Quantitation of RNA tumor viruses by spectroscopy of density gradient gels.

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Copyright i 1975 American Society for Microbiology _Printed_in16, No.U.S.A.

Quantitation

of

RNA Tumor

Viruses

by Spectroscopy

of

Density

Gradient

Gels

L. F.LIEBES,* E. F. RETZEL, V.M. MAHER, M. A. RICH, J. J. McCORMICK, I.SALMEEN, ANDL.

RIMAI

Department of Biology, MichiganCancerFoundation,Detroit, Michigan48201*andScientificResearchStaff,

FordMotor

Company,

Dearborn,

Michigan

48121

Received for publication23April 1975

We have developed a system for virus particle quantitation based on the

measurementoftheopticalabsorbance of stained viruses which first have been

banded at their

buoyant density

in an

equilibrium

24 to 53% (wt/wt) sucrose

density gradient, then fixed in position in the gradient by

photopolymerizing

an

acrylamide-riboflavin mixture in thesucrose,andfinally stained and destained.

Using plasma from mice infected with

leukemia

virus (Rauscher) or chickens

infected with avian

myeloblastosis

virus (BAI strain) or suitable controls, we

haveshown that thistechnique specifically detects RNAtumorviruses. Byusing virus stock solutions forwhich the absolute concentrations were determined by

laserbeat frequencyspectroscopy, wehave calibrated the absorbanceoftheviral

bands intermsofvirusparticle concentration. Using 0.8-ml gradients gels (4 by 45 mm) we can detect as low as 2 x 107 viral particles with

Coomassie

blue

staining and 6x 106 viralparticles witha moresensitive staining procedure using

amido black.

The

purpose

of this

paper isto

demonstrate

a

fast

and

simple quantitative

assay

for

RNA

tumor

virus

particles

present in

either

purified

or

nonpurified virus preparations. A number of

assays

for

such viruses

are

in

commonuse,

but

they

have disadvantages

for

quantitative

appli-cations.

Infectivity

assays,

although

necessary tomeasure

the

biological

activity

of

viruses,

are

time-consuming (1 week

to 12

months)

and

usually

cannot

be

analyzed

in terms of

the

number

of

virus

particles

in

the

sample (1,

8).

Assays for incorporation

of

radioactive uridine

into

particles

that band

at a

density of

1.15 to 1.18 in sucrose are

useful

as a

relative

measure of

virus

production

(11),

but

they

require that

the virus be

grown in

tissue

culture

(a condition

that

frequently

cannot

be

met) and

presume

that uridine

incorporation

into

viral

particles

is linear over the

iabeling

period.

Radioim-munoassays

or

immunofluorescent

assays are very sensitive and very specific tests for known

viruses,

but

the

absolute number

of virions cannot

be obtained

easily and,

to make anti-sera,

milligram quantities

of

homogeneous

vi-ral-specific

protein are

required

to serve asthe

antigen

(18).

Assay

for

RNA-directed

DNA

polymerase

(reverse

transcriptase) activity

isa

fast,

sensitive,

and

specific

test for all oncor-naviruses.

However,

as we

have shown

(9), such

enzymatic

assays

applied

to

preparations

of

virus can

be

inhibited by various

agents

and

cannot

be correlated

directly with the number

of

viral

particles. We have

shown,

for

example,

that

the

reverse

transcriptase activity

per virion of avian

myeloblastosis

virus

(AMV) is about

24

times

higher than that of

murine

leukemia

virus

(MuLV, Rauscher)

(unpublished data).

Finally, electron microscope

counting

methods

are

time-consuming and require careful

discern-mentof

viral

particles from cell debris (12).

Because

of

such

problems,

we

investigated

alternative

physical methods

for

determining

the number

of virus

particles

present in a

solution. Previously

we

demonstrated that laser

beat

frequency light scattering spectroscopy

can

be used

to

determine

not

only the diameter but

also

the number

of

viral

particles

in a

purified

sample (14). Here

we

describe

a

method

for

determining

the

number

of viral

particles

in a

preparation, using the

optical absorbance

of stained virus

particles which have been banded

at

their characteristic

buoyant density

in su-crose

and

spatially

immobilized

by

photopolym-erizing

an

acrylamide-riboflavin

mixture.

Other

workers

have used this

technique

to

immobilize

proteins

after

centrifugation

in a sucrose

density gradient (7, 13,

19),

to

im-mobilize

DNA in a cesium chloride

gradient

(4,

6),

and to

immobilize

bacteriophage

after

isopycnic

centrifugation

(20).

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In this

quantitation procedure,

the

oncor-navirus-containing sample

is first concentrated

into

avery narrow band

by

isopycnic

centrifu-gation.

Secondly, the position

of the virusatits

buoyant density

is fixed

by photopolymerizing

an

acrylamide-riboflavin

mixture which is

in-corporated

as part of the sucrose

gradient.

Finally, the

immobilized virus band is stained

and

destained,

and

by scanning

the stained

gel

the integrated optical

absorbance of the viral

band is determined.

The

quantity

of stain

absorbed

by

the viral

band,

and hence the

integrated

absorbance,

is

proportional

to the

virus

concentration. Since

the

proportionality

constant

depends

on the virus and the

stain,

exact

quantitation requires

that theabsorbance per virion must be calibrated for each

virus-stain combination.

For this calibration we

de-termined absolute

virus concentrations in the

purified

stocks

using

laser beat

frequency

spec-troscopy, but other suitable virus

quantitation

methods such

aselectron

microscopy

could be

used

aswell.

MATERIALS ANDMETHODS

Polymerizablegradients. Thepolymerizable

gra-dients were prepared from the following stock

solu-tions. Solution A(gelbuffer)consisted of 2.0 M

Tris-phosphoric acid (pH 7.0) containing 2.4%N, N, N',

N'-tetramethylenediamine; solution B (48% acrylam-ide stock) contained 24 g of acrylamacrylam-ide and 0.735 g of bis (N, N'-methylene-bis-acrylamide) in a final

vol-ume of 50 ml of water; solution C (riboflavin

photocatalyst [10-7M])was 0.4mg ofriboflavin in a

final volume of 100 ml of water; solution D was 20% (wt/wt)sucrose in 0.005Mphosphate buffer (pH 7.0);

and solution E was 65% (wt/wt) sucrose in 0.005M

phosphate buffer (pH 7.0). Just before the gradients

weremadesolution I (0.32 ml of solution A, 0.45 ml of

solution B, 0.15 ml of solution C, and 2.22 ml of

solutionD) and solution II (0.32 mlofsolution A, 0.45

ml ofsolution B, 0.15 mlofsolutionC,and 2.22 ml of

solution E) were prepared, and 0.4 ml of solution I and

IIwereaddedto agradient maker similarindesignto

thatdescribedbyBock andLing(2)toproduce 0.8-ml

linear gradients. We constructed a mixing device from two 1-ml plastic tuberculin syringe barrels

(Becton-Dickinson no. 5602) connected with an 18-gauge

syringe needle which was held in place with epoxy

cement. The outlet port was a 25-gauge needle.

Mixing was achieved using a Buchler vibration-type

stirrerassemblyfitted witha20-gauge rod. Gradient

formation wascontrolled using aHolter roller pump

(model 911, Extracorporeal MedicalSpecialties, King

ofPrussia, Pa.) set todeliverapproximately12ml/h.

Using this equipment linear gradientsof therange24

to53%(wt/wt)sucrose(as determinedfrom refractive

index measurements on a fractionatedgradient) were

generated using stock solutionsIandII.To

reproduci-bly generate such small gradients,wehavefound that

it is convenient tomakegradientsof 0.8mlandthen

remove avolume (0.1 ml or less) from the top of the

gradient equal to the volume of the sample to be

applied.

Viruspreparation.AMV (BAIstrain) and MuLV

(Rauscher) in plasma citrate weregenerously supplied

by Joseph and Dorothy Beard and University

Labora-tories, respectively, through the cooperation of the

Virus CancerProgram of the National Cancer

Insti-tute. The virus samples were purifiedby

discontinu-ous sucrose-gradient centrifugation as previously

de-scribed (14). After purification, the virus samples

were dialyzedovernight against three changes of 0.005

ionic strength NaH,PO3-K,HPO4 buffer, pH 7.0,

with the lastchange containing 0.02%(wt/vol) sodium

azide to preventbacterial growth. Prior to

quantita-tion by laser light scattering, the samples were

centrifuged at 19,600 x g (12 min) to remove any

aggregatedmaterial and dust.

Normalplasma preparations. Normal (i.e.,

non-viremic) mouse and chicken plasma citrate was

prepared by making a 1:1 dilution of plasma with

0.306 M sodium citrate (pH 7.0).

Gradient centrifugation and polymerization techniques. To maintain a uniform height, the sam-ple to be applied was made polymerizable by mixing

it with solutions A, B, and C, described above

(71:10:14:5 volume percent, respectively) and handled under the same conditions described for the gradient

components. Calibrated density beads (Reproductive

Systems, Inc., Menlo Park, Calif.) were added as markers for the 1.16 density region in the gradient. The gradient was then centrifuged (Spinco SW50.1

rotorwithadapters [Beckman no. 305527], 149,000 x

g for 2.5 h at 4C). The tubes were then removed and

photopolymerized by direct exposure to two daylight 40-W fluorescent lamps for about 1 h. The

polym-erized gels were removed by piercing the cellulose

nitrate tubes with a syringe needle and extruding

thegelbyforcing waterthrough the syringe.

Staining.The virusbands were stained with either

Coomassie blue R250oramidoblack, the later being

more sensitive because of a higher optical extinction

coefficient. Coomassie blue staining was carried out

by immersing the gels overnight in a 2% (wt/vol)

solution of Coomassie blue in water. Gels were

de-stained using several changes of an ethanol-acetic acid-water solution (30:5:65 volume percent) and then

werestored in 7% acetic acid. Because the virus was

locked into the gel matrix by the polymerization of the

7%acrylamide mixture, fixatives were not used. Since

the band intensities of Coomassie blue-stained

pro-teins areknowntofade with time after staining (3),

we developed a stable and more sensitive staining

method usingthe stain amido black. This was carried

outby modifyingthe procedure of Wray and Stubble-field(21).Prior tostainingwiththisdye, the gels were

soakedin3 M ureafor 3 hat 37 C.The gels then were

immersedovernightina 2%(wt/vol)solution of amido

blackin water.Destainingwascarried outonarotary

shaker at 37C with several changes of 1 M

H2SO,

containing 3 M urea. Amido black-stainedgels were

destained in 1 to 2 h. The absorbance profiles were

obtainedby scanning the gel on a Gilford model 2400S

recording spectrophotometer with linear transport

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with a slit (0.20by 2.36 mm)positioned between the

transport and the photomultiplier tube. This slit

allows for the center 35% of the gel to bemeasured,

which minimizes the effect oftheirregularities which

tend to occur onthe peripheral regions of the gel.

To determinewhether the amount of protein

stain-ing was proportional to concentration, several

dilu-tions ofbovine serumalbumin(Sigma, grade A) were

isopycnically

centrifuged in a5to 20%(wt/vol)sucrose

gradient (SW50.1 rotor, 149,000 x g for 20 h). These

gradients werephotopolymerized, stained with

Com-massie blue, and scanned as described above. The

integrated area of the stained bands plotted as a

function of protein concentration was found to be

linear.

Virusquantitationbylaser beatfrequency

spec-troscopy. Purified virus stock solutions were

quan-titated by laser beat frequency spectroscopy. We

have recently described thetheory and application of

this method to virus quantitation (14, 15).

RESULTS

The

gradient gel technique was initially

ap-plied

to preparations of purified virus which were quantitated

by laser

beat frequency spec-troscopy to determine the sensitivity of the system. Figure 1 shows typical absorbance

pro-files

of

Coomassie blue-stained

gradients gels

with purified

AMV and MuLV and identically

-treated material

from nonviremic animals. The

dual coordinates

on

the

abscissa

indicate

the

distance from the

top

of

the

gel (in

centimeters)

as

well

as thecorresponding sucrose

concentra-tion measured

in an

independent

liquid gradi-ent

before polymerization. A sketch of the

stained

gels

is

shown

at

the

top.

Only the

preparations from viremic

animals

contain

the

distinct stained

band

just

above the

1.16

den-sity region.

To

verify

that the integrated absorbance of

the viral

band

is

linear with virus concentration

and

to

estimate

the sensitivity

of

the

method,

we

prepared

a series of

gradient gels with

various

dilutions

of

purified virus stocks.

Figure

2

shows this

integrated

absorbance

at600 nm as a

function of the

particle

count.For

this

partic-ular

system,

the lower

limit of

detection

for

both

MuLV and AMV is

about

2 x

107 particles

per band. This staining method for detection

of

viral bands is

10

times

more

sensitive than

the scanning of the

same

gels for absorbance

at 260 nm

before staining.

The

system was

then tested

on

unpurified

plasma

preparations

(centrifuged

650 x g, 10 min to remove

cellular

debris)

from viremic

animals. The major

problems inherent with

such preparations

are

lower

virus concentra-tions

and the

presence of serumproteins.

Quan-titation

with the

gradient-gels requires that the

gradient-gels

separate

the

large

amount of

lower

density protein material from the relatively

dilute intact virus

particles. With the 0.8-ml

gradients,

one

is

generally limited

to

the

appli-cation

of a

maximum of

0.1

ml

of

sample

material. For preparations containing

less

than

108 particles/ml, this requires a more sensitive

staining

procedure. By modifying the amido

black staining procedure of

Wray and

Stubble-field (21)

so

that

it was

suitable

for

staining

gradient

gels, we

observed

viral bands with

applications

of

unpurified

virus-containing

plasma preparations.

Amido

black staining of

samples for which the virus concentration was

known

indicates that this

stain is more

sensitive

than

Coomassie blue. This

is

shown

in

Fig.

3

where the

plot

of

the

absorbance

area per

particle

count is

shown

for

AMV. The lower

limit

of

viral detection

is 6 x

106 particles with

this

amido

black procedure.

With this increased

sensitivity,

we were

able

to

quantitate virus

directly

from

plasma

of

leukemic chickens. Figure

4

shows

data for

applications

of 5

Al

of

plasma preparations from

normal and

leukemic chickens. As

we

have

seen

with

the

purified preparations, sharp

absorp-tion

profiles

are

found

in

the

1.16

density region

only

in

the virus-containing preparations. As

expected,

a

large increase

in

diffuse staining

is seenin

the lower

density regions

near

the

top of

the

gel.

DISCUSSION

We have demonstrated

that, with

as

few

as 2 x

107 particles,

the

gradient gel technique

can

be

used in conjunction with

Coomassie

blue

FIG. 1. Absorbance profiles (600 nm) of Coomassie blue R250-stained 0.8-ml gradient gels. (A) 20

Al

of

purified AMV ( )or 100

Ml

of normal chicken plasma(---)appliedto agradientgel. The peak0.5 cmfrom

the topofthenormalplasma-containing gel (density 1.08)iscausedbytheplasmaproteins.Thesharp peak1.6

cmfrom the top of theAMV-containing gel(density 1.15) is caused by the virus. The number of AMV particles

in the band is8.5 x 107asdeterminedbylaserbeatfrequencyspectroscopypriortoapplyingthe material to the

gradient. The peak 2.1 cmfrom the top of the AMVgradient gel is a 1.16density bead centrifuged toits

equilibrium position. (B) 100

jl

of MuLV ( ) ornormal mouseplasma (---) appliedtogel. The stained

materialextending from the topto0.9cminto the normalmouseplasma geliscausedbytheplasmaproteins.

Theband1.8 cmfromthetopof theMuLV-containing gelis causedby the virus. The number of virusparticles is 5.9 x 101. Sketches of the stained gels areshown at the top of thefigures. Theupper sketch is normal

plasma-containing geland the lower isvirus-containing gel.

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QUANT1TATION

OF RNA TUMORVIRUSES 549

PLASMA

E V "t DENSITY

-, BEAD

0

.200

E

U 0 0

CID

0 NORMAL

0 > ' ~MOUSE @

.100

PLASMA

TOP

l.Ocm,#3o

2.Ocm/40,

3.Ocm/50

-~~~

FIG. 1

VOL. 16,1975

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staining to measure concentrations of avian and

murine RNA

tumor viruses. With the more

sensitive amido

black staining procedure, the

gradient gel

technique can detect as few as 6 x

106 avian tumor

virus particles and equivalent

concentrations of murine RNA tumor

viruses

(data not shown).

We have observed that AMV

takes up twice as much stain as MuLV.

How-ever, for

both viruses the absorbance is linear

with respect to

concentration. The integrated

absorbances of viral peaks from duplicate

Coo-massie

blue-stained virus gels varied

by

about

15%, whereas for

amido black-stained gels they

varied

by less than 1%. This difference

appar-ently arises

because the amido

black-stained

gels can

be

completely destained without loss of

stain from

the viral

bands,

through use of the

modified staining

procedure, whereas the

Coo-massie

blue-stained

gels continuously

lose stain

from

the viral bands when destained with

ethanol-acetic

acid-water (30:5:65 volume

per-cent).

Despite

this

disadvantage, the

rapidity

with which this solution destains Coomassie

blue-stained gels

as

opposed

to

the time

re-.4V

r IL

4

.20

z S

4

vs

0

m

IL z

0

43._

I .40

:9

w

nr

2 4 6 a lo 12

PARTICLES (ixOe)

10 20 30

PARTICLES (xWVt)

FIG. 2. Plots of the areas under the 1.15 density viral peaks from a series of 0.8-mI gradient gels

containingvarious dilutionsofAMV and MuLV. The

gels were stained with Coomassie blue R250. Laser

beatfrequency spectroscopy wasused to quantitate

the numberorviralparticles.

quired to remove the stain

by diffusion in 7%

acetic

acid, or for

example in 12.5%

trichloro-acetic acid (3),

recommends it for such uses.

The use of

polymerizable gradient gels for

equilibrium centrifugation accomplishes three

purposes.

First, it enables the separation of

virus from nonvirus material on the

basis of

buoyant density and sedimentation

coefficients.

Using the tables formulated by McEwen (10) for

sedimentation of particles in sucrose

gradients,

we estimated the minimum

centrifugation time

(2.5

h) that would

be required for a particle in

the range of

600S to reach equilibrium. By using

this minimum centrifugation time, the virus

could

be

separated from components of similar

density

but lower sedimentation constant.

Sec-ond, it enables the concentration of the virus

into a narrow band. For example,

with the

4-mm

diameter gradient gel, the average height

of

the viral band is 1 mm, and thus the virus is

distributed

over

a

volume of 13

Al.

Thus, from

an

initial volume of 100

Al

applied to a

gradient

an

eightfold concentration is achieved. Third,

the

gradient gel (consisting of 7.5% acrylamide)

serves as a

support matrix to contain the virus

in

a concentrated band which enables

staining

and destaining without loss of virus.

The use of a 0.8-ml gradient gel limits

appli-cations of

material to

0.1 ml; however, larger

sample volumes can be added by choosing a

narrower range gradient of smaller

volume. An

alternative

is to

use a larger volume tube (i.e., 8

by

50 mm, 2-ml

tubes, Beckman no. 303369

6.0

4.0F

2.0

0

S._

p

._

4

49

D

49

o

0

In

m

20 40 60

PARTICLES (x10 )

FIG. 3. A plot of the areaunder the 1.15 to 1.16

density viralpeaks from a series of0.8-ml gradient

gels containingvariousdilutionsof AMVstainedwith

amido black. Laserbeatfrequencyspectroscopywas

usedtoquantitate the numberofvirusparticles.

A MuLV (Rauscher))

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OF RNA TUMOR

CHICKEN PLASMA

A VIREMIC B NORMAL

4

e

A B

lei

I

^

'Rdl 1

3 4

2

cm

FIG. 4. Absorbanceprofiles of 0.8-mi gradient gelstowhich 5

Al

of normal(---) orviremic(AMV) (-)

chickenplasmawasapplied. Gelswerestained withanamido blacktechniquemodified from that described by

Wray andStubblefield (21). The peakat2cmfrom thetopof the gel containing AMV is caused by 5x 107 virus

particles. The normal plasma (A) and virus-containing plasma (B) gelsareshownonthephotographat the

right.

with special adapters) for the gradient.

How-ever, the 0.8-ml gradient generally is more

convenient since itproducesa gel (4 by 45 nm)

which isideal forscanning.

Thegradient polymerization technique isnot limitedtosucrosegradients (4, 5). We also have

used unformed and

preformed polymerizable

CsCl gradients. Sarker and Moore (16) have shown that CsCl gradients can accentuate the

difference inbuoyant density betweenBandC forms of virus, and they suggest that CsCl gradients are better than sucrose gradientsfor purification of either Bor Cparticles. In

addi-tion, thedensity gradient technique enablesthe recognition of the other forms ofavirusthatare

sometimes found (e.g.,viralcores).

The stained gradient gels can serve as a

convenient and sensitive first-order screening method for the detection of knownorunknown

oncornaviruses. The sensitivity of the gradient gel technique described here is within anorder

ofmagnitude of themost sensitive form of the

reversetranscriptaseassay(i.e., that utilizinga

synthetic template). For a 1-h reaction using

thetemplate poly(rC) -oligo(dG) in avirus

opti-mized reverse transcriptase assay, we have

found that it is possible to detect 4 x 104

par-ticles/ml for AMV and 2 x 106 particles/ml

for MuLV (15). Thus, the sensitivity of virus detection with the gradient gel technique can

approach that of thereversetranscriptaseassay

while notbeing subject to the problems affect-ing the latter. Preliminary studies indicate thatfluorescence stains will increase the sensi-tivity of the gradient method by at least 100 timesoverthatobtained with absorption stains.

ACKNOWLEDGMENTS

This work was supported by National CancerInstitute

contractNO1-CP-33226, by Public Health ServicegrantsCA 13058 andCA 14060fromthe National CancerInstitute, and by an institutional grant from the United Foundation of GreaterDetroit.

1.2

.o

0.9

0.6 E

C

0 0

(D

LD

z

0

Cr)

0.3 X\

'I

1 _-_

VOL. 16,1975

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II!

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