Translation of mengovirus RNA in ehrlich ascites cell extracts

Vol. 57, No. 4, 1974

BIOCHEMICAL

TRANSLATION

OF MENGOVIRUS RNA IN EHRLICH ASCITES Bo F. 8berg

Department

of Microbiology, Roche Institute

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February

and Aaron

J. Shatkin

The Wallenberg of Molecular

CELL EXTRACTS

Laboratory,

Biology,

Uppsala,

Nutley,

N.J.

Sweden

and

07110

14,1974

SlJlmARY Mengovirus RNA was translated in Ehrlich ascites cell e itacts using as radioactive precursors f[35S]Met tRNAFMet and [35S]Met tRNAMEf to label the products in N-terminal and internal positions, respectively. Tryptic peptides were compared with those derived from purified [35S]Met-labeled mengovirus. The results indicate that the sequences corresponding to the viral coat polypeptides are preceded by a short "lead-in" peptide which is probably removed by a cleavage process in infected cells. INTRODUCTION The synthesis "lead-in"

peptide

a final that

of some proteins

product is

(l-3). in the

processed

not

precursor

virus

polyprotein

In this

MATERIALS

the

labeled

cleavage

It

(l-3).

where is

not

from post-translational the

we report

lead-in

another

of a lead-in

sequence

is

a short

to yield

either

or a polypeptide the

case of mechanism

the synthesis clear

if

the

processing

translation

is of the cleavage

of picorna-

has some special , the

example sequence

(l), in

with

off

The cleavage

systems

tRNAs were

was determined

by paper

by unformylated mengovirus,

prepared

and purified electrophoresis

Met tRNAFMet

mengovirus

was less

function

(1).

of mengovirus

indicated.

@j I Y 74 0~ Academic Press, Inc. of rcproductim in my form re.wrved.

as described (7). than

earlier

(2),

The contamination 3%.

RNA and SlO or S30 extracts

1186 Col)yri,ght All rights

cleaved chain

(2,3).

observed

starts

AND METHODS

[35S]Met

tRNAFMet

or if

cleavage

light

translating

different

(4-6)

communication

RNA, where

purity

is

apparently

subsequently

proteins

has been

peptide

is

by post-translational

in cell-free

polypeptide

of the short

peptide

(EMC) virus

active

eukaryotes

case of immunoglobulin

further

encephalomyocarditis probably

This

in

Preparation from

Ehrlich

and their of fMet

of

[35S]Metascites

Vol. 57, No. 4, 1974

cells

have been

BIOCHEMICAL

described

(8).

After

dissolved

in 1 ml of starting

pyridine,

15 ml of N-ethylmorpholine,

pH 9.4.

After

the

column

of AGI-X2

sample

30 ml/hr)

was performed

gradients

of the

adjusted

plus

with

acetic

acid,

Other

liter:

and acetic

Elution

at 35" C (flow

b = 150 ml buffer c = 150 ml buffer

a plus

to

to a 1 x 60 cm rate

consecutively

=

by linear 150 ml buffer

a at pH 6.4 plus

d = 150 ml 1 M pyridine-acetic

a

150 ml

acid,

pH 5.1

pH 5.1 and e = 150 ml of 2 M pyridineacid.

Fractions

scintillation described

acid

mesh resin.

a, followed

were

10 ml of

was applied

150 ml acetic

have been

per

the peptides

to pH 10, it

acid

in Triton-toluene

methods

includes

(8),

adjusted

acid;

pH 5.1;

pH 5.1 plus

and counted

a, which

75 ml buffer

150 ml 2 M pyridine-acetic acid,

digestion

composition:

to pH 6.4 with

acetic

trypsin

20 ml of a-picoline,

200-400

following

1 M pyridine-acetic

buffer

had been

(Biorad)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 3-3.5

mixture.

earlier

Recovery

ml were

collected

was better

than

85%.

(2).

RESULTS AND DISCUSSION As has been mengovirus

RNA starts

labeled

tryptic

elution

position

change

virus

is

peak is RNA is

tryptic

earlier,

when

in the

tRNA FMet ' eluting

virus

labeled

The amount

with

of

yields sequences (9),

the

of

peptide (2)

[35S]Met

in Fig.

2.

the It

that

be noticed

at about

fraction

220 at 1.3 M pyridine-acetic

in the

the peak

tryptic

for

some of the

pattern less tryptic

than

shown

proteins in Fig.

1187

found

3.

coding

obtained

is,

295 (Table

50% of the peptides

that

peptides

at fraction

[35S]methionine-labeled

with

label

labeled

coincides

label

the

ex-

of mengo-

resulting

should

The

an anion

translation

tRNAMMet,

fMet-

(2).

from

reveals

When the --in vitro

as shown

separation

account

tRNAFMet

tRNAMMet

and 11% in digest

1).

tryptic

and

to one predominant

[35S]Met

of terminal

fraction

genome

(Table

presence

f[35S]Met

protein

1.

rise f[35S]Met

Edman degradation

peak

mengovirus

is

shown

in Fig.

of EMC, mouse Elberfeld

giving

radioisotope

can be separated

The tryptic

site

N-terminal

done

this

--in vitro

of the mengovirus

the major

13% in

the

91% terminal

peptides

pH 5.1.

translation

at one initiation

peptide

column

in this

shown

that with acid,

however,

only

1).

from purified Since

virion

capacity

in the --in vitro

coat

of the picornaproducts

are

Vol. 57, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CPM 500400 300 200 -

k-a-b-c 100 FRACTION

Figure

1.

x;

500 400 300 200 100 . l-a-b-

=----%--

FRACTION

absent peptides Figs.

2.

from

the trypsin

is

translated

preponderance

digest

at lower

2 and 3).

of virions.

pH, i.e.

The virion

coat

by Edman degradation

Premature

dTe-

NUMBER

Anion exchange chromatography of tryptic labeled mengovirus proteins synthesized earlier for EMC virus proteins (2).

eluting

methionine

300

NUMBER

Anion exchange chromatography of tryptic digest of f[35S]Metlabeled mengovirus proteins synthesized in vitro. The method has been described earlier (2).

CPM

Figure

d-e-

200

termination in

This

fractions tryptic (Table

cell-free

of capsid

peptides,

about peptides

of [35S]Metas described

especially 210, contain

pronounced

220,

for

the

and 300 (compare

11% N-terminal

1).

of polypeptide

ascites

is

digest --in vitro

products

extracts in agreement

1188

takes

(2,3,8,10,11). with

place

when EXC virus

This

the EMC virus

results genetic

in a map

RNA

Vol. 57, No. 4, 1974

Table

1.

Edman degradation

Sample

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of tryptic

peptides

Terminal counts/min

;;y2;~~t2;;;~~~s labeled

BIOCHEMICAL

%

Internal counts/min

%

from 5634

10.8

46517

89.2

1636

91

155

9

66

13

452

87

75

11

631

89

56

8

680

92

mengovirus

Fraction Fig. 1

210-230

Fraction Fig. 2

205-230

Fraction Fig. 2

280-310

Fraction Fig. 3

185-200

in in in in

Lyophilized samples from fractions in Fig. l-3 and unfractionated tryptic digest of [35S]methionine-labeled mengovirus were subjected to Edman degradation and analyzed as described earlier (2).

CPM

1500-

1000-

500 -

looh-a-,,

100

c----%rFRACTION

Figure

3.

d---+T%-e--

NUMBER

Anion exchange chromatography labeled mengovirus.

1189

of tryptic

digest

of [35S]Met-

BIOCHEMICAL

Vol. 57, No. 4, 1974

which

places

the

The predominant dicates

they

RNA.

(Fig.

little

tryptic

also

(2),

from

cleavage

in mature

short

distance

previously peptide

be very It

during

likely

that

2, apart

from

the major the

for

tryptic

should

it

identical

the

with

from

labeled

with

the

f[35S]

products

containing

peptide

virions

obtained

from peptides

precedes

that

the N terminus

3).

coat

synthesized of about

2000

ENA, and perhaps

--in vitro

products

(Fig.

result

indicates

that

This

of the structural

proteins

sequences

at a

protein

of the viral

protein weight

(Fig.

the completion

and suggests

EMC virus

the mengovirus

polyprotein.

start This

was shown

--in vitro

where

the short

(2,

Thus,

the

other

3).

picornavirus

"lead-in"

translation

genomes,

of

appears

similar. has been

moved in viva be involved

reported

that

immunoglobulin

at the N terminus

a peptide

by cleavage

It

in intracellular of a short

sequences

lead-in

in picornavirus

(1).

chain

of about

was suggested

transport peptide

light

1500 daltons that

the

of immunoglobulin between

proteins

remains

Milstein, C., Brownlee, G.G., Nature 2, 117 (1972) fiberg, B.F. and Shatkin, A.J. Smith, A.E. Eur. J. Biochem.

Harrison,

the

synthesized

initiation

--in vitro

which

lead-in (1).

is

sequence However,

site

re-

and the

coat

to be elucidated.

2. 3.

T.M.

and Mathews,

Proc. Nat. Acad. 33, 301 (1973)

1190

Sci.

M.B.

USA69,

3589

may

the

EEFEEENCES 1.

the

methionine.

peptide

terminated

f[35S]

contribute

formylated

peptide

of the

with

which is

2) in-

5' terminus

labeling

tryptic

prematurely

genome (9).

(Fig.

to the

methionine

is

Fig.

with

tryptic

and EMC virus

contains

function

It

--in vitro

close

obtained

a formylated

expected

the virion

has a molecular

mengovirus

peptide,

coincided

peptides

by sequences

RNA translation,

peptide

from

for

the 5' end of the viral

of methionine.

of a short

found

for

222 in

a result

The N-terminal absent

near capsid

position.

at fraction

an even distribution

also

contains

elution

products

methionine

coded tryptic

case of EMC virus

--in vitro

is

also

l),

to its peptide

In the

are

sequences

of mengovirus

The N-terminal

Met tHNAFMet very

protein

synthesis

that

viral

coat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(1972)

to

1)

Vol. 57, No. 4, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4. 5. 6. 7.

Summers, D.F. and Maizel, J.V. Proc. Nat. Acad. Sci. USA 59, 966 (1968) Jacobson, M.F. and Baltimore, D. Proc. Nat. Acad. Sci. USA 6l, 77 (1968) Korant, B.D. J. Virol. l2, 556 (1973) Drews, J., Gresmuk, H. and Weil R. Eur. J. Biochem. 2, 416 (1972) a. Eggen, K.L. and Shatkin, A.J. J. Virol. 2, 636 (1972) 9. Butterworth, B.E., Hall, L., Stoltzfus, C.M. and Rueckert, R.R. Proc. Nat. Acad. Sci. USA 6&, 3083 (1971) 10. Boime, I. and Leder, P. Arch. Biochem. Biophys. 153, 706 (1972) 11. Kerr, I.M., Brown, R.E. and Tovell, D.R. J. Virol. I&, 73 (1972)

1191