Identification of viral ribonucleoproteins in the cytoplasm of murine cells chronically infected by a retrovirus

BIOCHEMICAL

Vol. 86, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

February 28, 1979

1110-1117

IDENTIFICATION OF VIRAL RIBONUCLEOPROTEINS IN THE CYTOPLASM OF MRINE CELLS CHRONICALLY INFECTED BY A RETROVIRUS E. Poitevin,

M.A.

D. Mathieu-Mahul

Auger-Buendia,

Laboratoire de Pharmacologic Experimentale, Saint-Louis, 2 place du Dr.A. Fournier, Received

November

and C.J.

Larsen

Centre Hayem, H8pital 75475 Paris Cddex 10

21,1978

SUMMARY Ribonucleoproteins were isolated from the cytoplasm of FriendEveline cells which produce the Friend virus complex, after a short labelling withc3H] uridine. These particles moved with a sedimentation coefficient of 53s in sucrose gradient and had a buoyant density of 1.46 g/cm3 in CsCl gradient. Analysis of their RNA content showed that they possessed a 35s major species having the size of the viral genome subunit. Moreover, a positive hybridization was observed when RNA of the 53s particles was annealed with viral complementary DNA. No such particles were found in cultures of uninfected murine cells suggesting that 53s RNPs have a viral origin. INTRODUCTION The multiplication synthesis which

by reverse

gets

integrated

RNA molecules POL genes 6,7,8).

35s RNA, which products,

polysomes mRNAs are plexes.

and 20s RNA which that

associated

state,

both

with

while

behave

ribonucleoproteins the relationship

called

similarly

to

if

by A.S.

The function

then

transcribed

into

(1,2,3,4)

code only

for

and cellular not

: GAG and

during

the

cytoplasmic

of ribonucleoprotein

com-

Spirin

under

to polyribosomes of informosomes of RNPs are

cells,

(5,

mRNAs on

identically

In eukaryotic

informosomes

the two types

the

the ENV gene information

the form

associated

involves

the provirus,

is

of viral

under

are

(11,12). between

It

expresses

synthesis.

proteins

others

cells

in the cytoplasm

the coexistence

in the protein

Some of them,

a free

genome.

characterized

has been demonstrated

cells,

suggests

involved

in mammalian

of a DNA intermediate,

the cellular

have been

In infected

events

versy

into

which

genome-sized

of retroviruses

transcriptase

still

(9,

10) exist as messenger

and the nature a matter

of

of contro-

(13,14,15,16,17).

We were interested in the characterization of cytoplasmic ribonucleoproteins which have a viral specificity. In this work, when virus produ1 cing cells were incubated for a short time with I 3Hjuridine, a free 53s RNP population its

could

size

virus

and its

be isolated, capacity

which

contained

to hybridize

genome.

0006-291X/79/041110-08$01.00/0 Copyrighr All rights

@I 1979

by Academic Press, Inc. in any form reserved.

of reproduction

viral

RNA species

with PI H DNA complementary

1110

as judged

by

to the Friend

Vol.

86,

No.

BIOCHEMICAL

4, 1979

AND

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RESEARCH

COMMUNICATIONS

MATERIAL AND METHODS Solution : Tris buffer : 1OmM Tris-HCl, acetate ; 3 yg/ml Sodium Polyvinylsulfate

pH 7.4 (PVS).

; IOOmM NaCl ; 2mM Mg

Cells : culture and labelling: Murine Friend-Eveline cells were grown in suspension cultures and maintained at a 2-8.105 cells/ml concentration range (3,18). For labelling experiments, exponentially growing cell were concentrated five fold and incubated for 30 min. with 50 pCi/ml of PlH uridine (29 Ci/mmole, from the CEA, Saclay, France). Parallel experiments were performed using uninfected D55 murine cells. Preparation of cytoplasmic extracts: At the end of incubation withpHI uridine, cells were disrupted in Tris buffer containing 1 % Nonidet P40 (3). The postmitochondrial supernatant was processed by two alternative methods. Procedure 1 : the postmitochondrial supernatant was spun at 50.000 rpm for 60 min., in order to pellet the polyribosomes (19). The postpolyribosomal supernatant was further centrifuged at 50.000 rpm for 5 hours and the postpolysomal pellet was resuspended in Tris buffer. Procedure 2 : the postmitochondrial supernatant was layered on a 75 % sucrose cushion in a SW50 rotor and run at 50.000 rpm for 5 hours. The upper part was discarded and the sucrose fraction was recovered and dialysed against. Tris buffer. Analysis of cytoplasmic particles: 0.6-1.0 0.D260 unit of the different extracts prepared as reported above was centrifuged through a lo-40 % sucrose linear gradient in Tris-buffer for I6 hours at 22.000 rpm at 4°C in a Spinco SW27 rotor. 405 and 60s ribosomal subunits were added as mar[I4 Cf labelled kers. Aliquots of the gradient fractions were precipitated with IO % trichloracetic acid and filtered on nitrocellulose membranes which were dried and counted in a scintillation Tricarb spectrometer. Particles isolated in sucrose gradients were analyzed in CsCl equilibrium gradients according to Spirin

(20). RNA analysis: Appropriate fractions recovered from the sucrose gradient were pooled and adjusted to 1 % SDS at room temperature for 30 min. RNA was then precipitated by ethanol with 50 pg yeast RNA as a carrier and stored overnight at -2O'C. It was analyzed by electrophoresis in 1.7 % polyacrylamide - 0.5 % agarose composite gels (21). Hybridization assays of RNA from particles with viral complementary synthetic DNA: The conditions of viral cDNA synthesis and RNA isolation have been described elsewhere (3). Annealing reactions were performed in small vials containing 0.6M NaCl ; 0.02M Tris-HCl, pH 7.4 ; ImM EDTA ; 0.1 % SDS and 12 pg/ml of denatured calf thymus DNA. 0.1 ml of mineral oil was added to prevent evaporation during incubation for 48 hours at 68°C. The hybrids were assayed for S , nuclease resistance (3).

RESULTS I - Isolation cells disrupted

viral

Cells since

were newly

plasm at this time. from cells incubated tivity

occured

in

particles

in the cytoplasm

RNA containing

at a 2mM Mg"

polysomes. 30 min.,

of subribosomal

: To isolate

concentration labelled

synthesized This for

of Friend-Eveline

particles,Friend-Eveline which

prevents

cells

the dissociation

of

with

for periods not exceeding c H 1 uridine ribosomal RNA had not yet entered the cyto-

point was verified by isolating cytoplasmic various periods of ,time. It was found that

the 18s region

were

of sucrose

1111

gradients

after

RNA radioac-

35 to 40 min.

Vol. 86, No. 4, 1979

BIOCHEMICAL

Friend-Eveline chondrial let

cells

supernatant

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

labelled

was prepared.

and postpolyribosomal

for

30 min.,

and a postmito-

It was fractionated

supernatant

(procedure

into

polysomal

I of Materials

pel-

and Methods).

Each fraction was then analyzed by velocity sedimentation gradients. Figure 1 shows the presence of radioactivity

in IO-40 sedimenting

% sucrose in a

peak located

The apparent

sedi-

mentation

between

the

coefficient

heterogeneous

60s and 40s ribosomal

value

labelled

of this

material

markers.

peak was estimated

were

visible

in the

to be 53s.

Other

lighter

regions

(10-305)

of 535 peak was reproducibly

found

in the

from

one expe-

of the gradients. Although

the material

postpolyribosomal

supernatant,

its

suggesting

that

riment

to another,

mented

in the polysomal

undissociated In order

polysomes were

chondrial

supernatant

in Materials a IO-40

not

tion

Indeed,

in the polysomal

for

all

gradient

the sucrose 2).

cells.

tained

were

2 did

rial

sedimenting

II

- Caracterization

not

obtained

procedures

cushion

(procedure

in

was resol-

recovery

second

2

was analyzed in the isola-

procedure,

1 as well 10-30s

were

from

it

was

repeated

retrovirus-infected

using

cytoplasm

murine

as that

et al.

supernatant

of the extract in the

D55 cells

(Ravicovitch

of the postpolyribosomal

show any radioactivity

in the

in which

The postmito-

content

a better this

starting

RNA in their

Analysis

by the procedure

procedure

using

shown).

experiments.

any viral

communication).

was used

structures.

cushion

not

a peak of radioactivity

Since

was obtained

The same experimental do not contain

personal

Again

of the gradient.

the following

method

with

(result

on a 75 % sucrose

(Fig,

or sedi-

co-sedimenting

pellet

subribosomal

Then,

The above results which

another

the

was centrifuged

of the 53s material

adopted

from

greatly

by degradation

53s material

recoveries,

separated

ved in the 53s region

varied be lost

fraction.

and Methods).

% sucrose

could

was found

to get quantitative

polysomes

importance it

resulting

53s region,

obfrom

but

-

only

the mate-

area.

of the 53s particles.

a) Analysis of the RNA moiety --by -------electrophoresis in ~olyacrylamide gels: -___-- --___-_-----_--~~-------------- --- ------The RNPs of the sucrose cushion were centrifuged in a IO-40 % sucrose gradient. Fractions containing 53s ribonucleoproteins were pooled and treated by SDS. The RNA was subjected to gel electrophoresis using 14C]-labelled 28s and 18s rRNA as markers. minant

peak moving

The results

at a slower

served.

A more precise

exactly

at the position

examination

rate

than of its

are presented mobility

of the 35s RNA isolated

1112

in Figure

3. A predo-

the 28s rRNA was consistantly showed

that

by denaturation

it

ob-

migrated of 70s viral

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1 : Velocity sucrose gradient of postpolysomal supernatant of A postpolysomal extract was isolated --Eveline cytoplasmic extract. from cells labelled for 30 min. withpHI uridine and analyzed in a IO-40 % sucrose gradient (Beckman SW27 rotor-22 000 rpm - 16 hours at 4°C). Aliquots of each sample were assayed for acid-insoluble radioactivity (v). The markers were centrifuged in the same tube (o-- -0). P4 C1 labelled Fig. 2 : Velocity sucrose gradient of the suc,rose fraction (procedure 2) from Friend-Eveline cells. A subribosomal particles extract was prepared as in 2 and anal zed in the same conditions ;;;y"$ _to the procedure markers run in the same H profile of RNPs (o---o). [ IX Cl-labelled tube (o---o).

20

60

40

fractions

of RNA extracted from 53 BNPs. Fig. 3 : Gel electrophoresis RNA was extracted from 53s material of the sucrose cushion and subjected to electrophoresis on a 1.7 % polyacrylamide0.5 % agarose gel. 1.5mm slices were incubated with water for 16 hours at 37°C and the radioactivity of the eluate determined as described (21).

1113

Vol. 86, No. 4, 1979

RNA. Hence, cies

it

BIOCHEMICAL

can be pointed

contained

by others

(1,4).

uninfected

cells

sence of 5-18s

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

out that

this

rapidly

in the 535 RNPs has the

size

of the viral

No such 35s RNA was found (not

shown).

RNA species

the same RNA species

suggesting

patterns

that

by others

major cushion

of D55

indicated

RNPs labelled

for

RNA spe-

35s mRNA described

in the sucrose

Electrophoretic

reported

labelled

mammalian

the pre-

in D55 cells cells

contain

(19).

b) Hybridization of total RNA of 53s RNP with viral cDNA: A cushion ex_ _____________---_-_-____________________----------tract was prepared starting from unlabelled cells and fractionated in a sucrose

gradient.

purified

virus

region

with

lighter

along

and Methods)

gradient its

part

(Figure

4).

and hybridized with

around

most

the lighter

the gradient

part

in three

regions

of

in the hea-

to viral degraded

of

in the 40-60s

The hybridization

represent

RNA was

quantities

present

corresponded

could

equal

RNA located

53s.

likely

and their

with

A peak was evidently

maximum centered

of the gradient while

polysomes,

pooled

cDNA. The cDNA hybridized

sucrose

viest

were

(see Materials

Friend the

Fractions

RNA bound viral

to

RNA or other

mRNAs.

c) Centrifugation in CsCl density - gradients: The 53s particles ----r-_--~-~~--~~-------_-_------red from a sucrose cushion as shown in figure 2 were pooled, fixed,

and ban-

ded in CsCl density

Major

part

gradient

of the radioactivity

density

(not

1.39-1.47

shown).

g/cm3

as described

in Materials

was concentrated Existence

range

has been

recove-

and Methods.

in a single

of free

ribonucleoproteins

reported

in previous

peak at a 1.46 g/cm3 banding

works

in the

(19,22,23,24).

DISCUSSION In the present precursor, a population 1.46

we found

work,

that

g/cm3 density

moving

value

content

ticles

revealed a very

that

subunit present in the Moreover, hybridization RNA sequences.

These

observed

in CsCl gradient

results

suggest

since

we used

the total

a RNA

infected

cells

contains

sucrose

gradient.

approximately

labelled

molecular

peak comigrating extracellular tests using

with

The

corresponds

and 25 % RNA (25).

of the rapidly

RNA or the viral genome itself. It protocol could detect other RNA than ling,

of chronically

most of the high

homogenous

conditions

at 53s in a velocity

of 75 % proteins

Gel electrophoresis ted in

pulse-labelling

the cytoplasm

of particles

to a relative

using

with

weight

RNA from

the 53s par-

radioactivity

was loca-

the 35s RNA of the

virions (in the form of a 70s complex). viral complementary DNA identified viral that

53s particles

contain

viral

genome-sized

should be noticed that our hybridization the 355 species vizualized by pulse label-

RNA extracted

1114

from

the 535 particles.

Vol.

86,

No.

4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

l

00

50

---+A++ FRACTIONS

Fig. 4 : Hybridization of the Friend virus cDNA with the RNA mned in the cytoplasmic RNPs of Friend-Eveline cells. An extract (procedure 2) was obtained from unlabelled cells and fractionated in a sucrose gradient as in Fig. 2. 14C labelled 40s and 60s ribosomal subunits were sedimented in a parallel tube. Adjacent fractions were pooled two by two along the gradient. Each combined sample was deproteinized by a SDSphenol procedure (3) and precipitated by 2 volumes ethanol in the presence of 20 pg of yeast RNA. RNA was dissolved in pure water and annealed with approximatively 800 cpm cDNA (see materials and methods). At the end of the incubation at 68"C, each sample was analyzed by S1 nuclease (3).

Cytoplasmic same experimental particles.

Eveline

of molecular

not contain region

a well

particles

the

nature

where

18-20s

peak of 53s

size

the only range.

unknown. Their

the

The ab-

in FriendIt

is

tempting

of RNPs which density

is

are

compatible

of RNPs with densities in the All the experiments presented here

polysomal

1115

under

showed

a population

proteins.

since the existence has been reported.

in conditions

resolved

of the peak found

represent

of the viral

prepared

of the gradient

not exceeding

the viral

in the synthesis

were performed

cells

of the 53s RNPs is presently

these

with such a function, 1.39-1.47 g/cm3 range

did

species

cells. The function that

of non infected

of this

of 35s RNA confirms

to consider involved

conditions

RNA analysis

presence sence

extracts

RNPs are not

released.

The 53s

Vol.

86,

No.

4, 1979

BIOCHEMICAL

RNPs recovered viral

in the postpolyribosomal

informosomes.

during

the

course

a very

early

step

of their

hypothesis.

be already

associated

protein

content

BIOPHYSICAL

RESEARCH

supernatant

could

COMMUNICATIONS

be considered

of some polysome-associated

preparation

cannot

be excluded.

In that

as RNPs

case,

the

a mixture of polysomal mRNPs and informosomes. 53s RNPs could be a precursor of the viral core

of its

this

their

the release

However,

53s RNPs would represent Alternatively, with

AND

Presence

synthesis.

In this with

case,

viral

should

of 35s RNA would

polypeptides

belonging

RNA in 53s particles.

distinguish

between

these

at

be compatible

to the core

Further

analysis

should of

possibilities.

AKNOWLEDGEMENTS This de la Sante

research

was supported

et de la Recherche

by a Grant

Medicale

(Contrat

from the libre

Institut

National

: 76.5.128.2).

REFERENCES 1 - Stacey, D.W., Allfrey, V.G. and Hanafusa, H. (1977) Proc. Nat. Acad. Sci. 74, 1614-1618. 2- Conley, A.J. and Velicer, L.F. (1978) J. Virology 25, 750-763. 3 - Michel, M.L., Roussel, M,, Poitevin, E,, Samso, A. and Larsen, C.J. (1977) Biochimie 2, 275-285. 4 - Gielkens, L.J., Salden, M.H. and Bloemendal, H. (1974) Proc. Nat. Acad. Sci. 2, 1093-1097. 5- Vogt, V.M. and Eisenman, R. (1973) Proc. Nat. Acad. Sci. 70, 1734-1738. 6 - Van Zaane, D., Gielkens, L.J., Dekker-Michielsen, M.J.A. and Bloemers, H.P.J. (1975) Virology 67, 544-552. 7- Vogt, V.M., Eisenman, R. and Diggelmann, H. (1975) J. Mol. Biol. 96, 471-493. 8- Wang, S.Y., Hayward, W.S. and Hanafusa, H. (1977) J. Virology 2, 64-73. 9- Spirin, A.S. (1969) Eur. J. Biochem. lo, 20-35. 10 - Spohr, A.G., Kayibanda, B. and Scherrer, K. (1972) Eur. J. Biochem. 31, 194-208. 11 - Perry, R.P. and Kelley, D.E. (1968) J. Mol. Biol. 35, 37-59. 12 - Lebleu, B., Marbaix, G., Huez, G., Temmerman, J., Burny, A. and Chantrenne, H. (1971) Eur. J. Biochem. 19, 264-269 13 - Zahringer, J., Baliga, B.S. and Munro, H.N. (1976) Proc. Nat. Acad. Sci. 73, 14 -

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Sonenshein, G.E. and Brawerman, G. (1977) Eur. J. Biochem. 13, 307-312. R.P. (1972) J. Mol. Biol. 63, 577-590. Schochetman, G. and Perry, 67, 335-352. Vesco, C. and Colombo, B. (1970) J. Mol. Biol. Yap, S-H., Strair, R.K. and Schafritz, D.A. (1978) Biochim. Biophys. Res. Comm. 2, 427-433. Seifert, E., Claviez, M., Frank, H., Hunsmann, G., Schwarz, I-I. and Sch‘a'fer, W. (1975) Zeitschrisftfur Naturforschung 30 C Serte 648-700. Gander, E.S., Stewart, A.G., Morel, C.M. and Scherrer, K. (1973) Eur. J. Biochem. 2, 443-452. N.V. and Lerman, M.I. (1975) J. Mol. Biol. 14, Spirin, A-S., Belitsura, 611-613.

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21 - Tiollais, P., Galibert, F., Lepetit, A. and Auger, M.A. (1972) Biochimie 54, 339-35 4. 22 - Kumar, A. and Pederson, T. (1975) J. Mol. Biol. 96, 353-365. O., Mandel, P. and Kempf, J. (1974) FEBS Lett. 23 - Egly, J.M., Krieger, 40, 101-105. 24 - cautard, J.P., Setyono, B., Spindler, E. and Kohler, K. (1976) Biochim. Biophys. Acta (Amst.) 425, 373-383. N., Morel, C. and Scherrer, K. (1970) Eur. J. 25 - Spohr, G., Granboulan, Biochem. 17, 296-318.

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