adenovirus methods and protocols

1996 The E3-11.6kDa adenovirus death protein ADP is required for effi- cient cell death: characterization of cells Infected with adp mutants... To permit collectton of medmm as stock whi

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

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

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

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

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

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

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)

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)

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

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

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

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

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

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

Coding sequence E3 protein

and killing of the virally infected cell by binding MHC class I molecules and inhibtting their transport

from Infected cells ($5)

Prevents TNF cytolysts Clears epidermal growth factor and msulm receptors from the infected cell surface

Clears Fas antigen from the mfected cell surface (6)

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

2 L of 4 A4 GuHCl m TEMP pH 8 0 4 A4 GuHCl made up n-t 10 mM Trts-HCl,

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

integral luer lock (cat no 420823-0016), and column reservotr 1000 mL (cat no

column (cat no 5820-39), Ace cylmdrtcal funnel (cat no 5822-15), couplmg

(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

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)

discussed m Chapter 1

10 15), HzndIII (cat no 1045), and calf intestinal alkaline phosphatase (CIP) (cat

no 2905)

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

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

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

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

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,

transferred to the viral genome by hgation of the mutated Ad2 EcoRI-D frag-

needed in the 14.5 K and 14.7 K protems, homologous recombmation has been

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

cleaved Ad5 TP-DNA (as described in ref II)

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

Gel purify the mutated viral DNA fragment and ethanol precipitate the Insert DNA to concentrate the fragment

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%)

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

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

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

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.)

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/

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

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-

Kpn 1 y / B I C IIIHI D IEI A IF Ad2

-A -B -c -D

-F -CI -H

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

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-

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

3 Roberts, R J , O’Netll, K E., and Yen C T (1984) DNA sequences from the adenovnus 2 genome J Blol Chem 259, 13,968-13,975

4 Tollefson, A E , Ryerse, J S., Scaria, A., Hermiston, T W., and Wold, W S M (1996) The E3 11 6-kDa adenovtrus death protein (ADP) is requtred for effi- cient cell death characterization of cells infected with adp mutants Vzrology

220,152-162

5 Tollefson, A E , Scat-la, A., Hermiston, T W , Ryerse, J S , Wold, L J , and Wold, W S M (1996) The adenovnus death protein (E3-11 6K) is reqmred at very late stages of infection for efficient cell lysrs and release of adenovnus from Infected cells J Vu-01 70, 229&2306

6 Tollefson, A E , Hermtston, T W., Ltchtenstem, D L., et al (1998) Forced degradation of Fas mhtbtts apoptosts m adenovnus-infected cells Nature 392,

726-730

7 Li, Y., Kang, J., and Horwitz, M J (1997) Interaction of an adenovirus 14 7- kilodalton protein inhibitor of tumor necrosis factor alpha cytolysls with a new member ofthe GTPase superfamily of signal transducers J Vzrol 71, 1576-l 582

8 Robmson, A J., Younghusband, H B , and Bellett, A J D (1973) A cucular DNA-protein complex from adenovnuses Vzrology 56, 54-69

9 Sharp, P A., Moore, C , and Haverty, J L (1976) The mfectrvrty of adenovuus 5

10 Chmnadural, G , Chinnadurai, S., and Green, M (1978) Enhanced mfecttvtty of adenovlrus type 2 DNA and a DNA-protein complex J Vzrol 26, 195-199

11 Ranhelm, T S., Shtsler, J., Horton, T M., Weld, L S , Gooding, L R., and Wold,

W S M (1993) Characterization of mutants wrthm the gene for the adenovuus E3 14.7-kilodalton protein which prevents cytolysts by tumor necrosts factor J Vzrol 67,2159-2167

12 Wold, W S M , Deutscher, S L., Takernon, N , Bhat, B M., and Magle, S C

the same mRNA m regron E3 of adenovu-us Vzrology 148, 168-180

13 Ausbel, F M , Brent, R , Kingston, R E., Moore, D D , Seidman, J G., Smtth, J A., and Struhl, K (1994) Introduction of DNA into mammalian cells, m Current Protocols zn Molecular Bzology, vol 1, Wiley, New York, pp 9.1 l-9 5 5

14 Trower, M K (1996) A protocol for site-directed mutagenesis employmg a uracrl- containing phagemld template Methods Mol BIOI 58,469-476

15 Deryckere, F and Burger-t, H -G (1997) Rapld method for preparing adenovrrus DNA Bzotechnzques 22, 868-870

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

1.7 Growth of Deletion Mutants as Mixed Stocks

composttion of a mixed virus stock changes over time In parttcular, a defec- tive mutant grown m the presence of replication-competent helper vn-us tends

to disappear from the stock as any cell infected by such a mutant alone yields

no progeny, whereas cells singly infected by the helper produce a normal yield of virus particles Three approaches can be used to minimize that ten- dency First, if a mutant helper that requires complementation for its own growth can be used, and if the deletion mutant of interest complements the helper, only dually infected cells will produce particles Second, the multi- plicity of infection (MOI) used to produce stocks can be made high enough

to ensure that virtually all infected cells contain both a helper virus and the defective mutant Finally, seed stocks can be enriched for the deletion mutant

by physical methods before use in preparing new stocks The use of all three approaches when possible maximizes the yield of mutant particles

1.2 Selection of Helper Virus

Three criteria should be used in selecting helper vnus:

1 If possible, the helper should be defective and require complementation by the mutant of interest for growth For example, helpers carrying temperature-sen- sitive (ts) mutations in late genes were used m the isolation of Ad2 E4 deletion mutants (10) The use of helpers partially defective m packaging has been described recently (13); these do not reqmre complementation, but then mher- ent growth disadvantage reduces the possibility that they will outgrow the mu- tant of interest m the stock

2 Because separation of the mutant and helper depends on differences m buoyant density that, in turn, reflect differences in DNA content, the difference in genome size between the helper and mutant should be as large as possible A helper with wild-type genome length can be used for mutants with deletions greater than approx 10% of the viral genome; helpers with genomes slightly larger than wild- type can be used for mutants with somewhat smaller deletions (24) It is impor- tant to ensure that no recombmants with a genome size nearer that of the mutant can arise during the growth of the mixed stock, as such recombinants make purr- fication of the mutant more difficult

3 The helper should have no properties that make low levels of contammation of

scheme IS completely effective

2 Materials

1 CsCl solutions, All CsCl solutions should be 20 mM Trrs-HCl (final concentra- tion), adjusted to pH 8.1 Adjustment of the pH should be made after dissolvmg the CsCl as some lots produce very acidic solutions

Defective Adenovirus Deletion Mutants 27

CsCl density 1.25, refractive index 1.3572; 33.8 g CsCl per 100 mL of solution CsCl density 1.34, refractive index 1.3663; 46.0 g CsCl per 100 mL of solution CsCl density 1.7: refractive index 1.3992; 95.1 g CsCl per 100 mL of solution

2 1,1,2 Trichlorotrifluoroethane (Sigma, St Louis, MO; cat no T5271)

3 200X IGEPAL (Sigma cat, no I-3021): 10% IGEPAL CA-630 solution m water

5 PBS: per liter: 160 g NaCl, 4 g KCI, 18.2 g Na*HPO,, 4 g KH,PO,, 37.2 g EDTA

pH should be 7.2

3 Methods

3.7 lsolation of Defective Mutants

by Complementetion PIaquing

3.7.1 With Defective Helpers (see Notes 1 and 2)

1 Prepare host cell monolayers m 6-cm tissue culture dishes If applicable, the host cells should be nonpermissive for the helper,

2 Determine the number of cells in one dish

3 Transfect monolayers with mutant DNA by the calcmm phosphate procedure (protocol elsewhere in this volume) DNA fragments can be used if mutants are being constructed by a recombinational strategy

4 After transfection, infect the monolayer at an MO1 of 2-5 PFU/cell with the helper virus Remove the medium from the transfected dishes, add the virus

m 1 mL medium, adsorb for 2 h, remove the inoculum, and till the dishes with agar medium

5 Contmue by a standard plaquing protocol If the helper is a ts mutant, incubate the dishes at the restrictive temperature

6 When plaques are vistble, pick and screen for the presence of the mutant (see Chapter 4)

3.1.2 With Nondefective Helpers

1, Prepare helper vu-us DNA-protein complex (DNAPC; see Chapter 4)

2 MIX mutant DNA and helper DNAPC in a molar ratio of 10: 1 to 50: 1 and trans- feet appropriate monolayers The optimal amount of DNA for transfection varies depending on the plaque-forming efficiency of the helper DNA; adjust the DNA level to produce approx 100 plaques per dish

3 Treat as a normal plaque assay

4 Pick and screen plaques for the presence of the mutant

3.2 Growth of Mixed Stocks

1 Prepare host cell monolayers in 6-cm tissue culture dishes If applicable, the host cells should be nonpermissive for the helper

2 Remove the medium from each dish Place the dishes with one edge slightly raised (for example, resting on a pencil) on a tray This makes it possible to restrict the inoculum to a small area of the dish and raises the MO1 in that region

3 Pipet 0.1-0.25 mL of a mixed moculum onto the lower edge of the monolayer The inoculum can be a ministock (Chapter 4), a portion of a previously made mixed stock, or a stock enriched for the deletion mutant by one round of CsCl denstty gradtent centrifugation (below), diluted with medium to approx log infectious units per mL

4 With the dish still tilted, incubate at 37°C in a humldlfied incubator for 2 h (Proper humidification IS important to prevent the raised side of the monolayer from drying out.)

5 Remove the moculum, refill the dish with medmm, place the dish flat, and mcu- bate until all of the cells have detached from the plate If a ts helper is being used, incubate at the restrictive temperature Feed twice weekly by replace- ment of the medium until evidence of viral infection 1s seen over a substantial portion of the monolayer

6 Harvest the infected cells and medium when all of the cells have detached from the dish This mixed stock can be stored at -80°C until use

3.3 Purification of Deletion Mutants From Mixed Stocks

Because adenovirus particles with differing DNA contents have differing

buoyant densities in CsCI, adenovirus deletion mutants grown in the presence

of helper virus can be separated from the helper by equilibrium sedimenta- tion in CsCl density gradients Only mutants with fairly large deletion

mutations (> 10%) can be efficiently purified from helpers with a wild-type genome size by this method, although helper virus with longer than wild- type genomes have been used to make the purification of mutants with smaller

deletions possible (14)

1 Prepare a mixed lysate One dish should be labeled with 32P as described below

2 To the mlxed stock, add IGEPAL to a final concentration of 0.05%

3 Extract the stock vigorously with l/5 volume of 1 ,1,2 trichlorotrifluoroethane Recover the aqueous phase after centrifugation at 4000g for 5 min in a Sorvall GSA rotor Re-extract the cell debris and organic phase with a small volwne of PBS; recover the aqueous phase and pool with the supematant from the previous centrifugation

4 Prepare a discontinuous CsCl gradient in a 35-mL polypropylene centrifuge tube

by adding (in order) approx 20 mL extracted virus suspension, 4 mL CsCl den- sity 1.25, and 5 mL CsCl density 1.7 Add each solution slowly through a plpet placed all of the way to the bottom of the tube After the CsCl solutions have been added, fill the tube to the desired level with extracted vn-us suspension

5 Centrifuge for 90 min at 17,000 rpm (29,000g) (Sorvall SV288 rotor or equiva- lent) or 3 h at 25,000 rpm (82,000g) (Beckman SW27 rotor or equivalent)

6 In a darkened tissue culture hood, illuminate the gradient wrth a narrow beam of light from one side A microscope lamp is a suitable light source The virus will form a sharp, blue-white, translucent band at the interface of the two CsCl solu- tions A broader, yellowish or tan, frequently granular layer of cell debris will appear at the top of the lighter CsCl cushion

Defective Adenovirus Deletion Mutants 29

7 Collect the virus, avoiding the cell debris (see Note 3)

8 Adjust the concentrated virus suspension to a density of 1.34 (refractive index 1.3663) with 20 mM Tris-HCl, pH 7.5 or with the density 1.7 CsCl solution Adjust to the required volume with CsCl density 1.34

9 Centrifuge the suspension at 35,000 rpm for 16 h in a Sorvall TV865 rotor (or equivalent) Two closely spaced virus bands should be visible m the center of the tube

10 Fractionate the gradient into single-drop fractions through a hole made in the bottom of the tube

11 Measure the radioacttvity m each fraction by Cherenkov counting Two more or less well-separated peaks should appear (Fig 1)

12 Pool the fractrons that comprise the lighter peak (see Fig 1) and repeat steps 8-11 (see Note 4)

13 Estimate the infectious titer of the virus suspension from its A,,, An A,,, of 1 corresponds to a plaque-formmg titer of 3.5 x 1 Og PFU/mL for Ad5 purified over three CsCl gradients

14 The purity of the mutant stock can be assessed by plaqumg under conditions permissive for the helper, or by restriction enzyme digestion of purified DNA

15 Purified virus is stable for months m buoyant CsCl at 4°C However, htgh-titer suspensions dialyzed against solutions of low ionic strength (for example, TE) frequently precipitate If it is necessary to remove the CsCl from a purified stock, first adjust the A 260 of the suspension to 0.5 or lower, and mimmize storage time

at low ionic strength

3.4 Preparation of 32P-Labeled Tracer Virus Particles (see Note 5)

1 Inoculate a IO-cm dish of cells with a mixed stock as described above

2 Examme the dish daily, replacing the medium every 3 d until one-fourth to one- half of the cells show evidence of viral infectron Gently remove the medium from the dish and replace it with 10 mL phosphate-free medium supplemented with 2% serum and 40 uCi/mL 32P orthophosphate

3 When all of the cells have become detached from the dish, harvest the cells and medium

4 Collect the cells by low-speed centrifugation Rinse the labeled cells twice by low-speed centrifugation and resuspension in PBS

5 After the second rinse, resuspend the cells in 5 mL PBS, add IGEPAL to 0.05%, and extract vigorously with 5 mL of 1,1,2 trichlorotrifluoroethane Centrifuge and recover the aqueous phase If intended for use as tracer, add this material to one tube of unlabeled virus and concentrate (step 3)

4 Notes

1 Some defective vrruses kill cells that they infect even though they do not form plaques If monolayers infected with helper at the MO1 recommended here do not survive, the following modification should be used

a Prepare monolayers m 24-well tissue culture dishes

b Determine the number of cells in one well

c Transfect the wells with mutant DNA by the calcium-phosphate procedure

d After transfection, infect the monolayer at an MO1 of 2-5 with the helper virus Remove the medium from the transfected dishes, add the virus in

Defective Adenovirus Deletion Mutants 31

0.5 mL of medium, adsorb for 2 h, remove the inoculum, and refill with medium If the helper is a ts mutant, incubate at the restrictive tempera- ture (see Note 2)

e 12-16 h after infection, trypsinize each well and reseed the transfected/ infected cells in a 6-cm dish along with enough uninfected cells to form a confluent or nearly confluent monolayer

f After the cells have attached (8-24 h), overlay with agar medium and proceed

as described In Subheading 3.1.1., step 5

2 If the mutant is available as virus particles (as in the isolatton of naturally occur- rmg mutants), use one of the protocols in Subheading 3.1.1., or m Note 1, replacing the transfection step with infection by the mutant stock at approx 50 mfectious particles per dish (or well)

3 It is convenient to collect virus through a hole made m the bottom of the tube with a pushpin Plug the tube with a rubber stopper pierced by a large-gage syringe needle, close the needle with a finger over its hub, and make the hole m the bottom of the tube The rate at which liquid flows out of the hole can be controlled by finger pressure on the hub of the syringe needle Alternatively, a needle can be inserted through the side of the tube and the virus band drawn out with a syringe For lysates from two or more lo-cm dishes, the virus band should

be visible in the drops as they leave the tube; for small preps, 32P-labeled virus tracer (see above) can be added before centrifbgation and fractions can be col- lected and the virus located by Cherenkov counting

4 Depending on the purity required, two or more gradient steps may be necessary

In the experiment shown in Fig 1, contamination of the deletion mutant stock by helper was approx 0.03% after three gradient steps

5 If labeled virus to be used as tracer 1s being prepared in parallel with a large unlabeled stock, the labeled cells should be harvested at the same time as the large stock, even if not all cells appear to be infected If the labeled dishes are ready for harvesting before the remaining dishes, collect and rinse the cells and store at -80°C until needed

References

1 Graham, F L., Smiley, J., Russel, W C., and Nairn, R (1977) Characteristics of

a human cell line transformed by DNA from human adenovirus 5 J Gen Vzrol

36, 59-72

2 Weinberg, D H and Ketner, G (1983) A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2 Proc Natl Acad Sci USA 80,5383-5386

3 Brough, D E., Lizonova, A , Hsu, C., Kulesa, V A., and Kovesdi, I (1996) A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions El and E4 J Viral 70, 6497-6501

4 Brough, D E., Cleghon, V., and Klessig, D F (1992) Construction, characteriza- tion, and utilization of cell lines which inducibly express the adenovints DNA- binding protein Virology 90,624-634

5 Amalfitano, A., Begy, C R., and Chamberlain, J S (1995) Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors Proc Nat1 Acad Sci USA 93,3352-3356

6 Ho, W Y., Karlok, M., Chen, C., and Ornelles, D (1995) Adenovirus type 5 precursor terminal protein-expressing 293 and HeLa cell lines J Vwol 69, 4079-4085

7 Kroughliak, V and Graham, F (1995) Development of cell lines capable of complementing El, E4, and protein IX defective adenovirus type 5 mutants Human Gene Ther 6,157~1586

8 Wang, Q., Jia, X.-C,, and Fmer, M H (1995) A packaging cell lme for propaga- tion of recombinant adenovirus vectors containing two lethal gene region dele- ttons Gene Ther 2,775-783

9 Yeh, P., Didieu, J.-F., Orsim, C., Vigne, E., Denefle, P., and Perricaudet, M (1996) Efficient dual transcomplementation of adenovirus E 1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit J Vzrol 70,559-565

10 Challberg, S S and Ketner, G (198 1) Deletion mutants of adenovirus 2: tsolation and initial characterization of vuus carrying mutations near the right end of the viral genome Virology 114, 196-209

11 Fisher, K J., Choi, H., Burda, J., Chen, S.-J., and Wilson, J M (1996) Recombi- nant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis

12 Mitani, K., Graham, F L., Caskey, C T., and Kochanek, S (1995) Rescue, propa- gation and partial purification of a helper virus-dependent adenovirus vector Proc Nat1 Acad Sci USA 92,3854-3858

13 Kochanek, S., Clemens, P R., Mitani, K , Chen, H -H., Chan, S., and Caskey,

C T (1996) A new adenoviral vector: Replacement of all viral coding

dystrophin and P-galactosidase Proc Natl Acad Scz USA 93, 573 l-5736

14 Falgout, B and Ketner, G (1987) Adenovirus early region 4 is required for effi- cient virus particle assembly J Virol 61, 3759-3768

Manipulation of Early Region 4

Julie Boyer and Gary Ketner

1 Introduction

Early region 4 (E4; Fig 1A) occupies the right-hand 3000 bp of the human adenovirus genome The sequences of E4 and E4 cDNAs indicate that E4 encodes seven polypeptides (I-41, most of which have been detected m infected cells Analysis of E4 mutants has implicated E4 products m a variety

of processes that occur in Infected cells, including viral early and late gene expression, DNA repllcatlon, the shutoff of host-cell protein synthesis, trans- formation, and the ability to stimulate replication of adenovlrus-associated vn-us (AAV) Specific E4 products have been implicated in many of those pro- cesses and, in some cases, mformatlon on the molecular mechanisms of E4 function IS emerging Current knowledge of the functions of E4 products 1s summarized below

ORFl mutants of adenovirus type 5 (Ad5) are viable in standard hosts and

no mutant phenotypes have been detected in cultured cells However, the prod- uct of ORF 1 of adenovirus 9 (Ad9) is necessary for the induction of mammary tumors m mice by that virus, and Ad9 E4 ORFl alone can transform cells m culture (5) The mechanism of transformation by Ad9 ORFI is unknown ORF2 mutants of Ad5 are viable in standard hosts and have no known mutant phenotypes in cultured cells No evidence implicating ORF2 in events in infected cells has been published

The products of ORFs 3 and 6 are required for viral late-gene expression and thus for viral growth ORFs 3 and 6 are genetically redundant; either prod- uct is sufficient for normal (ORF6) or near-normal (ORF3) late-protein syn- thesis and growth m cultured cells, but mutants lacking both are profoundly defective (6-8) When constructing virus with mutations that simultaneously disrupt both ORFs, an E4-complementing cell line such as W 162 (ref 9; see

From Methods m Molecular Medmne, Vol 21 Adenovrrus Methods and Protocols

Edlted by W S M Wold 0 Humana Press Inc , Totowa, NJ

33

A

Fig 1 (A) The orgamzatlon of adenovuus early regton 4 E4 open reading frames (ORFs) are indicated by open boxes above a scale that Indicates position m map units (0 to 100; 1 map unit is approx 360 bp) and nucleotide numbers ORFs 1,2, 3,4, and

6 are colmear with the viral DNA; ORFs 3/4 and 6/7 are the result of inframe sphcmg

of the E4 mRNA precursor E4 is transcribed from right to left, (B) Reconstructton of

an intact viral genome by ligation in vitro (C) Reconstructton of an intact viral genome by overlap recombmation (D) Construction of an E4 mutant by recombma- tion between a plasmid and an E4 deletion mutant In C and D, the X indicates a potential homologous recombmation event In B, C, and D, the position of a hypo- thetical E4 mutation is noted by a V

below) must be used for propagation The viabiltty of viruses expressing at least one or the other of these ORFs may be exploited when constructmg mutants by the homologous recombinatton-based method described below It should be noted that for all of the methods described, the slightly reduced abil- ity of ORF3+/ORF6- viruses to form plaques on normal hosts, while not abso-

genotype dtfficult to recover

In transfection experiments, both ORF3 and ORF6 stimulate both nuclear and cytoplasmic RNA accumulation from cotransfected transcriptional units

Early Region 4 Manipulation 35 contammg 5’ mtrons Then modes of actton apparently differ; ORF3 specifi- cally stimulates the accumulation of messages containing optional exons such

as the adenovnus ‘1’ leader, whereas ORF6 stimulates accumulatton of mes- sages with or without such exons The Elb 55-kDa protein is not required, nor are viral nuclerc acid sequences required m the target RNAs (10,11,39) The ORF6 product has roles m the transport of late viral mRNAs from the nucleus

to the cytoplasm of infected cells (81 and 1s required for completion of second- strand DNA synthesis m the life cycle of AAV (12) ORF6 forms a physical complex with the 55-kDa product of adenovnus early region 1 b (E 1 b) and the complex is the functtonal unit m at least some steps in late gene expression (13-15) The ORF6 product also bmds the cellular p53 protein, preventing activation of transcription by p53 in transfection systems (26) Whether this activity is mvolved in late message production is not known Both ORF3 and ORF6 affect the architecture of the nucleus in infected cells and may mediate some of then effects in that way (I 7,181

ORF4 mutants are viable m standard hosts Expression of ORF4 depresses transcription from at least some promoters that contam CREB protem-bmdmg sites, mcluding cJunB (19) and the adenovirus E2 and E4 promoters (20,21) The effect of ORF4 is indirect; an ORF4-induced change in the phosphoryla- non state of cFos results m a reduction m cJunB transcriptton and consequently JunB levels and AP- 1 activity Reduced AP- 1 activity presumably reduces E2 transcription (21) The ORF4 protein bmds to and modulates the activity of protein phosphatase 2a, providmg a plausible btochemical mechamsm for its effect on cFos phosphorylation (22) As a result of its effects on E2 transcrip- tion and, perhaps, on protein phosphorylatton, ORF4 downregulates viral DNA synthesis This activity IS antagomzed by the E4 ORF 3 and 6 products by an unknown mechanism (23)

ORF6/7 mutants are viable m standard hosts The ORF6/7 product binds to transcription factor E2F and confers cooperattvity on E2F binding to promot- ers, such as the E2 promoter, that contain appropriately positioned E2F bmd- ing sites (24,225) As a consequence, ORF6/7 expression stimulates E2 transcription in infected cells (26) In most host-cell types, this effect requires the E 1 a 289R protein, which dissociates E2F from pRB and makes it available for binding by ORF6/7 (27) ORF6/7 mutants are not obviously defective m viral DNA synthesis in cultured cells

1.1 Complementing Cell Link

E4 mutants lacking both ORF3 and ORF6 are defective for growth m nor- mal adenovirus hosts and must be propagated on cell lines capable of supply- ing required E4 functions in trans The most commonly used such cell lme is

W 162 (9) The W 162 cell lme was constructed by mtroducmg the Ad5 EcoRI

B fragment (map units 86.3-100) mto Vero cells as part of a plasmrd carrying the selectable marker gpt W 162 cells contain intact E4 and supply the E4 func- tions required to support the growth of deletion mutants lacking all identified E4 products E4 is attached to its natural promoter in W 162 cells and it is presumed that E4 expression 1s off until induced by Ela expression from an infecting virus Both WI62 and Vero cells (which are of monkey origin but permissive for human adenovnuses) support plaque formation by wild-type adenovirus approx lo-fold less efficiently than do other adenovnus hosts, such

as 293 cells It IS therefore important when comparing defective E4 mutants titrated on W 162 cells to other viruses on a PFU basis, that W162 titers be obtained for all stocks

In addition to W 162 cells, several 293-derived cell lines that complement mutants with lesions m both El and E4 have been isolated These lines, devel- oped primarily for use in construction of gene-delivery vectors, make it pos- sible to grow multiply-defective mutants Because of its role m mhtbtting host-cell protein synthesis, E4 IS presumed to be toxic if constitutively expressed Ela expression induces the E4 promoter, and regulable heterolo- gous promoters therefore have been used to control E4 expression in each of the 293-derived complementing cell lines E4 expression thus remains low until

it is deliberately induced The E4 complementing cell lines described so far are listed in Table 1

1.2 Isolation of Mutants

7.2.1 Naturally Occurring Mutants

The first E4 mutants were isolated from a stock of adenovnus 2 that had accumulated a substantial proportion of deletion mutants, presumably as a result of many undiluted serial passages All of the mutants lacking E4 sequences were defective and were originally cloned and propagated by the use of a LS helper virus For characterization, deletion-mutant parttcles were purified from the resulting mixed stocks by repeated CsCl density gradient centrifugatton (ref 29; see Chapter 3) The development of E4 complementmg cell lines made the use of helper viruses unnecessary, and most of what is known of the phenotypes of E4 mutants has been learned using mutants con- structed in vitro and propagated on W 162 cells

1.2.2 Construction of Mutants in Vitro

Most E4 mutants are made by in vitro manipulation of E4-containing plas- mids and subsequent mtroduction of the mutations into an intact viral genome The mutations themselves can be made by any of a large variety of standard technrques Three methods have been used to incorporate E4 mutations into

Early Region 4 Manipulation 37

Table 1

Cell line complemented Mutations Promoter/inducer (if applicable) Ref

Ketner (9) VK2-20; VKl O-9 El, pIX, E4 MMTVldexamethasone Kroughak and

Graham (33)

A2 El, E4 Metallothtonein/metal Ions Brough et al (35)

unpublished

infectious virus: assembly of intact viral genomes by m vitro ligatton of a mutant E4 DNA fragment to one or more subgenomic viral DNA fragments, recombina- tion between a mutant E4 DNA fragment and an overlapping subgenomic frag- ment (or fragments) of the vu-al genome m transfected cells, and recombmation between a mutant E4 DNA fragment and an intact viral genome (Fig lB-D)

A variety of viral and plasmid DNA fragments have been used for constructron

of E4 mutants by ligation (Table 2) Both two-fragment and three-fragment schemes have been used In most cases, a large restriction fragment covering the left-hand portion of the genome, derived from virion DNA or from virion-dertved DNA-protein complex (DNAPC; ref 30), 1s ligated to smaller plasmid-derived fragments that make up the remainder of the genome The ligated DNA is then introduced into appropnate cells by transfection The virion DNA fragment can be purified; alternatrvely an unpurified fragment can be used if the chance of reassem- bling parental virus 1s reduced by digesting the vnion DNA with an enzyme that cuts the right-hand end of the genome repeatedly Neither of these approaches 1s completely effective m eliminating plaques produced by virus with the genotype of the left-end donor and plaques that contain the desired recombinant are usually mixed Therefore, plaques must be screened to identify those that contam recombi- nants and recombinants usually must be plaque purified Constructrons expected to yield defective E4 mutants must be done m complementmg cell lines

1.2.2.2 OVERLAP RECOMBINATION

For the construction of E4 mutants by overlap recombmation, a subgenomic left-hand terminal fragment (or fragments) of the viral genome 1s transfected

Table 2

Construction of E4 Mutants by Ligation and Overlap Recombination

83 6 (EcoRI) to 100 from pEcoRIBAd5 76.0 (EcoRI) to 100 from ~75-100 None

76 0 (EcoRI) to 100 from ~75-100

60 0 (BarnHI) to 100,

79 6 @&I) to 84.8 (XbaI) deleted m constructton

0 to 76.0 (EcoRI), 73 2 (findII1) to 89 0 (kzzndIII),

83 6 (EcoRI) to 100

Brtdge and Ketner (6)

Huang and Hearing (7)

Marton et al (37)

Huang and Hearing (7)

Halbert, Cutt, and Shenk (8)

Hemstrom et al (38)

Genome posltlons are given m map units (MU); 1 mu IS approx 360 bp

into cells along with a plasmrd bearing a mutant version of E4 The mutant plasmrd must share sequences with the left-hand genomrc fragment(s) so that homologous recombmation m the region of overlap can regenerate a full-length viral genome Consrderatrons such as purrficatron of the left-hand fragment, the necessity for screening plaques, and the use of complementmg cell lines are the same as those described for the hgatron protocol, above

1.2.2.3 RECOMBINATION WITH INTACT VIRAL DNA

Many E4 mutants are viable in normal adenovirus host cell lines Such mutants can be can be selected after cotransfectron of normal hosts (such as

293 cells) with a mutant E4 DNA fragment and intact viral DNA prepared from a E4 deletion mutant that will not grow m those cells (for example, H5d11011, ref 6) Because no plaque-forming virus IS involved m this proto- col, the background of unwanted plaques is much lower than with the above methods, and when this technique can be used rt IS probably the most efficient

of the available approaches

Early Region 4 Man&w/at/on 39

2 Materials

1 TE: 10 mM Tns-HCl, 1 mA4 EDTA Adjust to pH 8.1

2 Buffered 8 h4 guamdmmm hydrochloride solution: 8 A4 guanidinium hydrochlo- ride, 50 mM Tris-HCl, pH 8.1

taming either 3.03 M CsCl (51.0 g of CsCl per 100 mL of solution) or 4.54 M CsCl (76 5 g of CsCl per 100 mL of solution), buffered with 20 mM Tns-HCl Final pH should be 8.1 Confirm the pH before use, as some lots of CsCl produce very acidic solutions unless neutralized

4 PMSF* 100 mM Phenylmethylsulfonylflourlde in isopropanol Store tightly

HBS (HEPES-buffered salme) 8 g/L NaCl, 0.37 g/L KCl, 0.125 g/L Na2HP0, 2H,O,

1 s/L glucose, 5 g/L N-2-hydroxyethylpiperazine-W-2-ethanesulfomc acid (HEPES), final pH 7.05 The pH of the HBS 1s cntlcal for the success of transfectlons

Herring sperm DNA Dissolve herring sperm DNA in TE at approx 5 mg/mL Somcate on ice m 30-s bursts with an immersed probe until the viscosity no longer changes appreciably with each burst (6-10 bursts) Determine the concentration

of the solution spectrophotometrlcally and adjust to 2.5 mg/mL with TE

Plasmlds (see also Table 2)

a pBN27 (6): pBR322 containing Ad5 DNA from map unit (mu) 60 (BarnHI) to

mu 86.4 (NdeI) One map unit corresponds to 1% of the viral genome, or approx 360 bp

b pEcoRIBAd5 (28) pBR322 containing Ad5 mu 83 6 (EcoRI) to mu 100 (right genomic end)

Vu-uses

a Adenovlrus 5

b H5d11011 (6) a defective E4 mutant that lacks Ad5 nt 33091 to 35353

c H5d13 lOXba+ (7) carries a single XbaI site at mu 93.5

Sucrose solutions* 5,20, and 60% sucrose solutions prepared m I MNaCI, 1 mM EDTA, 10 mM Tns-HCl, pH 8.1

200X IGEPAL (Sigma, St LOUIS, MO; cat no I-3021) 10% IGEPAL CA-630 solution in water

Phosphate-free medium MEM formulated without sodium phosphate can be purchased from media suppliers (for example, Glbco-BRL, Gaithersburg, MD, cat no 21-097-027) For labeling viral DNA, supplement with 2% fetal bovine serum (FBS) (dialysis is not necessary) and 40 pCi/mL carrier-free 32P orthophosphate

Lysls buffer: 0 6% sodium dodecyl sulfate (SDS), 200 pg/mL Protemase K, 10 mM Tris-HCl, pH 8 1, 1 mM EDTA

1,1,2 Trichlorotrifluoroethane (Sigma cat no T527 1)

25% glucose solution 25% (w/v) glucose m HBS; filter sterilize

Agar medium MEM without phenol red, supplemented with 2% FBS, penicillin, and streptomycm, and solidified with 0.9% Difco (E Molesley, Surrey, UK)

Bacto-agar Prepare and filter stenhze 2X MEM without sodium bicarbonate, glutannne, and phenol red Supplement with sodium bicarbonate, FBS, penicillin, streptomycm, and glutannne, and store tightly closed at 4°C Note that thts mednnn 1s 2X, and supplements should be added accordmgly Prepare and autoclave 1.8% agar m water To prepare agar overlays, melt the agar tn a steam bath or mrcrowave oven, mix with an equal volume of supplemented 2X MEM, equihbrate to 48°C and use to overlay plates

3 Methods

3.1 Preparation of Adenovirus DNA-Protein

Complex (DNA PC; ref 30)

Dialyze CsCl density-gradient purified virus particles (see Chapter 3) against

TE Highly concentrated virus suspensions sometimes precipitate during dtaly- sis, this does not affect the procedure

Mix equal volumes of virus suspension and buffered 8 M guanidmium hydro- chloride (GuHCl) solution Immediately add PMSF to 1 mM final concentratton Incubate on tee for 5 min The turbidity of the suspension will decrease as the vu-us particles are dtssoctated

To the disrupted virus, add 4 A4 GuHCl, 4 54 M CsCI, 20 mA4 Tris-HCI, pH 8.1 The volume should be four times the original volume of the virus suspenston Adjust to a convement volume with 4 A4 GuHCl, 3.03 M CsCl, 20 mA4 Tris- HCl, pH 8 1

Centrifuge the solutton overnight at 45,000 rpm (177,OOOg) and 15°C m a Sorvall TV865 rotor or equtvalent

Collect 0 25- to 0 5-mL fractions and locate fractions containing DNA by UV spectrophotometry

Pool DNA-contammg fracttons and dtalyze 4 h vs TE, 1 M NaCl then overnight

vs two changes of TE Store the DNAPC m a plasttc tube, as the termmal protein adheres to glass The yield should be approx 10 pg of DNAPC per IO-cm Petrt dish of infected cells

3.2 Construction of E4 Mutants by Ligation

1 Introduce the destred E4 mutation mto the plasmtd pEcoRIBAd5

2 Prepare Ad5 or H5dZlO 11 DNAPC The use of H5d110 11 reduces the background

of plaques contammg parental vnus when constructmg viable E4 mutants m noncomplementmg cell lines

3 Prepare pBN27 and mutant pEcoRIBAd5 DNAs by CsCl-ethtdmm bromide den- sity gradient centrifugation, Qiagen (Valencia, CA) column, or similar method

4 Digest 9 pg of DNAPC with BumHI and EcoRI m a mtcrofuge tube This ytelds

a large left end fragment (O-60 0 mu) and cleaves the right end mto three pteces

5 Precipitate the digested DNA by the addmon of 2.5 volumes of ethanol directly

to the digestion reaction Chill on tee for 15 mm; centrifuge for 15 mm at 4°C m

a mtcrofuge, and remove the supernatant with a mtcroprpet Invert the tube and air-dry the pellet for 15 min at room temperature