Vol. 65, No. 4, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

PURIFICATION OF POLYOMA D. PAULIN(+),

T

ANTIGEN FROM TRANSFORMED CELLS

P. GAUDRAY(~), and F. CUZIN(~)

D6partement de Biologie mol6culaire, Institut Pasteur, 75015 Paris(+), and Centre de Biochimie, Universit6 de Nice, 06034 Nice, France(%)

Received Julv 9,1975 SUMMARY. The T (tumor) antigen from transformed mouse cells hat been purified 400-fold and prominent polypeptides of molecular weights 85-90,000 and 70-75,000 resolved. Polypeptides exhibiting same molecular weights are adsorbed specifically on insolubilized anti-T antibodies. Study of temperature sensitive early mutants of polyoma and SV 40 viruses has led to the conclusion that the protein product of an early viral gene (ts-a gene of polyoma, A gene of SV 40) plays a critical role in the initiation of autonomous viral DNA replication cess

(3,4).

(1,2), as well as in the transformation pro-

It seems very likely that the protein product of

this gene carries the antigenic site for the virus specific intranuelear T antigen

(5).

As a preliminary step toward the ana-

lysis of the function of polyoma virus ts-a at the biochemical level, we devised a purification procedure for T antigen, using complement fixation

(CF) as an assay. MATERIAL AND METHODS

Polyoma transformed PY6 cells, derived from Swiss mouse 3T3 fibroblasts were obtained from Dr.T.Benjamin. transformed mouse cells

Spontaneously

(3T6) were u s e & as a control. Conditions

for cell growth and the procedure for radioactive labeling were described previously

(6).

T antigen was assayed according to

Sever (7). Immunoadsorbants.

Immunoadsorbants were prepared by the

Cuatrecasas method

(8).

Copyright©19~ ~ Aca~mwPress,~c. AHngh~ ~repro~c~onma~rmrese~ed.

At least 80 to 90% of the proteins were 1418

Vol. 65, No. 4, 1975

found

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

coupled

to the

Sepharose

Gel e l e c t r o p h o r e s i s . Studier

(9), using

The analysis 0.1%

sodium

lyacrylamide

for r u n n i n g

Purification

of T antigen.

and r e s u s p e n d e d

same buffer. homogenized

The

gels

for

PY6 and

a specific trations

homogenizer

radioactivity

not d e t e c t a b l e

and

was

sulfate 40%

was

discarded.

about

40 mg/ml

Mann)

was

discarded.

up to a final

units

contained

with concen-

and were

no d e t e c t a b l e

The p r e c i p i t a t e

was

collected,

sulfate

solution,

pH 7.4,

and d i a l y z e d

DE 52) column

containing

against

the

of T antigen)

gradient

containing

sulfate

fraction

was a p p l i e d

of NaCI

0.05

to the

the a d s o r b e d

concentration

1419

sulfate of

20%

CF a c t i v i t y

to 40% a m m o n i u m

dissolved

(1.2 x 20 em) was

pH 7.4,

ammonium

Ammonium

adjusted

buffer,

buffer,

:

was

50 ml of the same buffer, linear

T antigen

concentration

w~shed

with

a

in 1 ml of

0.05 M NaCI

and 10%

same buffer.

~ = ~ 9 ~ _ ~ 9 ~ 9 ~ g D ~ Y _ ~ D _ ~

The d i a l y z e d

twice

of protein,

supernatant

ammonium

glycerol,

phosphate

times,

sonicated

in PY6 extracts

D 2)

added

The

saturation.

(Whatman

three

Supernatants

Polyoma

from 128 to 256 CF units

0.01 M p h o s p h a t e (w/v)

in the

at 100 W, and c e n t r i f u g e d

of 106 cpm/mg.

The p r e c i p i t a t e

saturated

of 108 cells/ml

in 3T6.

Grade,

saturation.

15% po-

saline

(10 strokes),

A m~_~Di~_~if~_p~i~2~_~ (Enzyme

with

of

in Tris

and thawed

apparatus

contained

(SDS),

were w a s h e d

frozen

The pellet

3T6 cells

ranged

Cells

was

Sonifier

g.

the p r o c e d u r e

(10 cm long).

at a c o n c e n t r a t i o n

in a Potter

30 m at 30,000

from

:

suspension

for 15 s in a Branson

followed

dodecylsulfate

slab

Ce~_ex~ra~s_~s~e~_~ buffer,

beads.

: a DEAE-cellulose equilibrated M NaCI

and

with

0.01 M

10% glycerol.

(4 mg p r o t e i n

and 256 CF

column.

washing

material (0.05

After was

with

eluted w i t h

to 0.6 M)

a

in 100 ml of

Vol. 65, No. 4, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.01 M p h o s p h a t e the T antigen NaCI,

buffer,

activity

pH 7.4,

was

eluted

and II at 0.20 M NaCI.

justed

to 0.15 M NaCI,

of T antigen fractions

were

~

!

selective sheep

was

~

~

extensively

sample

were

~

of contact

!

~

_

~

purified

_

was

~

!

_

pH 7.4,

~

_

prepared

and coupled

I or II, a d j u s t e d

1,

i at 0.12 M

the peaks

~

were

ad-

The ratio

I.

The two

2

to 0.15

T antigen

was r e c o v e r e d

unadsorbed

protein

of the flow through

(6).

4 B.

The

A 2 ml

was

antibodies.

with

from a

protein

M NaCI,

applied

After

1 h

2 ml of 0.01 M phos-

10% glycerol

Polyoma

: a

antibodies

cellular

was w a s h e d

remained

_

using

coupled

0.15 M NaCI,

~

to Sepharose

0.2 ml/m.

protein

under

in Fig.

by CF for T antigen.

with mouse

at 4 ° , the column

of the total

peaks

to 1 in f r a c t i o n

1 x 3 cm of Sepharose

phate buffer,

As shown

separately.

immunized

of fraction

to a c o l u m n

Fractions

and assayed

immunoadsorbant

antibodies

in two main

2 in f r a c t i o n _ l l

pooled _

10% glycerol.

at a flow rate

quantitatively

and wash,

while

of

in the

80 to 90%

on the column.

RESULTS Puri~cation Table

of two d i s t i n c t

1, m a t e r i a l

was p u r i f i e d

with

correspond

to d i f f e r e n t

to one antigen to a viral equilibrium antigen same

antigenic

chromatography

between

seems

were

likely,

of isolated

obtained

separated (Fig.

recognized

complexes

in d i f f e r e n t

the most

conditions

species

antigens

different

As shown

in

as in step 4

with a 25 to 50% recovery.

on D E A E - c e l l u l o s e

in d i f f e r e n t

peptide

species.

T immunospecificity

300 to 400 fold,

Two distinct exchange

antisenic

with

1).

by ionThey might

by the immune cellular

aggregates.

components,

The h y p o t h e s i s

molecular

configurations

because

rechromatography

form

I or II also

1420

serum,

generates

or

of an

of a unique under

the

a bimodal

Vol. 65, No. 4, 1 9 7 . 5

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

O -H

~J 0 O~ •H O

I

O

o-~

O

~.o

O i.o oq

co

P~

bO

-.-I-

q~ . H - H -P .H>CO O H D D

co

~

oo

co c,q

0 oo

x-I

O O co

c~

D. O P ~ co ~ J O ~ "-" 0

LO D

I

O O

LO Oq

aO C~

O ~, O 0.)

(D b0 .~1 4~ r6 E~

,--4 - H 6 O

CO

co

~-4

LO

J

J

H

H H

co

(.D

O

~D t~ -H -p rd

-.d-

0 0 ~ 0

P~

_dO

O I° O

O

E~ q-4 O

!

O .H rd O -H q~ .H ,q P-,

n3

O .H

,-~ ,-i (9

I o

O

~

o

4~

o

¢) ~ [>u~ ,-4 r-I

~ ,-4 r-I

ml ~ 14H

,-H ,-4

O .,-I -tJ CJ nJ

I--t H

I--I ,--t I r6~0 •H ~

~ O

rd •H

Oq-t

~l I 6"0 . H mi

O

n3

nJ .,-40q~

0 ~ 4

P~

(D

~

~

I

~

I

J

43 l ~ - 4 0

P~

oq

co

4~ cO

1421

~g44~

nJ ~u~

O

Vol. 65, No. 4, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

m

I

I

z I 0.60.

/

16

s*f /s* 1 , ' /

V

o

7~ =,

//¢ t

j"

0.40-

j"

82

j' 0.20. ,/

4 ~

i

O.05J

40 6O FRACTION NUMBER

20

80

I00

Figure 1. C h r o m a t o g r a p h y on DEAE-cellu!ose. Fractions were collected and analyzed for CF activity and 14C r a d i o a c t i v i t y on 25 pl aliquots.

elution profile with peaks c o r r e s p o n d i n g trations

to the same NaCI concen-

(0.12 and 0.20 M).

Characterization

of purified

sition from fractions

polypeptides.

The p o l y p e p t i d e

compo-

I and II was analysed by S D S - p o l y a c r y l a m i d e

gel electrophoresis. As shown in Fig. contained fraction

2, the material

8 main peptides

derived from fraction

with low background,

I was more heterogeneous.

II

while that from

The m o l e c u l a r weights

corres-

ponding to the 4 main bands of I (A to D) and to the 5 bands of II (a to e) are indicated

in Fig.

2.

Similar values

are found for

w

peptides ~ (fraction B~

I) and ~ (fraction

II)

(85-86,000)

and for

b (72-74,000).

Binding of T antigenic labeled protein

material

to i n s o l u b i l i z e d

containing T antigenic

step 2 was adsorbed on i n s o l u b i l i z e d to Del Villano and Defendi

(10).

14

C

activity at p u r i f i c a t i o n

anti-T antibodies

No T antigen

1422

antibodies.

according

could be detected

Vol. 65, No. 4, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

BSA

BSA

OV

BLG

dimer B

I

I

I

t

I

I

I

a

I

0

|

I

2

86 72 68 62 52 I

~

I

4

MIGRATION

I

I

5

i

6

7

IN CM

Figure 2. Gel e l e c t r o p h o r e s i s in SDS of p u r i f i e d fractions (step 4) derived from fractions i (top) and II (bottom) of Fig. 1. 10,000 cpm of labeled p r o t e i n o b t a i n e d after immunoa d s o r p t i o n of host p r o t e i n w e r e a n a l y z e d and the autoradiograms scanned w i t h a densitometer. M a r k e r proteins are d e n o t e d by BSA and BSA dimer : b o v i n e serum a l b u m i n e (MW : 68,000), 0V : o v a l b u m i n e (MW : 43,000), BLG : 6 - 1 a c t o g l o b u l i n e (MW : 18,000).

in the first

supernatant

quantitatively material 0.05%,

was

bound eluted

dissolved

ammonium

to the with

respectively,

or in the wash,

suggesting

immunoadsorbant.

0.2 N H C l - g l y c i n e ,

of the r a d i o a c t i v i t y sulfate

tracts.

Two main bands

phoresis

w i t h MW 7 0 - 7 5 , 0 0 0

precipitates

are r e s o l v e d and

that

The bound pH 2.2

About

(Fig.

0.3 and f r o m re-

3T6 cell

in p o l y a c r y l a m i d e

80-90,000

antibody

were r e c o v e r e d

of PY6 and

it was

gel

exelectro

3).

DISCUSSION Polyoma dure using

virus

T antigen

complement

fixation

was p u r i f i e d (CF)

1423

in a f o u r - s t e p

as an assay,

proce-

and by direct

ad-

VoI. 65, No. 4, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DIMER

FiGure 3. Gel electrophoresis in SDS of material eluted at pH 2.2 from Sepharose-coupled anti-T immunoglobulin. Marker proteins are the same as in Fig. 2. r

sorption on insolubilized antibodies.

The purified fractions have

in common two polypeptides with molecular weights in the ranges 70-75,000 and 85-90,000. tes

These values agree with previous estima-

(10,12) of the size of the early viral peptide of polyoma and

SV 40 viruses. During a lytic infection, the early viral messenger can eode for 90-100,000 daltons of protein.

RNA complementary to the early

viral region in PY6 cells, however, only corresponds to 70-75,000 daltons of protein.

Additional

sequences are transcribed from the

1424

Vol. 65, No. 4, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E strand in the late region communication). not known.

(antilate)

(13 and R. Kamen, personal

The size of the viral messenger in these cells is

Nor it is known if viral and cellular sequences are

transcribed into a single mRNA molecule. Two hypothes~s can

be advanced

: (i) the 70,000 MW peptide

constitutes the only viral gene product.

The 90,000 MW peptide

could then be a cellular component associated with the viral protein.

(ii) A mRNA corresponding to 70 to 80% of the early viral

region is translated into the 90,000 MW peptide, which is, in turn, partially converted into a smaller antigenic component.

Finger-

prin~ analysis of the peptides is in progress to decide between these hypotheses.

Acknowledgements.

The technical assistance of C. Maczuka, N. Montreau and J. Perreau is gratefully acknowledged. This work was made possible by grants of the Centre National de la Recherche Scientifique, the Commissariat l'Energie Atomique, and the Institut National de la Sant~ et de la Recherche M~dicale (ATP 18 Contracts to F.C. and to G. Meyer). For one of us (P.G.), this work was performed in partial fullfilment of the requirements for the Doctorat degree at the Universities of Paris and Nice.

REFERENCES 1. Tegtemeyer,

P.

(1972)

J.Virol., 10, 591-598.

2. Francke, B., and Eckhart, W. 3. Osborn, M., and Weber, K. 4. Tegtmeyer,

P. (1975)

(1973)

(1975)

7. Sever, J.L.

(1962)

J.Virol., 15,

636-644.

J.Virol., 15, 613-618.

S. Paulin, D., and Cuzin, F. (1975) 6. Pauiin, D., Perreau, 699-705.

Virology, 55, 127-135.

J.Virol., 15,

J., and Cuzin, F. (1974) J.Immunol., 88,

1425

320-329.

393-397. J.Virol., 13,

Vol. 65, No. 4, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. Cuatrecasas, P. (1970) 9. Studier, F.W.

(1973)

J.Biol. Chem., 245, 3059-3065. J.Mol. Biol., 79, 237-248.

10. Del Villano, B.C., and Defendi, V. (1973) 34-46. 11. Tegtmeyer, P. (1974) 39, 9-15.

Virology, 51,

Cold Spring Harbor Symp. Quant. Biol.,

12. Carroll, R.B., Hager, L., and Dulbecco, R. (1974) Acad. Sei. USA, 71, 3754-3757.

Proc. Nat°

13. Kamen, R., Lindstrom, D.H., Shure, H., and Old, R.W. (1974) Cold Spring Harbor Symp. Quant. Biol., 39, 187-198.

1426