Capsid proteins of simian virus 40

Vol. 40, No. 1, 1970

BIOCHEMICAL

CAPSID Marc

AND BIOPHYSICAL

PROTEINS

Girard,

Unite de Physiologie ques sur le Cancer,

OF SIMIAN

Louise

Marty

RESEARCH COMMUNICATIONS

VIRUS

40

and Frangoise

Suarez

des Virus, Institut de Recherches B. P. 8, 94 - Villejuif (France)

Scientifi-

Received May 25, 1970 Summary Purified SV40 virions were disrupted by sodium dodecyl sulfate (SDS), B-mercaptoethanol and heat, and the proteins analyzed in polyacrylamide gels in the presence of SDS. Six different polypeptide chains were found. Agregation of proteins was ruled out by treatment with 8 M urea. The major protein had a molecular weight (M. W. ) of approximately 45,000, two other proteins M. W. ‘s of 35,000 and 25,000 respectively. In addition, three minor components were found with M. W. ‘s of approximately 18, 000, 14, 000 and 10, 000.

Introduction Simian

Virus

40 (SV40) is a small

a DNA of approximately can carry

only

2, 5 x lo6 daltons

a relatively

As a prerequisite synthesized their

limited

the number

M. W. ‘s.

M.W.

amount

pH,

of six polypeptide

of information.

proteins

which

are

desirable

in the virion

and

polyacrylamide

in the presence chains

containing

(1, 3), which

it was considered

be shown below,

at neutral

the existence

cells,

of capsid

As will

electrophoresis

virus,

to the study of the proteins

in SV40 infected

to investigate

oncogenic

gel

of SDS, revealed

in the mature

virion.

Methods CVI

cells

the large

grown

plaque

0.1 pfu/cell.

strain Virus

in 2 liter

roller

flasks

of SV40 at an input and cells

were

were

multiplicity

a gift from

From

the 5th to the 7th day after their infection, 14 labeled with a mixture of C labeled amino-acids a 1’Energie

Atomique,

Saclay

- France).

97

infected

Labeled

P.

with

of Tournier.

the cells

were

(Commissariat virus

was first

Vol. 40, No. 1, 1970

purified NEB for

BIOCHEMICAL

by centrifugation

Spinco.

Fractions

(saturation 25,000

in 0.01

rev/min.,

22”,

with

mg/ml

chloracetic

acid

in the cold, phosphate

with

then purified

lo5

cpm/O.D.

Nuclear,

sucrose

gradient

previously

with

acetone,

of 5 % tri-

then resuspended Virions

were

and l/3

further

volume

of

by two

6000 in the cold

radioactivity

was

(2, 12),

of the purified

virus

was grown

of tritium

labeled

Mass. ).

centrifugation

amino

Virions

were

in the presence

Capsid

(6).

in HeLa

proteins

acids purified

cells

in

(New by

of 0. 5 % SDS as

were

then prepared

as

above.

was added

prior

again

After

to electrophoresis,

to the samples

then heated

8 cm long,

at 100” for

2.8 hours

l-2

solution

gels made

as previously

layered

according

at 90 volts,

were

These

to be analyzed.

They were

min.

electrophoresis slices

1 % a-mercaptoethanol

of proteins

7. 5 % polyacrylamide

and 1 mm thick

and Bray’s

to Maize1

the gels were

cut and counted

described

over

in NH40H

(5).

and Discussion

Six polypeptide SV40 virions A,

twice

at

virus

in the presence

glycol

of poliovirus

Boston,

Immediately

Results

M EDTA

Purified

pH 7. 4.

15 ml

260

strain

described

over

SV40 was concentrated

Specific

of a mixture

England

frozen,

buffer,

5 % polyethylene

The Mahoney the presence

with

of the

4. 5 hours

of 1 % 6-mercaptoethanol

as above.

was usually

for

washed

twice

rotor

layered

rotor.

acid

albumine,

In one experiment,

precipitations

described

and centrifuged

in

0. 02 M EDTA)

pH 7. 5, 0.01

in the SW 25.1

by addition

10 % SDS.

(8).

M Tris-HCl

serum

in 0. 01 M sodium disrupted

peak were

5 % trichloracetic

bovine

gradients

4”, in the SW 25.1

the virus

was done at 4”),

precipitated

were

rev/min,

from

KBr

5 to 20 % sucrose

pH 7. 5, 0. 01 M NaCl,

at 25,000

of saturated

0.6

through

(0. 01 M Tris-HCl 90 min.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Fig.

B and C in order

D, E and F.

chains

were

repeatedly

The three

1).

major

detected components

of decreasing

M. W.,

material

heavier

In addition,

98

the minor

in purified were

labeled

components

than component

A

BIOCHEMICAL

Vol. 40, No. 1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C.P.M.

-2000

.lOOO

Fig. 1 : Gel electrophoresis pattern of SV40 capsid proteins. SV40 capsid proteins labeled with ‘“C amino-acids were prepared as described under Methods and analyzed by electrophoresis in a 7. 5 ‘$0 polyacrylamide gel in 0.1 M sodium phosphate buffer, is from left to right. pH 7. 4, 0.1 % SDS. Migration was also observed varied

from

(first

experiment

peak to the left, to experiment.

was found in the presence

it very

represented

ment

shown in Fig.

undissociated

It was however

experiments

(see Fig.

were

not actual

trypsin

and sonication

as well

as repeated

of 8 M urea agregates.

quite

distinct

from

no

(see below), In the experi-

to the light

side

peak A in most

2).

the possibility part

in addition,

1, peak B ran as a shoulder

of peak A.

To exclude

1) but its amount

Since,

such material likely

Fig.

that

some of the proteins

of SV40 virions, for various cycles

treatment

lengths

of purification 99

observed

of the virus

of time

were

through

sucrose

with

performed, gradient

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

u =

VP1

200

250

100

20

50

40

Fractions

Fig. 2 : Determination of the molecular weights of *SV40 major SV40 capsid proteins labeled with “‘C aminocapsid proteins. acids were mixed with tritium labeled poliovirus capsid proteins and analyzed by gel electrophoresis as in Fig. 1. centrifugation samples

and band sedimentation

of SV40 virions

cenfrifugation

in CsCl.

were

out eventual

SV40 capsid at loo”, this gel.

agregation

Electrophoresis agregate

of proteins,

respect

their

isopycnic

had any effect

a sample

to urea.

buffer

also

as in Fig.

contained

1 were

Electrophoresis

8 M urea.

observed

of

was heated

in a 5 % polyacrylamide-8 again,

of

M urea The same but the

had disappeared.

The M. W. Is of the SV40 proteins ring

through

in SDS and 6-mercaptoethanol

was performed proteins

purified

Also,

pattern.

then made 8 M with

sample

six capsid heavy

proteins

further

KBr.

None of these treatments

on the gel electrophoresis To rule

in saturated

electrophoretic

mobility

100

were

to that

determined of poliovirus

by compacapsid

Vol. 40, No. 1, 1970

proteins, mide

BIOCHEMICAL

since

gels,

M. W.

it is known

proteins

poliovirus

proteins proteins

and VP3 having ly (9),

were (Fig.

determined.

There

2).

28,000

correct

too few counts

determination

capsid

components

patterns

obtained

in other

M. W. ‘s determined

14,000

However,

experiments

A,

D, E and F were

respective-

respectively. to allow

for

minor

SV40

on the basis

(see Fig.

VP2

B and C were

of the three

above for components

M. W. ‘s of components

A,

labeled

VPl,

and 23,000

experiments

of the M.W.‘s

poliovirus

of 14C labeled

and 23-25,000

in these

D, E and F.

major

proteins

components

32-35,000

to log of

with tritium

The poliovirus

M. W. ‘s of 35,000,

as 45-50,000,

were

related

Samples

co-electrophorized

the M.W. Is of SV40 capsid

determined

l),

of

and of the

B and C, the

evaluated

to 18,000,

and 10, 000 respectively.

In six repeated A amounted

experiments,

each proteins

minor

Work

of polypeptide

A,

major

of 45,000

protein

(7).

and Koch

(lo),

agreement

were

in the

recovered

their

results

between

for SV40 (see Fig.

to determine

of the capsid

DNA.

It is clear

of the SV40 capsid

of which

in

M. W. has also been found Anderer

respectively. our results

wether

already, is constituted 45,000. for

A

the capsid

et al (1) and Schlumberger,

on another

and ours

D,

or internal

is approximately

hand,

the M. W. ‘s of which

and 16,400

between

sub-unit

the M. W.

virus

in SV40 virions,

are part

with the virus

that the major

Anderer

recovered

is in progress

components

associated

of polyoma

radioactivity

of protein

B and C, and 3 70, 1. 5 % and 1 % in proteins

three

however,

radioactivity

11. 5 % of the counts

E and F respectively.

proteins

average

to 72. 5 % of the total

Approximately

gels.

16,800

is inversely

pH polyacryla-

the M. W. ‘s of the three

have been accurately

SV40 capsid

these

mobility

and since

RESEARCH COMMUNICATIONS

that in the SDS-neutral

electrophoretic

(4, ll),

AND BIOPHYSICAL

described

were

estimated

The reasons are unknown. and the pattern

2 in reference

8).

101

three

for

proteins

at 16,900,

the discrepancy

There

is good

shown by Maize1

Vol. 40, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Acknowledgments This investigation a 1’Energie Atomique Recherche Scientifique

was supported in part by the Commissariat and in part by the Delegation G&x%-ale a la et Technique.

References 1. Anderer, F. A., M.D. Schlumberger, M.A. and H. J. Eggers, Virology -’32 511 (1967). s2. Calothy, G. , M. Digeon Paris 265, 1764 (1967). 3. Crawford,

L. V.

4. Dunker, (1969).

A. K.

5. Girard, (1969).

M.

6. Granboulan, 7. Kass,

-J.

and M.

and P.H.

Raynaud,

Black,

Marty,

N. and M. Virology

C. R.Acad. ----

SC.,

Sot.

Chim.

J. Virol. --

Biol.

51, 31

A, 475 (1969).

-5, 381 (1970).

8.

Maizel, J. V., In : Fundamental Techniques in Virology, K. Habel and NF Salzman ed, Acad. Press(1969).

9.

Maizel,

J. V.

Frank

-24, 388 (1964). J. Biol. Chem. 244, 5074 ----

Bull. -----

Girard,

H.H.

Virology

and R. R. Rueckert,

and L.

Koch,

and D. F.

Summers,

Virology

-’36 48 (1968). and M.A. Koch, Virology

10. Schlumberger, H. D., F.A. Anderer 36 42 (1968). -’ 11. Shapiro, A. L. , E. Vinuela and J. V. Maizel, Biophys. Res. Commun. -28, 815 (1967).

12. Yamamoto, K. R., B. M. Alberts, R. Benziger, and G. Treiber, Virology 40, 734 (1970). -

102

Biochem. L.

Lawthorne