Inhibition of Vesicular Stomatitis Virus transcriptase complex by the virion envelope M protein

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 14, 1979

Pages 117-123

INHIBITION

OF VESICULAR STOMATITIS VIRUS TRANSCRIPTASE BY THE VIRION ENVELOPE M PROTEIN Arlette

Institut

COMBARD and Christiane

de Microbiologic, Orsay

91405

Received

March

PRINTZ

COMPLEX

AN6

(B3t. 409), Universit6 Cedex, France

Paris-Sud,

2,1979

SUMMARY. An inhibition of the RNA-dependent RNA polymerase associated with Vesicular Stomatitis Virus occurredwhen the reaction was stimulated by whole virions and not by enzymatically active ribonucleocapsid (RNP) cores. The matrix (M) protein and the glycoprotein (G), both components of the virion envelope, were purified and added separately to the polymerase reaction stimulated by RNP cores. The G protein fraction had no effect on the reaction, except in some experiments ; in that case, the inhibition was demonstrated to be the consequence of a NTPase activity. The M protein fraction never showed such an activity, but induced a strong inhibition of the polymerase reaction. The data presented suggest that the M protein is the transcriptase inhibitor present in the whole virions. Previous

studies

transcription

on ts'

rate

of viral

an altered

matrix

(1).

the report

Since

the envelope, tion sured protein virus, reaction.

Sendai

virus,

We report activity

inhibiting These

the envelope

viral

suggested

complex

from

the effects

the presence,

effect

of another

of

negative

VSV meaboth

M

strand transcriptase

of purified

of the virion

within transcrip-

that

on the in Vitro

of the addition

protein

proteins

a possible

activity

(5) have demonstrated

of the

synthesizing

envelope

the in Vitro

et al.

the second

(2),

observations

Marx

had an inhibitory below

(G),

4).

by mutants

one of the virion

on the transcriptase

Moreover,

and glycoproteins

and glycoprotein merase

(3,

infected

is

and Kingsbury

component

of the M protein in Vitro.

which

of Perrault

of a viral

of VSV have shown an enhancement

mRNA in cells

(MI protein,

has been documented

effect

mutants

M protein

envelope,

on the poly-

of VSV-RNP cores. MATERIALS AND METHODS

Viral material : The wild type strain of VSV (Indiana serotype) was grown on chick embryo cells as previously described (6). Fresh stocks were purified by centrifuging to equilibrium (Spinco rotor SW 27, overnight at 5' C, 25,OOC rpm) in a preformed glycerol (30-O%)-potassium tartrate (O-SO%) gradient containing Tris 10 mM, NaCl 1 M, EDTA ImM, pH 7.6. RNP cores were extracted from purified virions according to Breindl and Holland (3) and used immedia' Abbreviationsused : ts, temperature sensitive ; VSV, Vesicular Stomatitis Virus ; RNP, ribonucleocapsid ; SDS, sodium dodecyl sulfate ; NTP, nucleoside triphosphates ; NTPase, nucleoside triphosphates phosphohydrolase. 0006-291X/79/090117-07$01.00/0 117

Copyright All rights

@ 1979

by Academic Press, Inc. in any form resewed.

of reproduction

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tely. Viral envelope proteins were extracted following a modified procedure of Mac Sharry et al. (7). Purified virions at a concentration of = 0.5-1.0 mg of protein per ml in 0.01 M Tris buffer pH 7.6 were treated with Triton X-100 at a final concentration of 0.5%. The solubilized proteins were separated over a 20% glycerol cushion from the insoluble viral proteins by centrifugation (Spinco rotor SW 65, 1 h at 5' C, 45,000 rpm). The supernatant fluid was decanted : it constituted the G protein fraction. The pellet was resuspended in Tris 0.01 M, NaCl 1 M, pH 7.6, at a protein concentration of = 0.5-1.0 mg/ml and was treated with Triton X-100 at a final concentration of 0.5%. The supernatant fluid separated by the same centrifugation step as above, was decanted and thoroughly dialyzed against 0.01 M Tris buffer, pH 7.6 at 4' C. The material which precipitated during dialysis was collected by low speed centrifugation (Spinco rotor SW 65, 20 min at So C, 10,000 rpm) and resuspended in Tris buffer 0.01 M : this was the M protein fraction. In Vitro polymerase assay : The in Vitro polymerase reaction mixture consisted of 50 mM Tris-HCl, pH 8.4, 100 mM Na acetate, 100 mM K acetate, 8 mM Mg acetate, 5 mM dithiothreitol, 1.2 mM each of ATP, GTP and CTP, 0.12 mM of UTP and 0.004 mM c3~] IJTP (12 Ci/mmol). Triton X-LOO was added to a final concentration of 0.02%, when entire virions were to be tested. Viral material was present at a concentration of 0.250 mg/ml of whole virion proteins (or equivalent to for RNP cores), and the temperature of incubation was 30° C. The reaction was followed by measuring the amount of radioactivity in TCA insoluble material, each aliquot being in duplicate and each cpm value being corrected for the value measured at time 0 (= 500 cpm in 25 ~1). ATPase assay : The release of free 32Pi from [y-32~]ATP was measured by incubating the viral material and this nucleoside triphosphate (3,000 Ci/mmol) under the same conditions as described for the RNA polymerase assay, except for the absence of other unlabeled nucleoside triphosphates. Aliquots were removed at intervals during the course of incubation at 30° C and analyzed by ascending chromatography on 3MM Whatman paper. The paper was developed with a mixture of n-butanol, pyridine and ammonia 0.880 (40:20:40, v/v) at 28' C for 18 h. The ATP and free Pi were located by autoradiography of the chromatogram, then the 32P radioactivity of the corresponding spots was determined by Cherenkov counting. RESULTS AND DISCUSSION The existence was first

suggested

dependence the

of an endogenous

sed above

by the observation

of the yield

reaction

which

a determined

and RNP core

Controls

of Triton

(data

not

shapes

value,

the VS virion

of an unexpected

virion

concentration

in Vitro

was not observed (3).

activities

This

was also

ruled

out

of the kinetic

curves

virions

the more rapidly

reached

differed

virion

in our effect

were

between experiments relevant

of was increaused in whole (Fig.

to the pre-

and not in the RNP cocktail that in many experiments

one another.

Fig.

1).

the

1 illustrates

RNA synthesis stimulated by RNP cores was always linearity appeared as soon as the concentration of

a precise

the curve

from

The inhibition

difference

observed

some trivial

(2).

of whole when RNP cores

striking

X-100 only in the virion assay it appeared shown). But, in addition,

such an example : whereas linear, a divergence from whole

in

when the concentration

range

were done which

sence

inhibitor

of RNA synthesized

occurred

the same concentration virion

transcriptase

bent

range.

The to a plateau.

118

more

concentrated This suggested

the virions a possible

were, li-

BIOCHEMICAL

Vol. 88, No. 1, 1979

Virions

. I 1250

RNP

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

C

20

250

01

30 60 Time (min)

30 60 90

90

Fig. 1. Effect of viral protein concentrationson the in Vitro RNA synthesis stimulated by whole VS virions or RNP cores. In Vitro transcription reaction mixture (described in Materials and Methods) were prepared with virions or RNP cores at concentrations equivalent to 125 us/ml, 250 us/ml, 500 pg/ml and 1.25 mg/ml of whole virion proteins. The reactions were incubated in stoppered tubes at 30' C, and duplicate aliquots were removed from each reaction at 30, 60 and 90 min. Acid insoluble RNA was measured by scintillation spectrometry. Fig. 2. Comparative analysis of the proteins in whole virions (a), M protein fraction (b) and G protein fraction (c). M and G protein fractions, extracted as described in Materials and Methods, were samples of materials used in experiments illustrated by Fig. 3. Protein samples were precipitated overnight at 4O C by the addition of 2 volumes of ethanol and the pellets were dissolved in a solution of 62.5 mM Tris-HCl pH 6.8 with 1 M urea, 1% SDS and 1% mercaptoethanol. After boiling for 3 min the samples were subjected to electrophoresis on discontinuous Tris-glycine-buffered SDS-slab gel consisting of 10% resolving gel and 3% stacking gel according to Laemmli's procedure (12). Electrophoresis was from top to bottom. The protein bands were stained with Coomassie blue.

mitation cores

of substrates. under

In view

of the

the same conditions,

linear

the only

curves

available

be the disappearance

of NTP occurring

when the virions

It

VSV possesses

the location for

most

would

of which

viruses

which

be critical

for

reaction.

We therefore

conducted

to test

and M protein

fractions A sample

gel

might

that

be within

bud out

from

the demonstration paid

attention

the

viral

the host

as is

surface.

of an inhibitor to it

in

Its

the following

electrophoresis

and the

gel

was analyzed was stained

119

to

their (S),

the case presence

of the RNA polymerase

on the polymerase reaction, isolated from virions as described

of each fraction

seemed

have kept

envelope, cell

the RNP

a NTPase activity

the effect,

and Methods. slab

is known

for

explanation

membrane

components.

observed

experiments of the G protein in Materials

by SDS-polyacrylamide (Fig.

2).

The densitometer

BIOCHEMICAL

Vol. 88, No. 1, 1979

M protein

I

0

,G protein

30 60 (min)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

$O

90

04

Time

(min)

Fig. 3. Effect of the addition of G or M protein fraction on the in Vi&O Identical 200 ~1 reaction mixtures were each RNA synthesis by VSV-RNP cores. prepared with fresh RNP cores equivalent tc5Oug of whole virion proteins. One sample served as control and did not receive M or G protein fraction with the M protein fraction (res(curve 0). Three samples were supplemented pectively 10, 30 and 100 pg per assay) and 2 samples with the G protein Incorporation of ["H] UTP into acid prefraction (10 and 20 Vg per assay). cipitable RNA was determined as described in Fig. 1. The dotted curve (Gal was obtained in another experiment in which the G protein fraction (25 pg) did not exhibit any effect on the reaction. Fig. 4. ATPase activity in different protein fractions extracted from rions. Three equivalent(200 ~1) reaction mixtures were prepared on the and no other of the RNA polymerase reaction, but containing [y- 32P]ATP cleoside triphosphate. One sample contained VSV-RNP (equivalent to 50 the second contained 20 pg of the G protein of whole virion proteins), tion and the third 20 pg of the M protein fraction. During incubation aliquots were removed at intervals and subjected to paper chromatography autoradiography as described in Materials and Methods. The percentage over the totality of label recovered was recorded. C3' P 3 phosphate

VS vibasis nuug fracat 30° C and of

traces

us to

corresponding

estimate Fig.

that

3 illustrates

merase

reaction.

results

were

was depressed protein for

to the electropherogram

the M protein

reaction illustrates

the results Concerning

always

of their

unequivocally

of time

(data

95% pure

not

addition

enabled >98%

to RNP cores

in

pure.

the poly-

of the M protein fraction, the reaction clear-cut : the RNA polymerase

of inhibition

followed

Moreover,

the kinetic

examined.

On the other

the increase

curves hand,

were various

with

the G protein fraction : either no effect or various degree of inhibition was observed, an experiment

shown)

and the G protein

the addition

and the degree

concentration.

the period

obtained

was >

in which

the

G protein

120

fraction

strictly results

of the M linear were

on the polymerase occurred.Fig. 3 exhibited

a strong

BIOCHEMICAL

Vol. 88, No. 1, 1979

inhibitory

effect

plateau.

presence account

complex.

(8)

which

acted

Fig.

4 that

fraction ver,

for

by splitting

the NTPase activity

and,

in contrast,

we noticed,

to a decreased

sis

measured

results

to the addition

the slope

of the curve

contrary,

in a polymerase

by comparison

in

the polymerase

of

with

in which

C” 1 H

Howein

the

with

reaction

or without

mixture

a diminution

to the presence argued

polymerase

for

complex for

a direct

data

tion

to the RNP cores

bovine

serum

2'min,

modified

inhibition

this

of a NTPase activity

leading

tary

nor

HeLa cells

with

because were

a restriction

in the presence

enzymic

not degraded

activities

the M protein

on the RNP

neither

the addi-

VSV proteins,

i.e.

at 100"

Secondly,

not appear

VSV mRNAs extracted when added

effect

Complemen-

heated

did

fracVitro

In contrast,

fraction

reaction.

fraction

the

to be lin-

from

to a polymerase

C for

wild

type

assay

in

fraction. of the in Vitro

of the M protein directly

in

6. First,

VSV polymerase

fraction.

such as NTPase or RNAse in acted

We therefore

activity.

of Mprotein

purified

On the

the presence

the fraction.

to the

of the polymerase

alone,

of the G protein

in Fig.

5

was observed

sample.

of its

unrelated

the M protein

of the M protein

In conclusion,

given

the addition

the kinetics

in

of the M protein

of a protein

ked to a ribonuclease, the presence

effect are

Fig.

was perfor-

an artifactual in

to an attenuation

assumption

albumine,

obtained

was rather

synthe-

pre-incubation.

pre-incubated

after

would

the

incubation.

of the RNA synthesis addition

the

cocktail with

a 40 min-incubation

complex

that

reaction

0 of the

observed

observed

fraction. of activity

UTP. In the case of the RNP cores

was identical

fraction,

at time

effect

infected

RNP

the M protein

by comparison

to the RNP polymerase data

from

levels

companion

related

of free

One can see in

the G protein

the not pre-incubated

concluded

that

in

of Roy and

containing

proteins.

various

of RNA synthesized

of an experiment

of the G protein

ble

fraction

when [" H ] CJTP was added

shows the

all

hiqh

which

NTPase activities

absent

experiments,

amount

med prior

tion

the results

mixture

was virtually

the

fraction,

the release

envelope

for

fraction, even an absence. If the degradation of NTP was responthe inhibitory effect of the G protein fraction, then pre-incubation

at 30“ C of the G protein lead

since the four

reaction

viral

especially

on repeated

we looked

a

on the polymerase

We followed

the polymerase

each of the

above,

effect

was checked, the y-Pi.

seem to reach

the M or G protein

enzyme bearing

only in

in

RESEARCH COMMUNICATIONS

the curves

reported

inhibitory

activity

a unique

and without

G protein sible for

apparent

the ATPase

from [y-32 P]ATP with

that

of the observations

an only

argued

cores

be noticed

of a NTPase activity

for

Only

Bishop 32Pi

can also

On the basis

possible would

; it

AND BIOPHYSICAL

on the polymerase

121

activity

The absence this fraction complex

was

of detectasuggested

and attenuated

BIOCHEMICAL

Vol. 88, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IRNP RlfPi D-

:

l

I 0

30

4

4

I

P

I’

I

_

-3” c

f

00

06

90 120

0

5 TIME

306090

(min)

TIME

(min)

30

60

90

Fig. 5. Effect of a pre-incubation of the G protein fraction on the RNA polymerase assay. Four polymerase reaction mixtures (200 ul) were each prepared with RNP equivalent to 50 ug of whole virion proteins. To two samples, 20 ug of the G protein fraction were added. One sample with only RNP (RNPi) and one sample with G (Gi) were incubated at 30' C for 40 min prior to the addition of [3H]HTP (+). The two other reaction samples (RNP and G) were incubated immediately after the addition of the radioactive precursor as usual. Fig. 6. Control experiments of the in Vitro RNA polymerase reaction. Reac-mixtures (200 ~1) were each prepared with VSV-RNP e uivalent to 50 ug of whole virion proteins. One sample did not receive ["H ‘I UTP but 25,OOG cpm of r3n]uridine labeled purified mRNA isolated from HeLa cells infected with VSV wild type : aliquots (40 ~1) removed at intervals, were TCA precipitated to follow a possible degradation of the radioactive RNA. Other samples served to determine the incorporation of [3H]HTP as usual. Two samples were independent controls (RNP), another received 30 ug of fresh M protein fraction (i M), another received 30 ug of the M protein fraction boiled for 2 min prior to its addition (+ heated M), the last one received 30 ug of bovine serum albumin (+ BSA).

its

efficiency.

syntheses trated

Vitro

is

to the

the in Vitro reaction.

thors

(2,

envelope occasionally

protein

The M protein However,

and which hamper

observations

the

ts defect

(9,

of VSV infected

of the 4).

M

by our (1)

if).

and in ViVo results

in the cytoplasm

the inhibition

supported

by ts mutants

to be linked

between act

This

induced

appears

thus

VSV transcriptase the NTPase activity

together

the in Vitro

polymerase

122

the

in a similar far

reaction

was solubilized

that

which with

ViUo

RNA

has been demons-

the good agreement

Moreover,

suggest

cells

on the in of which

protein

M

would

way as in the in

as the best

candidate

for

detected

by various

au-

in the

VSV

was present the G protein

reaction.

Since

may also we always

BIOCHEMICAL

Vol. 88, No. 1, 1979

observed

a good correlation

G protein

fraction

polymerase bit

the in Vitro

glycoproteins

we conclude polymerase

from

the strong

inhibitory

tion

previously

reported

the NTPase activity various

a true

intrinsic

packed

during

was not

degrees virion

the cell procedure,

remains

thought role

it

of

currently

the mechanism

rather

of virions.

favors

the presence

that

on the nature

investigation.

of the inhibition,

a virion

This

aspect

be reported

of the

it

not

be

reaction,

we

or our pu-

The important envelope also

as well

of the RNAs synthesized

will

case,

was present,

stocks,

might

in Vitro

be

reac-

as such an activity

virus

VSV protein, complex

might

contaminant

of the activity.

a structural

transcriptase

it

conceivably

be a cellular

our

inhiof

In our

on the polymerase

growing

is now demonstrated

of the

and when it therefore However,

working

used for

the M protein under

might

strain

to ours,

on the polymerase

detected, but

on the does not

of preparation

similar

fraction

in the

to a NTPase activity.

: it

process

up to now to be only

at the level

The effect is

that

regularly

by authors

fraction by itself

the procedure

was related

component,

the budding

of this

was essentially of this

RESEARCH COMMUNICATIONS

of NTPase acticity

the G protein Since

of activity

suppose rification

virus (5),

been noticed

point

of effect that

effect

has scarcely that

the absence

reaction.

Sendai

that

showed

between

and the absence

reaction,

AND BIOPHYSICAL

work,

which

protein, play

a

as in ViVo. in Vitro deals

with

elsewhere.

ACKNOWLEDGMENTS : This work was supported in part by grants from the Centre National de la Recherche Scientifique (L.A. no 136) and the Fondation pour la Recherche Medicale Francaise. We would like to thank Dr. J.C. PATTE for helpful discussions of the data presented in this report. Note added in proof : During the preparation results of A. Carroll and R. Wagner (in press) with ours.

of the manuscript we learnt which are in good agreement

REFERENCES 1. Martinet, C., Combard, A., Printz-Ane, C. and Printz, P. (1979). J. Virol. (in press). 2. Perrault, J. and Kingsbury, D.W. (1974). Nature 248, 45-47. 3. Breindl, M. and Holland, J.J. (1976). Virology 73, 106-118. 4. Carrel, A.R. and Wagner, R.R. (1978). J. Biol. Chem. 253, 3361-3363. 5. Marx, P.A., Portner, A. and Kingsbury, D.W. (1974). J. Virol. 13, 107-112. 6. Printz And, C., Combard, A. and Martinet, C. (1972). J. Virol. 10, 889-895. 7. MC Sharry, J-J., Compans, R.W., Lackland, H. and Choppin, P.W. (1975). Virology 67, 365-374. 8. Roy, P. and Bishop, D.H.L. (1971). Biochim. Biophys. Acta 235, 191-206. 9. Lafay, F. (1971). J. Gen. Virol. 13, 449-453. 10. Lafay, F. (1974). J. Virol. 14, 1220-1228. 11. Knipe, D.M., Lodish, H.F. and Baltimore, D. (1977). J. Virol. 21, 1140-1148. 12. Laemmli, U.K. (1970). Nature 227, 680-685.

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