Protamine—A potent inhibitor of vesicular stomatitis virus transcriptase invitro

Vol. 98, No. 3,198l February

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 875-883

12, 1981

PROTAMINE--A

POTENT INHIBITOR OF VESICULAR STOMATITIS VIRUS TRANSCRIPTASE --IN VITRO Sohel

Roche

Received

December

Talib

and Amiya

K. Banerjee

Department of Cell Biology Institute of Molecular Biology Nutley, New Jersey 07110

11,198O SUMMARY

stomatitis virus The virion-associated RNA polymerase activity of ves&yular is inhibited by protamine at a concentration as low as 10 M. The inhibition is reversible, appears to be at the level of initiation and competitive with respect to ATP. Histone IIA and IV are also inhibitory whereas other fractions The endogenous protein kinase activity is significantly inhibited by are not. Virion-associated RNA or DNA polymerases of several animal viruses protamine. are also inhibited by protamine. INTRODUCTION The transcribing (VSV)

contains

negative

a single-stranded

polarity

two other species

tightly

minor are

reaction

ribonucleoprotein

sequentially the

of replication,

transcription

to replication

the

triphosphates. control

with

in vitro been

analogue These

also

shown

L protein (4).

synthesized

--in vitro

results --in vitro.

It

to be present

that

has been

NS proteins in the purified

are with

Under

three

(3).

normal

that

appropriate

different

degrees differential

required

switch

from

the presence

of

ribonucleoside of proteins

the phosphorylated

components

with

mRNA

of Triton-disrupted

states

necessary

virion

This

in

phosphorylated shown

(N),

genome RNA, the

incubation

and the

protein

distinct

by preinitiation

of REP and then

suggested

(2).

--in vitro

was achieved

and RNP template

Moreover,

Five

of the

virus

4 x 1D6) of

nucleocapsid

NS (1).

strand

of ATP (AdoPP(NH)P)

transcription

along

is

ATP and CTP, reisolation

B,y imido

the principal

positive

stomatitis

weight,

by the RNP --in vitro

full-length

intermediate

VSV with

with

L and the phosphoprotein

synthesized

of vesicular

genome RNA (molecular

associated

proteins

conditions

(RNP) core

for

may

NS protein

transcription

of phosphorylation affinity

have for

the

0006-291X/81/030875-09$01.00/0 875

Copyright 0 1981 by Academic Press, Inc. All rights of reproducfion in any form reserved.

Vol. 98, No. 3,198l

BIOCHEMICAL

Purified

RNP (5,6). predominantly

RNP also

the NS protein

in the multiplication stand tion

the

role

transcription

that

a low molecular

polypeptide

from

a potent

trout

inhibitor

activity the effect

activity

that

and also

of the virion-associated

basic

acceptor

RNA polymerase

MATERIALS

acceptor

proteins

we demonstrate

highly

a phosphate

to under-

of phosphoryla-

phosphate

communication

daltons)

kinase

In an effort

of various

(5000

phosphorylates

of the protein

unclear.

In this

weight (9)

kinase

COMMUNICATIONS

in VSV and the processes

process.

testis

RESEARCH

The role

(7,8). is presently

studied

in the --in vitro protamine,

a protein

--in vitro

kinase

we have

in general,

contains

of the virus

of protein

AND BIOPHYSICAL

arginine-rich

protein

(lo),

is

of VSV.

& METHODS

In vitro transcription assay: VSV RNA polymerase reactions (0.2 ml) --In vitro 4 mM dithioth?jeitol, contained 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 5 mM MgCl 0.05% Triton NlOl, 1 mM each of ATP, CT32 and GTP, 0.1 m&'HTP and 10 nc H UTP (specific activity = 660 cpmfpmole), a- P LJTP (1.8 Ci/mmole) and 10 to 15 ng of purified VSV (Indiana serotype grown in baby hamster kidney cells - 21, clone 13 adapted to suspension culture) (11). The incubations were at 30°C for desired lengths of time as described in legends. RNA synthesis by --In vitro purified vaccinia virus (kindly provided by Dr. J. Hurwitz), cytoplasmic polyhedrosis virus (kindly provided by Dr. Y. Furuichi), WSN strain of influenza virus (kindly provided by Dr. R. M. Krug), and reovirus (kindly provided by Dr. A. J. Shatkin) were done under standard conditions as described in references 12, 13, 14 and 15, respectively. Reverse transcriptase activity of Rous sarcoma virus (kindly provided by Dr. A. M. Skalka) was determined using standard assay conditions virus used was approximately lo-15 ug, (16). In each case the amount of purified except for Rous sarcoma virus 100 ug was used. The incubation time was 2 hrs. Protamine, casein, and phosvitin were purchased from Sigma Chemicals and different fractions of histones were purchased from Boehringer Mannheim. In vitro protein kinase Tria-HCl (pH 8.0), 0.1 ATP (final-specific Y- “P Incubation was at 30°C according to the method gel electrophoresis as

assay: The reaction mixture (0.2 ml) contained 50 mM M NaCl, 5 mM M&l,.. 4 mM dithiothreitol. 50 uM ATP. and activity = 4006.cpm/pmole) and 50 ug-of purified RNP. for 15 min. The phosphorylated proteins were processed of Moyer and Summers (8) and analyzed by polyacrylamide described (10).

RESULTS --In vitro various

RNA synthesis

concentrations

RNA synthesis corresponds

of protamine.

was achieved to a molarity

the RNA synthesis

by purified

RNP was carried As shown

at a protamine

concentration

of approximately

was virtually

in Fig.

lo-'.

abolished.

IA,

in the presence a 50% inhibition

of 0.45

At concentrations

The inhibitory

876

out

effect

rig/ml

of of

which

of 1 ug/ml of protamine

Vol. 98, No. 3,1981

BIOCHEMICAL

PROTAMINE

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TIME (mid

(pg /ml 1

Fig. 1. Inhibition of --in vitro transcription by protamine. (A) Standard transcr f ption reaction mixtures were set up as described in Materials and Methods using [ H] UTP as the labeled precursor. Protyine was added at different concentrations and trichloroacetic acid insoluble H radioactivity was measured (B) In separate standard transcripusing toluene based scinttilation cocktail. tion reaction mixtures i rig/ml of protamine was added at different times during RNA synthesis as indicated by arrows. The RNA synthesis was then followed by taking aliquots (20 ~1) from the reaction mixtures at different times and assaying for trichloroacetic acid-precipitable radioactivity.

was quite (Fig. it

striking

1B).

when added

Although

the

can be seen in Fig.

5 to 10%) continued inhibition

at the

RNA chains

were

We next,

amounts

1B that

indicate

level

in Fig.

gradient. reversible,

In order

the

kinetic

the

of the

inhibition

effect

experiment

the

amount

with

the

are

where

previously

inhibitor

characteristic

typical

might

of initiated

although rates.

RNA products

found

were

decreased These

the

effect

have been

preparation.

may be at or near

inhibitory

was performed.

877

of protamine

of RNA (approximately

patterns

the mRNA products

terminated

whether

the addition

inhibitor.

of protamine

no prematurely

of transcription

of RNA synthesis

that

and sedimented

onset

after

reproducible

These

indicate

to determine

following

rapidly

by RNase contaminating

2 clearly

since

after

of RNA synthesis

whether

the inhibitory

the

but

in the presence

undegraded

of RNA synthesis

a small

of initiation

determined

that

ceased

to be synthesized.

completed

remained

times

RNA synthesis

due to degradationofmRNA Data shown

at different

RNA synthesis

resuits

initiation

of protamine --in vitro

in

in the was was

Vol. 98, No. 3,1981

BIOCHEMICAL

FRACTION

02

AND BIOPHYSICAL

RESEARCH

NUMBER

FRACTION

COMMUNICATIONS

NUMBER

Fig.

2. Velocity sedimentation analyses of VSV mP.NA synthesis in the presence of varying concentrations of protamine. Standard transcription reacgion mixtures containing different concentrations of protamine were set up using [ HI-UTP as the labeled precursor and incubated for 2 hr at 3O'C. The reaction was terminated by addition of SDS and the reaction mixtures were directly layered onto a 15-30X SDS-sucrose gradient. The centrifugation was at 33,000 rpm for 17 hr at 23'C in a SW40 Spinco Rotor. Fractions (40 drops) were collected from the bottom of the tubes and trichloroacetic acid insoluble radioactivity measured. $4, no protamine; A-A, 0.5 pg/ml protamine; O-O, 0.75 pg/ml protamine; A-A, 1.00 pg/ml protamine. Arrows indicate the sedimentation position of VSV mRNA speices. Fig.

3.

Reversibility

of protamine

inhibition

of VSV RNA synthesis.

Two transcription assay mixtures were set up in 0.02 ml in the presence of protamine (1 ug/ml) containing unlabeled precursors and incubated for 15 min. The first sample was diluted to 0.2 ml while maintainjng the concentrations of the reaction components including protamine (1 rig/ml). H UTP was added and the reaction continued for 45 min (O-O). The second sample was similarly diluted and incubated while reducing protamine concentration at 0.1 pg/ml (A-A). A third sample (0.02 ml) initially received low protamine (0.1 ug/ml) after 15 min was similarly diluted maintaining protamine concentration at 0.1 rig/ml (A--I). RNA synthesized by three samples were individually analyzed by velocity gradient as described in Fig. 2.

carried

out

in duplicate

In one of the while

synthesized

the reaction These

results

containing indicate

that

of the

by velocity

ugfml the

components

sedimentation.

of protamine

activity present

effect

878

for

was reduced of reaction

45 min following

RNA synthetic

inhibitory

(1 us/ml)

concentration

an additional

regained 0.1

of protamine

the protamine

for

RNA was analyzed mixture

the presence

of the rest

was continued

reaction

in

mixtures

the concentrations

The reaction

diluted

reaction

tubes

virtually

3, the

identical

is

totally

unchanged.

and the

in Fig.

at the beginning

of protamine

ten-fold

remained

reduction

As shown

15 min.

to

of the

reaction.

reversible.

Vol. 98, No. 3,198l

BIOCHEMICAL

AND BIOPHYSICAL

0.5

I.0

RESEARCH

COMMUNICATIONS

2.cI

NTP MA)

Pig. 4. Synthesis of VSV mRNA in the presence of protamine at varying ATP and GTP concentrations. Standard transcription r3action mixtures were set up as described in the Materials and Methods using [ H] UTP as the labeled precursor excepting that ATP and GTP concentrations were varied between 0.1 n&f to 2.0 &I and 0.5 pgfml of protamine was used. The RNA synthesis after 2 hr incubation were determined by trichloroacetic acid insoluble radioactivity. ATT, A-A; ATP + protamine, A--d; Cl?, O--O; Gl'P + protamine, 0-O.

The mechanism by varying

the

reaction

of protamine.

experiments

protective

(not

effect

a competitive Since

is

basic

We also

polymerases

with

proteins

of several

proteins

such as phosvitin

of vsv.

Interestingly,

significantly

Similar

inhibitory

was observed

was shown These

basic

the effect animal

effects

in vaccinia

for

protein,

viruses.

transcription reversed

to 2 mM.

UTP nor

suggest

that

the

In

CTP had a

protamine

it

was of interest

acceptor

proteins

may be

histone that

the observed

on VSV RNA polymerase

As shown

1, neutral

in Table

on --in vitro

proteins,

only

the basicity

and histone

IIA

transcription IIA

and IV were

of a protein

effect

polyhedrosis

879

the

on the virion-associated

inhibitory

cytoplasmic

to study

same proteins

have no effect

indicates

of protamine virus,

of 0.1

neither

results

of the

and casein

This

the

of GTP, had virtually

range that

was studied

to ATP only.

among the basic

criterion

addition

and phosphate

other

inhibitory.

absolute

it

in

of ATP effectively

concentration

respect

a highly

studied

4, addition

the

to ATP.

inhibitor

of other

over

by protamine

triphosphates

In contrast,

shown)

similar

protamine

--in vitro.

be the

in Fig.

on RNA synthesis

similar

effect

of ribonucleoside

As shown

effect

no effect

of VSV RNA polymerase

concentration

mixture.

inhibitory

of inhibition

of protamine.

on --in vitro virus,

may not

RNA synthesis

influenza

virus,

Vol. 98, No. 3,198l

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE1 Effect

of some basic

and neutral proteins several animal

on virion-associated viruses.

polymerases

Of Proteins Required For 50% Inhibition (M/ml) Protamine Histone Phosvltin

of

Concentration Viruses

I

IIA

IIB

0.5

(-)

2

(-)

1

(-)

1

C-1

1

C-1

10

virus

1

(-)

Rous sarcoma virus

5

N.D

C-1

C-1

vsv Vaccinia

Virus

Influenza

Virus

Cytoplasmic Polyhedrosis

Reo virus

IV (-)

C-1

(-)

(-)

C-1

C-1

(-)

C-1

C-1

10

C-1

(-)

C-1

N.D

N.D

N.0

N.D

N.D

t-1

C-1

C-1

C-1

C-1

10

The --in vitro transcription of the viruses were carried conditions as described in Materials and Methods. N.D denotes not determined; (-) denotes no effect.

and the reverse where

no protein Finally,

kinase

we studied using

little,

if

at all.

contamination a residual

of protamine abolished

(Fig.

the L protein

this

labeled protein

1).

donor

of the NS protein

of the L protein

in purified

It

band migrated band has not

RNP.

of the NS protein the --in vitro

(20%)

had no effect

concentrations

tested.

slightly been

faster

characterized.

880

than

is

interesting

remained

on the It

very due to

to note

even at 1 ug/ml was virtually phosphorylation

was also

NS protein

at protamine

was possibly

transcription

protamine

5, significant

was inhibited

of

at the various

the phosphorylated

was observed

M protein

Moreover,

protein

and analyzed

of phosphorylated

concentration

was reovirus

--in vitro.

The appearance

phosphorylation at which

The exception

As shown in Fig.

Phosphorylation

the protein

standard

on the endogenous

electrophoresis. (80%)

of 0.5 pg/ml.

of protamine

as the phosphate

gel

of phosphorylation

concentration

second

y-32 P-ATP

out under

virus.

on RNA transcription

the effect

by polyacrylamide

inhibition

trace

of Rous sarcoma

had any effect

activity

proteins

that

transcription

Casein

observed

(lanes

of that

B, C, & D);

a

Vol. 98, No. 3,198l

BIOCHEMICAL

AND

A

BIOPHYSICAL

B

RESEARCH

C

COMMUNICATIONS

D

5. Effect of protamine on the endogenous protein kinase activity. VSV cores (50 up) were incubated In the complete reactiog2mixture (0.2 ml) at varying with 50 UM ATP and [y P] ATP (final specific activity = protamine concentrations 4000 cpm/pmole) for 15 min at 30°c. The samples were precipitated with 20% trichloroacetic acid containing 1 mM ATP, 1 mM EDTA. The precipitated proteins were treated with 0.1 N NaOH in the cold for 1 min, reprecipitated with 20% trichloroacetic acid washed, solubilized, and analyzed by 10% polyacrylamide slab gel as described (8). Autoradiogram of the gel was made using Kodak SB5 film. A - without protamine, B - 0.25 ug/ml protamine; C - 0.5 ug/ml protamine; D - 1 ug/ml protamine. Fig.

DISCUSSION In this highly

conrmunication

basic

activity

of purified

ATP in

either

by dilution

the

inhibitors

polymerase.

which

3) or by adding

(Fig.

4).

Of the

the

interaction

of the viral

and ARA-ATP

nucleotide that

Since

In the

protamine inhibitory with

RNA polymerase

for

appears

which

effect the viral

binds is

at

protamine been

protein(s)

proteins

(L,

881

shown

of behaves

to be

of VSV (17). --in vitro

(18),

the initiation

reversible

can be

concentrations

RNA transcription

VSV RNA synthesis

possibly

effect

respect

have also

was achieved

to be at the

increasing

latter

weight

RNA polymerase

of RNA synthesis

The inhibitory

1).

(Fig.

of ATP in --in vitro

initiating

Seem to indicate

(Fig.

a low molecular

the viron-associated

The inhibition

as lo-'M.

to phosphonoformate

competitive

nature

mixture

protamine,

A 50% inhibition

of RNA synthesis

the reaction

similarly

ATP is

of as low

that

inhibit

VSV --in vitro.

of initiation

reversed

shown

can effectively

protein,

at a concentration level

we have

it

site

appears

is weak.

NS, or N (1))

It binds

Since

the results of the

that is not

RNA

the known

to protamine.

Vol. 98, No. $1981 The NS protein

appears

and protamine NS protein

band 4) is

(19)

and

phosphorylation

is more sensitive

of the

full-length

results

suggest

that

the primary

and not

the replicase.

Isolation

will

be important

transcription

it

seems that

regions

that

interact

with

RNA or DNA synthesis associated (Table

1).

interaction

histone

work

histone

of protamine

specific

stimulated

polyribonucleotide-directed leukemia

on the role

VSV in particular

virus

(20).

There

of these

Further

fractions also

viruses

that

viruses

if in

than

These

transcriptase

histone (Table

1).

shown protamine

inhibits

contain

virionprotein

is mediated or by inhibition

this

IIA

some specific

that

however,

along

in viral

only

an endogenous

proteins

any,

the -in

may contain

inhibition

one report,

studies

--in vitro

proteins

that

synthesis line

will

kinase by the of the

showed

polydeoxyribonucleotide

of phosphorylation, and animal

is

viral

of

inhibition

shown).

may be the

and in some cases

with

to the

VSV transcription

this

the

inhibition

that

not

to note

animal

show whether

kinase.

(data

We have

will

protein

light

or these

by several

endogenous

murine

inhibit

RNA or DNA polymerases

Whether

is

of the protamine-binding

interesting

proteins

as NSl

by protamine

by protamine

(3)

the role

VSV proteins.

--in vitro

Further

is

designated

observed

and characterizaton

It

conditions

related

of protamine

to understand

protamine

recently

strand

target

inhibitory

inhibited

to inhibition

plus

and replication.

and IV among the basic Thus,

We have

phosphorylates

the additional

of NS protein,

directly

a phosphoprote

(a)

as NS2 (19). is

is that

whether

is not

by protamine

COMMUNICATIONS

it

activity

under

component that

because

clear

phosphorylation

unclear.

RESEARCH

kinase

not yet

of NS, designated

is also

mRNA synthesis

protein,

is

NS protein

of NS protein

synthesis

protein

phosphorylated

form

of transcription

vitro

It

during

the residual

hyperphosphorylated

capped

5).

appears

the lesser

(b)

BIOPHYSICAL attractive

the endogenous (Fig.

that

AND

to be particularly

inhibits --in vitro

protein (Fig.

BIOCHEMICAL

histones by

shed some

on transcription/replication

of

general.

REFERENCES 1. 2.

Wagner, R. R. (1975) in Comprehensive Virology R. R. Wagner, eds. (New York: Plenum Press), Moyer, S. A. and Banerjee, A. K. (1975) Cell&,

882

4, H. Fraenkel-Conrat pp. l-80. 37-43.

and

in

Vol. 98, No. 3,198l

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A. K. (1980) Proc. Natl. Acad. Sci. Testa, D., Chanda, P. K. and Banerjee, 77, 294-298. S. U. and Yu, Y. H. (1975) J. Virol. l5, 1348-1356. Emerson, Clinton, G. M., Burge, B. W. and Huang, A. S. (1978) J. Virol. 7, 340-346. Kingsford, L. and Emerson, S. U. (1980) J. Virol. 33, 1097-1105. Inblum, R. L., and Wagner, R. R. (1974) J. Virol. l3, 113-124. Moyer, S. A., and Summers, D. F. (1974) J. Virol. 13, 455-465. Ando, T., and Watanabe, S. (1969) Int. J. Protein Res. 1, 221-224. Roux, L. and Kolakofsky, D. (1974) J. Virol. 13, 545-547. Banerjee, A. K., Moyer, S. A. and Rhodes, D. P. (1974) Virology 61, 547-558. Spencer, E., Shuman, S., and Hurwitz, J. (1980) J. Biol. Chem. 255, 5388-5359. Smith, R. E., and Furuichi, Y. (1980) Virology 103, 279-290. Plotch, S. J., and Krug, R. M. (1978) J. Virol. 25, 579-586. Furuichi, Y., and Shatkin, A. J. (1977) Virology 77, 566-578. A. (1980) Proc. Natl. Acad. Sci. USA 77, 847-851. Boone, L. R., and Skalka, A. K. (1980) Virology (in press). Chanda, P. K., and Banerjee, Testa, D., and Banerjee, A. K. (1979) J. Biol. Chem. 254, 2053-2058. Clinton, G. M., Burge, B. W. and Huang, A. S. (1979) Virology 99, 84-94. Manly, K. F. (1974) J. Virol. 13, 305-311.

883