29,49 1 29,770 29,468 29,77 1 Required for efficient cell lysis and release of adenovuus
from Infected cells ($5)
10.4 K (RID CL)~ 29,784
30,057 29,78 1 30,054 10.4 K and 14.5 K proteins form a complex that.
Prevents TNF cytolysts
Clears epidermal growth factor and msulm receptors
from the infected cell surface
Clears Fas antigen from the mfected cell surface (6)
14.5 K (PYIDP)~ 30,062 30,458 30,059 30,449
14.7 K 30,453 30,837 30,444 30,828 Inhibtts TNF-mduced apoptosis
GTPase bmdmg protein (7)
‘ADP, adenovirus death protein
*RID
3
receptor mtemallzatlon and degradation complex, made up of 10.4-K (RIDa) and 14 5-K (RIDB) protems
14 Hermlston, Tollefson, and Wold
10
11
12
13.

14.
15
16.
17.
18.
4 L of TEMP pH 8.0: 10 mM Trts-HCl, pH 8.0 (20’(Z), 1 mA4 EDTA (EDTA at
pH 8.0-8.5), 2 mMP-mercaptoethanol (P-ME), 2.0 mMPefabloc, filter sterthze
2 L of 4 A4 GuHCl m TEMP pH 8 0 4 A4 GuHCl made up n-t 10 mM Trts-HCl,
pH 8 0, 1 mA4 EDTA (pH 8 O-8 5) 2 mM P-ME, 0 2 mMPefabloc, filter sterthze
200 mL Sepharose 4B-200 m 4 M GuHCl in TEMP pH 8 0. 250 mL of
Sepharose 4B-200 (Stgma cat no 4B-200) mixed wtth 750 mL of 4 MGuHCl
m TEMP pH 8 0
Column chromatography assembly Ktmble/Kontes (Vmeland, NJ) flex-column
(cat. no 420401-2550), l-way stopcock (cat. no. 420163-0001) female luer (cat.
no 420407-0000), ferrule for l/16-in OD tubmg (cat. no 420822-0116), 1/4-m
tubing nut for 1/16-m OD tubmg (cat. no 420821-0116), 5-ft PTFE tubing w/
integral luer lock (cat. no. 420823-0016), and column reservotr 1000 mL (cat no
420406- 1025). Alternatively, Ace Glass (Vmeland, NJ). Ace chromatography
column (cat. no 5820-39), Ace cylmdrtcal funnel (cat no 5822-15), couplmg
(cat no 5841-50), bottom-drip adapter (cat no. 5838-53), male nut connector
(cat. no 5854-09), ferrule connector (cat. no 5854-26), TFE Teflon tubing (cat
no 12684-28) and filter disk (cat no 5848-25)
Fraction collector.
16&200 silicomzed stertle glass test tubes (13 x 100 mm) 13 x 100~mm tubes
(VWR cat. no 60825-923) can be stltcomzed with Stgmacote (Stgma cat no
SL-2), which 1s resistant to autoclavmg
Markers for mcluston and excluston volumes wtthm the column. 0 1% (wt/vol)
phenol red and dextran blue (Sigma cat nos P4758 and D575 1) m 4 M GuHCl-
TEMP pH 8.0, respecttvely
8 L 5 mA4 Trts-HCI, pH 7.5, 1 mM EDTA, autoclaved or filter sterthzed

Dialysis tubing and closures. Spectrum (Laguna Hills, CA) Spectra/Par stertle
CE membranes, MWCO 15,000 (cat. no 130562), and closures, (cat no. 132735)
Tissue-culture reagents for propagation of A549 cells and for plaque assays IS
discussed m Chapter 1
Restriction enzymes, New England Biolabs (Beverly, MA): EcoRI (cat no
10 15), HzndIII (cat. no 1045), and calf intestinal alkaline phosphatase (CIP) (cat
no. 2905)
2X HEPES-buffered saline (HeBS) solution. 42 mM HEPES (N-2-hydroxy-
ethylptperazine-Ar-2-ethanesulfomc acid), 270 mMNaC1, 10 mM KCl, 1 0 mA4
Na2HP04, 0.2% dextrose pH to 7 05 with 5 NNaOH and filter sterdtze through
a 0 45-pm mtrocellulose filter (see Note 1) After functtonal testmg (descrtbed
m Note l), altquot the solutton m lo-mL ahquots and store frozen at -70°C
2 5 MCaCl* dihydrate (Sigma. cat no. C7902) Filter sterthze through a 0 45-pm
mtrocellulose filter (Nalgene) and store at -20°C m lo-mL ahquots (can be fro-
zen and thawed repeatedly)
Somcated salmon-sperm DNA (Stratagene, La Jolla, CA, cat no 20 1190). 10 mg/mL
stored frozen at -20°C
25% Glycerol shock solution: (for 10 mL) 7 5 mL of Dulbecco’s modtfied Eagle’s
medmm (DMEM)/lO% fetal bovine serum (FBS), 2 5 mL glycerol (hgma cat no
2025)
Adenowrus E3 Mutations
15
19 1 8% Noble agar. 1.8 g ofNoble agar (DIFCO, Detroit, MI, cat no. 0142) m 100 mL
ddH,O Sterilize by autoclavmg
20 Hirt lys~s buffer. 0 6% SDS, 0 01 M EDTA, 0.01 M Tris-HCl, pH 7.4
21. Proteinase K: (Boehringer Mannhelm cat no. 1 373 196) 15 mg/mL stock
22 5 MNaCl.
23 Loading dye. 6X buffer 0 25% bromophenol blue, 0.25% xylene cyan01 FF, 30%
(w/v) glycerol in water, store at 4O C.
24. 0 8% agarose. Electrophoresis-grade agarose (Glbco-BRL, Galthersburg, MD,

ultrapure agarose, cat no 15510-027). Prior to pouring the gel, add ethldium
bromide (Sigma cat. no E875 1) to a concentration of 0.5 clg/mL for DNA visual-
lzatlon under UV light
25 50X TAE buffer (per liter) 242 g Tris base, 57.1 mL glacial acetlc acid, 100 mL
0.5 M EDTA (pH 8.0), brought up to 1 L by the addition of ddH,O (workmg
concentration of 40 mM Tris-acetate, 2 ti EDTA)
26. Plasmld-containing mutated E3 gene(s) of choice
3. Methods
3.1. Adenovirus TP-DNA Preparation
Adenovlruses contam two origins of replication located in the inverted ter-
mmal repeats. The E2-coded terminal protein, along with the adenovirus DNA
polymerase, bind wlthin the origin of replication where the terminal protein 1s
cleaved to a polypeptlde of 55 kDa. This smaller polypepttde is then covalently
attached to the vu-al genome, where it serves to initiate viral DNA replrcatlon.
Purified adenovlrus DNA that has retained the terminal protem produces vu-al
plaques at a 40-100 times higher frequency than viral DNA lacking the
termmal protein followmg transfection (8,9). Because of its enhanced
Infectivity, TP-DNA has been used as a starting point toward constructmg
recombinant adenoviruses mutated in the E3 region. The following IS a proto-
col for the purification of TP-DNA from the point where the mvestlgator has
CsCl-purified adenovirus. Preparation of CsCl-purified adenovlrus is described
m Chapter 1.
3.7.1. Column Preparation
1 Fill the column with 200 mL of 4 M GuHCl-TEMP.
2. Mix 250 mL of Sepharose 4B with 750 mL 4 MGuHCl-TEMP, degas for 10 mm.
3 Add the Sepharose 4B/GuHCl-TEMP solution to the column A glass rod may be
used to stir the Sepharose 4B solution m the column to remove any air bubbles
4 Move the column to 4°C and connect the column to a fraction collector Estabhsh
a flow rate of 18 mL/h Elute excess column buffer, being careful to retam enough
column buffer so that the packing material 1s not exposed to air. Allow the

Sepharose 4B to settle (usually overnight) at 4°C. The final column bed volume
will
be approx 35 cm.
76 Hermiston, Tollefson, and Wold
5 Add 200 mL of 4 M GuHCl-TEMP to wash the column agam retammg enough
column buffer so that the packing material is not exposed to au
3.1.2. Virion Lysis and Collection of Ad TP-DNA
1. Collect CsCl-banded Ad5 virus prepared from 6-7 L virally infected KB spmner
cells (as described Chapter 1).
2 Place the CsCl-banded adenovirns mto dialysis tubing and dialyze against two
changes of TEMP at 4°C (allow 3 h/change of buffer) Normally, a white precipl-
tate will appear (the adenovlrus vmons)
3 Remove the dialyzed adenovu-us from the tubing Measure the vlrns volume and
add an equal volume of 8 M GuHCl-TEMP at 4”C, mix gently, and incubate at
least 10 min on ice The white precipitate of adenovirus vlrions ~111 clear upon
the addition of the 8 M GuHCl-TEMP
4 Using a pipet, gently layer the lysed adenovlrus vlrlon solution onto the pre-
pared Sepharose 4B column described above, bemg careful not to disturb the
column bed.
5. Begin runnmg the column, collectmg 2-mL fractions. Immediately add 1 mL 4 M
GuHCl contammg 0 1% dextran blue and 0.1% phenol red as markers for exclu-
sion and inclusion volumes.
6 Add 5 mL 4 MGuHCl-TEMP After these solutions have run into the column and
the volume above the column nears the column bed, add 300 mL of 4 MGuHCl-
TEMP to the column reservoir and continue running the column The full run
will take approx 6-8 h
7. Pool the first 20 mL from the column and save
8. Test the additional fractions (usually every other one) for the appearance of the
viral TP-DNA. This can be done by takmg absorbance measurements at 260 nm
and 280 nm, usmg the column buffer, 4 M GuHCl-TEMP, as a blank After plot-

ting these data (a typical graphed experimental run appears in Fig. 2), pool the
peak fractions (to maintain a maximum concentration) and subsequent shoulders
These fractions are then dialyzed at 4°C against two 2-L volumes of 5 nuW Tns-
HCl, pH 7.5, 1 mMEDTA.
9 Read the absorbance at 260 nm and 280 nm to get a final concentration of the TP-
DNA pools (the 260 280 ratio should be 1.8 to 2 0). The DNA-protein complex
(normally 30-50 pg/mL for the pooled peak fractions) can be stored at 4°C for up
to 10 yr However, for maximal mfectivlty, DNA-protein complex should be
used within 1 yr Do not ethanol-precipitate TP-DNA.
3.2. Transfecfion of A549 Cells
Thts se&on presents a standard protocol used for transfecting A549 cells to
generate recombmants m the E3 region. Mutations m the E3 12.5 K, 6.7 K,
gp19 K, ADP
(11.6 K), and the majority of the 10.4 K-coding sequence can be
transferred to the viral genome by hgation of the mutated Ad2 EcoRI-D frag-
ment to EcoRI-digested Ad5 TP-DNA (12). In cases in which mutations are
needed in the 14.5 K and 14.7 K protems, homologous recombmation has been
Adenovirus E3 Mutations
17
0.8
0.7
0.6
E
x x
X
X
0
Xo .o x 0 0
0 0
Y “XX 0

X0
l x
l A A
X
X’
X0
xxxxxxxx
by;: T X0
xxx
0 15
30 45
loo
x = 260
l
= 280
Fraction
tubes
(2mVsample)
* Only an npproxlmatton, elution times and peok levels WIII
vary
dependent on column packmg and startmg viral matcrud
Fig
2. Schematic representation of a typical graph of 260 and 280 absorbance readmgs
from an adenovirus
TP-DNA
isolation using Sepharose 4B column chromatography.
employed, using the KpnI-A fragment of Ad2 for recombmatron with EcoRI
cleaved Ad5
TP-DNA (as described in
ref. II).

3.2.1. Preparation of Cells
Propagation and handling of A549 cells (a human lung-carcmoma cell line
from ATCC, Rockvtlle, MD, cat. no. CCL 185) is detailed elsewhere (Chapter
I). These cells should be plated at approx 5 x IO5 cells per 60 mm dish (2
dishes per viral construction) the day before the transfection. This will put the
cells at 60-80% confluency at the time of transfection. Correspondmg adjust-
ments to accommodate 35- or loo-mm dishes can be calculated accordmgly.
3.2.2. Preparation of DNA
3.2.2.1.
LIGATION PROTOCOL (SEE NOTE
2)
1. Calculate the amount of plasmid containing the E3 region mutated gene(s) needed
to generate 10 pg of insert DNA by EcoRI cleavage. Followmg cleavage of the
plasmid, add calf-intestme phosphatase to the reaction, treating for 30 mm at
37°C This treatment Inhibits the formation of multtmers m the ensumg step of
ligating the mutated Ad2
EcoRI-D fragment into the EcoRI-cleaved TP-DNA
Gel purify the mutated viral DNA fragment and ethanol precipitate the Insert
DNA to concentrate the fragment.
18 Hermlston, Tollefson, and Weld
2 Cleave 2.5 ug of Ad5 TP-DNA wrth 20 U of EcoRI per viral construct The
EcoRI cleavage of the Ad5 TP-DNA will create three fragments, 2733 1, 5886,
and 2718 bp m size. Check for adequate cleavage of the viral TP-DNA by run-
ning 0 5 pg of the DNA on a 0.8% agarose gel The larger two fragments are
from the termmt of the vnus Because of then association with TP, they ~111 not
migrate mto the gel and will appear retained m the well of the gel followmg
exposure to UV light Smce the EcoRI fragment of 2718 bp IS an Internal frag-
ment, it will migrate easily into the gel The EcoRI-digested Ad5 TP-DNA will
be used directly m the ligation reaction with the mutant viral DNA (Overnight
restriction-enzyme dtgestton usually results m Me detectable restriction endo-

nuclease activtty the followmg day ) Do not ethanol-precipitate the restrtctron
endonuclease-cleaved TP-DNA
3 Combme 2 pg of the EcoRI-cleaved Ad5 TP-DNA with 5 pg of the mutated E3
insert DNA (Ad2 EcoRI-D fragment) and hgate overmght at 16°C This puts the
insert at a 33-fold molar excess over the equivalent wild-type sequence that ~111
still be present in the EcoRI-cleaved TP-DNA. The large molar excess, however,
favors the msertion and wild-type vnus IS rarely regenerated (~10%)
3 2 2 2. OVERLAP RECOMBINATION PROTOCOL
In this method, the mutated viral DNA segment 1s not gel purified. Instead,
a plasmtd with the KpnI-A fragment of Ad2 (bp 25,881 to 33,594) containing
the mutant E3 gene(s) 1s cotransfected with the EcoRI-digested Ad5 TP-DNA
The EcoRI digestion ensures that the wild-type vu-us 1s not regenerated and
retains enough overlappmg sequence for homologous recombmatton to occur
between the EcoRI-cleaved Ad5 TP-DNA and the KpnI-A adenovtrus
sequences in the plasmtd. This protocol IS similar to the hgatton technique
described previously but elrmmates the need to isolate the mutated viral
DNA sequence from a plasmtd and the ligatton step, making tt more time
effective. Simply calculate the amount of the E3-mutated plasmid required
to generate 10 pg of the mutated viral DNA segment and cotransfect tt with
the restriction-enzyme-drgested viral genomtc TP-DNA followmg the proto-
col described below.
3.2.3 Transfection Protocol
1 Plate A549 cells at 5 x lo5 per 60-mm dish the day before the transfectton Be
sure to include control plates for transfecting uncut TP-DNA ( 100 ng) to ensure
that your TP-DNA is good (this should give a lawn of plaques on a 60-mm dish at
approx d 5-7 posttransfection) and cleaved TP-DNA (2 pg) (this ~111 mdtcate the
cleavage efficiency of the TP-DNA by EcoRI and serve to determine the plaque
background level for the experiment)
2 Change the medmm on the A549 cells 3 h prtor to the transfectton solutton bemg
Introduced onto the monolayer

Adenovirus E3 Mutatrons 79
3 Combme the ligand DNA with 20 clg of salmon sperm DNA and add sterile
ddH,O to a volume of 450 pL
4. Add 50 & of 2.5 M CaCI,.
5 Place 500 pL of 2X HeBS mto a sterile IS-mL comcal tube. While mtxmg the 2X
HeBS (either mechanically by bubbling air through the solution usmg a mecham-
cal pipettor or by hand shakmg the solution) add the DNAKaCl, solution dropwise
(using a Pasteur pipet or Ptpetteman). Immediately vortex the solutton for 5 s
6 Allow the tube to stand at room temperature undisturbed for 20 mm while a pre-
cipitate forms
7 Aspirate the medium off the cell monolayer and add 0.5 mL transfection mixture
onto cell monolayer in each 60-mm dish Incubate for 20 mm m a 37°C 5% CO2
mcubator
8 Add 4 mL complete medra (DME + 10% FCS) and Incubate for an additional 4 h
m a 37°C COz incubator
3.2.4. Glycerol Shock
1. Remove the medmm on the plate and add 1 mL glycerol shock solution (warmed
to 37Y) to the cells for
1 mln
2. Aspnate off the shock medium and wash 2X wtth 5 mL of DME/lO% FBS
Alternattvely, the mittal wash of DME/lO% FBS can be added dtrectly to reduce
the exposure of the cells to the solutton if a number of transfecttons are being
done at one time
3.3. Agar Overlay
1 After all transfected cells have been shocked (with either protocol), an agar over-
lay 1s apphed
2. Make up 50 mL agar overlay solutton m the following fashton. 25 mL 2X DME,
I mL of FBS, 2.5 mL 7 5% NaHCO,. Warm the solution to 37°C. Immediately
prtor to overlaying the cell monolayer, add 2 1 5 mL sterile 1 8% Noble agar that
has been melted and cooled to 56°C.

3 Add 5 mL of the agar overlay medium per 60-mm dish Allow the solution to
sohdify at room temperature (approx 3 min) and then return the plates to a 37°C
CO, incubator.
4 At d 3 posttransfectlon, add a second agar overlay. Add a third overlay on d 7.
The d-7 agar overlay should contam neutral red (Gtbco-BRL cat no. 15330-012)
at a concentration of 0 45 mL/50 mL agar overlay to enhance the vtsuahzatton of
the viral plaque on the cell monolayer The cell monolayer contams hve cells that
will stain red and viral plaques, made up prmcipally of lysed and dymg cells, that
will not take up the stam and consequently will appear clear or white (discussed
m Chapter 1) Plaques will appear on transfected control plates with uncut TP-
DNA as early as d 3 (see Note 3). Transfectrons contammg DNA from the liga-
non or overlap recombmation methods will begin showing plaques startmg at d
7-14, with clearly visible plaques by d 14 (see Subheading 3.).
20 Hermiston, Tollefson, and Wold
3.4. hitid isolation and Storage of Viral Plaques
Pick well-isolated plaques from transfected cultures by punching out agar
plugs with a sterile Pasteur pipet. Store agar plugs in 0.5 mL sterile PBS with
calcium and magnesium and 10% glycerol at -70°C. Ideally, however, plaques
are propagated on A549 cells immediately after they are picked and viral
supernatant containing the candidate virus IS stored
(see Subheading 3.5.).
3.5. Initial Propagation of Viral Plaques
1. Plate A549 cells at 5 x lo5 cells per 35-mm dish the day before picking plaques
This will place the cells at 80% confluency the day of the infection
2. Remove medium from cells and add 0.2 mL vuus (agar-plug suspenston solutton
described in the previous step). Adsorb at room temperature for 30 mm and then
add 2 5 mL complete medta and incubate at 37°C. Cells should not need to be
refed during the imtial propagatton.
3 Viruses are ready to harvest when all cells are rounded (due to the viral cyto-
pathic effect or CPE) and most have detached from the dish (usually 4-7 d)

4. To permit collectton of medmm (as stock) while retammg the majority of the infected
cells (for analysis), leave dishes undisturbed in the tissue-culture hood for 30 mm
5 Gently remove 2 mL medium and add tt to a sterile veal containmg 0 25 mL
sterile glycerol Gently mix and store these candidate viruses at -70°C
6 Slowly aspirate any remammg medium from the plate If this IS done carefully,
the majority of cells ~111 remam m the dish and can be used m the next section for
analysts of adenovirus plaques.
3.6. Analysis of Adenovirus Plaques
The vast majority of E3 viral mutants has been constructed by mutating the
Ad2 E3 region and then placing this mutated E3 region mto an Ad5 back-
ground. This type of methodology takes advantage of the Ad2 E3 region’s
additional Hind111 restriction enzyme sites for screening recombinant plaques
(see
Fig.
3). The followmg is a protocol for isolating viral DNA and usmg a
restriction-enzyme polymorphism to screen for the mutated adenovn-us E3
recombinant (see
Note 4).
An alternative method for isolation of viral DNA
has recently been described (15).
1 Prepare confluent monolayers of cells by plating out A549 cells at 5 x 1 O5 cells/
35-mm plate m 2 mL DME/lO% FBS.
2. The next mornmg, remove the medium and add 200 pL serum-free DME and
1 O&200 pL viral supernatant from the mittal plaque propagation or 1 to 2 pL of
high titer (~10’~) CsCl purified Ad5 vuus (approx 50 PFU/cell) Incubate 1 h at
37°C m a CO2 Incubator. Followmg the mcubatton, add 2 mL DME contammg
2% FBS and incubate overnight
3 Check the cell monolayer the next day. If the monolayer 1s intact, remove super-
natant and proceed with step 4 If the cells have detached, collect cells and super-
Adenovirus E3 Mutations

21
A
map
units 0 10
20 30 40 50 60 70 80 90 100
76 E3 86
57 23.7 39.8 42.8 47.5 61.9 72.0 93 5
Kpn 1 y /
B I C IIIHI D IEI A
IF Ad2
A IGIEIKIJI B IDIFICIH
Ad5
31.4 80.2
7.8 173 32.2
Hand 111 0” ’ D
+‘ 51.0 73.4 79.8 80.7 89.7 97 3
I B 1 IlMiJI D 1
I AAI
1 HiLiE IG IK
1 E I c I HIJI D B IFI1
73.3 89.1
59.4 71.4 762 83.6 89.8
Eco RI
A B
IFIDIEIC
A 1 Cl9
B
Ad5
-A
-B

-c
-D
- E
-F
-CI
-H
- I
- J
Ad5-Ad2-Ad5
El mutant
-A
-c
-D
-E
- B*
-F
-G
=i
-1
- B*
-J
Ad 2
Ad5
Ad 2
Ad 5
Fig. 3. Analysis of adenovnus E3 mutant plaques (A) Schematic illustrations of
the Ad2 and Ad5 genomic restriction endonuclease cleavage patterns for restriction
endonucleases HlndIII, KpnI, and EcoRI. (B) Schematic illustrations of the agarose
gel analysis of HwrdIII-cleaved genomic DNA from Ad5 and an AdS-Ad2-Ad5 E3-
mutated adenovnus genome.

22 Hermiston, Tollefson, and Weld
4
5
6.
7
8.
9
10
11
12
13.
natant m an Eppendorf tube and pellet for 1 mm m a mtcrofuge, dtscard the
supernatant and proceed to step 4
Pnor to use, premtx 800 pL Hnt lys~s buffer and 35 pL of protemase K per plate Add
835 pL of the solution to each monolayer or pellet and incubate at 37°C for 1 h
Draw off the viscous cellular lysate (approx 800 pL) and transfer it to a sterile
Eppendorf tube.
Add 200 p.L of 5 MNaCl, invert three times, and Incubate at 4°C for at least 4 h
Allowmg the incubation to proceed overnight will result m a tighter pellet when
the cellular debris is centrifuged, allowing for easier manipulation and a higher
yield of viral DNA.
Centrifuge for 15 min at 4°C m a microfuge and transfer the supernatant to a
fresh Eppendorf tube.
Extract with 700 pL of phenol*chloroform~isoamyl alcohol at a ratio of 25 24 1
Spin for 5 mm m the microfuge and take the aqueous (upper) phase, sphttmg it
into two tubes and precipitating the viral DNA by adding 2 5 vol of 100% ethanol
Spm down the viral DNA for 15 mm m a microfuge, wash with 500 pL of 70%
ethanol, an dry, and resuspend m 60 pL of ddH,O
Take 26 p.L viral DNA, add 3 p.L HzndIII restriction enzyme buffer, and 1 pL
of HzndIII.

Incubate for 2-5 h at 37’C Add loadmg dye
Run the samples overmght on a 0 8% agarose TAE-buffer gel contammg ethrdmm
bromide to enhance the separation of the viral restriction-enzyme fragments
Identification of the E3-mutant adenovn-us can be done by selecting the virus
with the altered stammg pattern of the DNA fragments (The normal and mutant
staining patterns are depicted in Fig. 3 for an HzndIII restriction-enzyme diges-
tion.) In situations m which DNA levels are low, the agarose gel can be prepared
for Southern blot analysis (23) and probed with Ad5 DNA
4. Notes
1. Transfection reagents: An exact pH for the 2X HeBS solution is extremely
important for efficient transfection There can be wide variability m the effi-
ciency of transfectton obtained between batches of 2X HeBS The efficiency
should be checked with each new batch. The 2X HeBS solution can be rapidly
tested by mixing 0.5 mL 2X HeBS with 0.5 mL 250 ti CaCI, and vortexing
Place a drop onto a glass shde with a cover slip. A fine precipitate should develop
that is readily visible on the microscope. Transfection efficiency must still be
confirmed, but if the solution does not form a precipitate in this test, there is
something wrong Alternatively, kits of these reagents are available commer-
cially from Promega (Madison, WI, cat no. E1200), Sigma (cat. no. CA-PHOS),
and Clontech (Palo Alto, CA; cat. no. K2050-1). The Clontech kit also includes a
reagent, CalPhos Maximizer, which enhances the transfectton efficiency by 3 5-
to 12.7-fold, dependent on the cell type bemg used. Alternate cell lines may be
used, however, transfectton procedures need to be refined to the cell type used
and an optimization protocol has been described (13)
Adenovirus E3 Mutattons 23
2 Plasmtd DNA preparatton. The quality of the plasmtd DNA used m transfectton
expertments IS crucral for success. Plasmid DNA should be prepared by CsCl
banding (13) or by commerctally avatlable kits designed to reduce the quantity of
lipopolysacchartde (LPS) present in the completed plasmtd preparation (Qtagen,
Chatsworth, CA, cat no 12362)

3. Plaque assay. The plaque appearance may be greatly delayed (2 1 d or longer) in
cases m which the adenovnus E3 ADP (11.6 K) protein expression is altered by
deletion or mutation These plaques will typically be small in size.
4 Screenmg adenovirus plaques* An alternattve method to determme the E3-mutant
adenovnus utrhzes m vtvo labelmg of the vtral DNA wtth 32P. Much of this pro-
cedure parallels that previously discussed with the followmg exceptrons Seven
to nme hours postmfectton, wash the cell monolayer with phosphate-free DMEM
(Gtbco-BRL, cat no 1197 I-025); then add 1 mL of phosphate-free DMEM con-
taming 2% FBS and 32P-orthophosphate (NEN-DuPont, Wilmington, DE; cat.
no. NEX-053) at 50 @I/ 35-mm plate. Incubate overnight in a 37°C CO, mcuba-
tor (32P orthophosphate IS a p emitter. The mvesttgator’s radtatton safety officer
should be nottfied for proper handling and dtsposal of all matertals ) Harvestmg,
processmg, and restrtctton-enzyme digestion of the vtral DNA can contmue as
prevtously descrtbed. After the restrictton-enzyme fragments are separated by
gel electrophorests, the gel can be drted at 80°C for 2 h under a vacuum (be sure
to have a cold trap installed to capture 32P-radtoacttve waste) and then exposed to
autoradiography film for 5 mm or more at room temperature
References
1. Wold, W. S. M , Tollefson, A E , and Hermiston, T W (1995) E3 transcriptton
unit of adenovu-us, in The Molecular Repertowe of Adenovwuses, Current TOPICS
zn Mzcroblology and Immunology, vol. 199 (Doerfler, W. and Bohm, P , eds ),
Springer, pp 237-274
2. Chroboczek, J., Bteber, F , and Jacrot, B. (1992) The sequence of the genome of
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3
Isolation, Growth, and Purification
of Defective Adenovirus Deletion Mutants
Gary Ketner and Julie Boyer
1. Introduction
Adenovirus mutants that lack essential genes must be grown by complemen-
tation, the products of the missmg genes supplied by a source other than the vtral
genome. Two methods are available for the growth of defective adenovirus
mutants by complementation. For mutations confined to E 1, E4, or portions of
E2, complementmg cell lines that contain segments of viral DNA and that can
supply the missing viral products can be used to produce pure stocks of mutant
particles (1-9). This approach will probably be extended to other regions of the
viral genome, but may prove difficult to adapt to genes such as the late genes,
whose products are required in large amounts by the virus. Alternatively, defec-
tive mutants can be grown as mixed stocks with a second helper virus that can
supply
in
truns functions required by the mutant (10). Providing that a mutant
contains all of the c&-active elements required for viral growth and is large
enough to be packaged into an adenoviral capsid, there are in principle no restric-
tions on the DNA sequences that can be deleted from a mutant grown by comple-
mentation with helper virus. In addition, because the helper virus replicates, even
products needed in large amounts can be effectively supplied
in trans.
Recently,

adenoviral genomes constructed for gene therapy purposes and lacking nearly all
viral sequences have been propagated in this way (11-13).
Growth of mutants by complementation with helper virus requires that, for
most purposes, the mutant and helper be physically separated before use. This is
done by CsCl equilibrium density gradient centrifugation. The following proto-
cols were developed for propagation of defective mutants with modest deletions
(l O-20% of the viral genome), but are applicable to larger deletions and to sub-
stitutron mutants with genome sizes that differ from that of wild-type virus.
From Methods In Molecular Me&me, Vol 21 Adenovms Methods and Protocols
Edited by W S M Wold 0 Humana Press Inc, Totowa, NJ
25