The effect of methylglyoxal bis(guanylhydrazone) on vaccinia virus replication

Vol. 73, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFFECTOF METHYLGLYOXAL BIS(GUANYLHYDRAZONE) ONVACCINIA VIRUS REPLICATION

J.D. Williamson Department of Virology, St. Mary's Hospital Medical School, Paddington, London W2 1PG Received

September

8,1976

SUMMARY:

Methylglyoxal bis(guanylhydrazone) , an inhibitor of S-adenosylL-methionine decarboxylase, inhibits partially the yield of infectious, progeny virus in vaccinia-infected HeLa cells. This effect is reversed by the simultaneous addition of spermidine showing that this polyamine is required for optimal virus growth. Although DNA, RNA and protein synthesis are unaffected, the formation of DNA-containing, cytoplasmic inclusions characteristic of vaccinia infection occurred at a reduced frequency compared with non-inhibited controls. Thus, the association of virus DNAwith cytoplasmic inclusions is decreased in the absence of spermidine synthesis. The nature of residual virus replication in the presence of the inhibitor is discussed.

Previous studies in this laboratory cells with vaccinia in ornithine

virus results

have shown that infection

in both quantitative

decarboxylase (EC. 4.1.1.17)

the formation of putrescine,

the initial

activity

(1).

of HeLa

and qualitative

changes

This enzyme catalyzes

step in polyamine biosynthesis.

Two

polyamines, spermidine and spermine, have been shown to be present in vaccinia virions

and the synthesis of these polyamines continued after

The biosynthesis

hydrazone) (MGBG)is a potent inhibitor

the partial

the effect inhibition

decreased yield

(2).

of spermidine and spermine in mammaliancells is affected

S-adenosylmethionine decarboxylase (EC 4.1.1.50).

investigates

infection

Methylglyoxal

of this enzyme (3).

of MGBGon vaccinia

virus replication

of the production of infectious,

is accompanied by a reduction

of DNA-containing inclusions

by

bis(guanyl-

This paper and describes

progeny virus.

This

in the frequency of formation

in the cytoplasm of vaccinia-infected

cells.

METHODS: The laboratory line of HeLa cells used (4) was grown in Eagle's minimum essential medium (MEM) containing 5% calf serum. Confluent monolayers of HeLa

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

120

Vol.73,No.

1, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cells in test-tubes(1 to 2 x lo6 cells/tube) were infected with the Lister strain of vaccinia virus using 5 plaque-forming units (p.f.u.)/cell. After washed with adsorption for one hr the inocula were removed, the monolayers Hanks' balanced salt solution and the infected cells maintained subsequently in medium containing various concentrations of the inhibitor. The maintenance medium used was MEM supplemented with 2% calf serum. All procedures were carried out at 37'C. After incubation for 18 hrs the infected cells were disrupted by two cycles of freezing and thawing followed by ultrasonic treatment. Infectivity titres were determined by plaque formation in cultures of Vero cells. The synthesis of DNA, RNA and protein in infected and control cell cultures was examined by incorporation of C3H]-thymidine (sp.act. 5 Ci/mmol), C14Cj-uridine (sp.act. 492 mCi/mmol) and C3H3-leucine (sp.act. 57 Ci/mmol), respectively. All labelled compounds were obtained from The Radiochemical Centre, Amershem, Buckinghamshire. Infected and sham-infected HeLa cell monolayers were maintained in medium containing the appropriate labelled precursor together with a suitable concentration of the inhibitor. After 6 hrs the amount of incorporated radioactivity was determined (4). The incorporation of c3Hl-thymidine into the cytoplasmic fraction of infected cells was measured as described previously (5). The appearance virus-infected cells

of cytoplasmic, was visualized

DNA-containing inclusions by acridine orange staining

in vaccinia (6).

Methylglyoxal bis(guanylhydrazone) dihydrochloride monohydrate was obtained from Aldrich Chemical Co. and spennidine trihydrochloride from Sigma Chemical Co. RESULTS: The production cells

was reduced

of infectious progressively

0.1 mM-MGBG.

Maximal

concentration

of the

(Table

1).

inhibition

resulted of this

to maintenance

cultures

immediately

inhibitory

effect

was obtained

required

that

virus Eagle's ation

for

synthesis

production of this

Since

higher

yield

and similar

medium,

was observed

inhibitor

HeLa cell

is

results

present

virus in

with

yield

continues

obtained

replication intracellular

121

by the

0.5 mM-MGBG supplied

to

results

not using

yield of

reversal

effect

of the

show that

spermidine

progeny

in vaccinia-infected

virus

further

reductions

a serum-free,

modified

at the

and

cells.

may have resulted pools

than

addition

of infectious,

of MGBG did were

of virus

Complete

These

greater

0.5 mM-MGBG and this

was examined

infection. 1).

HeLa

of concentrations

medium containing

(Table

concentrations

of spermidine

effect

after

polyamine

in vaccinia-infected

in a 70% reduction

of the full

residual

virus

in the presence

The specificity

1.0 mM-spennidine

, progeny

from time

the

utiliz-

of infection.

of

Vol. 73, No. I, 1976

BIOCHEMICAL

Table 1. infectious

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The effect vaccinia

MGBG hnM)

of MGBG on the production of virus in HeLa cell monolayers

Infectivity (p.f.u./ml)

0 0.10 0.25 0.50 1.00 0.50 + mM spermidine

1.00

Consequently, presence

confluent

of 0.5

in the

of the

released

difference

from

from

cells

x lo6

infection

compared

the

maintained

time

p.f.u./ml

any cytotoxic

cell.

plasmic

fraction

91

for

42 hrs

inhibitor.

after

infection

released

cells

from

in

exposed titre

and after

with

Microscopic

of

to the of virus

infection

the inhibitor

examination

containing

subsequently

showed no significant cells

before

the

Titration

The infectivity

treated

in medium

maintained

and maintained

of the

of MGBG both from

were

infected

only.

of vaccinia

virus

in

the

occurs

incorporation

of vaccinia-infected

presence

from was

after

of uninfected

0.5 x&l-MGBG did

system

cells

used,

in the

the

incorporation

C3H.J-thymidine

is

a measure

is complete

to control into

cells.

infected

of thymidine Neither

or control

not

reveal

122

of the into

the

cyto-

of virus-specific

DNA

by 6 hrs post-infection was unaffected. into

whole,

the incorporation cells

cytoplasm

of

of 0.5 mM-MGBG such incorporation

same conditions

L3HJ-leucine

x lo6

virus

p.f.u./ml.

Consequently,

which,

was similar

9.95

effects.

infected

In the

100 100 65 32 34

then

of infection

x lo6

The replication

synthesis

with

and that

maintained

lo7 lo7 lo6 lo6 lo6

at 18 hrs

in the presence

was 2.85

cultures

cells

yield (%I

x x x x x

of HeLa cells

24 hrs,

Virus

1.10 1.12 7.10 3.62 3.83

same concentration

these

in yield

inhibitor

2.74

mM-MGBG for

presence

virus

monolayers

titre

was affected

(4). Under

uninfected of

cells

[ 14C1 - uridine

by the

inhibitor

the

nor

BIOCHEMICAL

Vol. 73, No. 1, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of MGBG on macromolecular synthesis 2. The effect uninfected and vaccinia virus-infected HeLa cells

Table

Incorporated

radioactivity

(dpm x 10

C3Hl -thy-midine

Control

Inhibited

6,559

6,558

-3

)

Infected

Uninfected Precursor

in

Control

Inhibited

100

534'

550*

103

% control

% control

C14 Cl-uridine

940

914

97

615

653

106

C3HJ-leucine

633

679

107

676

685

101

* Radioactivity

(Table

2).

incorporated

These

results

maximum inhibition or protein

virus

of 0.5 mM-MGBG cytoplasmic

cultures

were

guishable occupies

under

in MGBG-treated The inclusions diffuse of virus MGBG were results

these

in every a large

at 6 hrs

DNA is

inclusions.

proportion

of the

cell

was about

40% of that

in both

inhibited

and control

a stage

of development

without show that

in unstained effect the

on the association

cultures

of virus

123

observed

cells

that

were

subsequently

of cytoplasmic

DNA is

not distinnucleus their

cultures

with

Concentrations

DNA with

virus

However,

associated

In the

when infected

the HeLa cell

volume.

can

cells.

in non-inhibited

(7).

frequency

by the

inclusions

inclusions

because

cells

particles

normally

confirming

cell total

HeLa cells.

These

were

produces on DNA, RNA

of vaccinia-infected

Cytoplasmic

infected

effect

accompanied

post-infection

control,

in appearance,

of MGBG which

measurable

inclusions

conditions.

only

or vaccinia-infected

staining

presence

synthesized

is without

orange

fractions

a concentration

DNA-containing

by acridine

examined

cytoplasmic

uninfected

of vaccinia

of cytoplasmic,

be visualized

growth

in either

The synthesis formation

show that

of virus

synthesis

into

(Table

became

3).

more

the maturation less

inclusions.

cytoplasmic

frequency

than

0.1 mMThese

inclusions

is

Vol. 73, No. 1, 1976

Table containing,

BIOCHEMICAL

3. The effect of canaline on the frequency cytoplasmic inclusions in vaccinia virus-infected

Number of cells examined

Treatment Without MGBG With 0.5 mM-MGBG

reduced

markedly

the yield

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Number of cells showing inclusions

572 631

in the presence

of infectious,

Cells showing inclusions(%)

207 90

of concentrations

progeny

of DNAHeLa cells

36.2 14.3

of the

inhibitor

that

affect

virus.

DISCU.SSION: Polyamines ing

have been

two groups

could

function

when

available

of large,

before

cells

and,

However,

ornithine

infection

in vaccinia

virus

(2).

virions

of infectious,

the

effect

is

reversed

These

results

virus

and confirm

Increased transformation polyamine

virus

show that

inhibited

before

shows that

simultaneous

spermidine

previous

is

observations

virus

virus. (9).

since

of exogenous

reduced the

with

yield

inhibitory

spermidine.

the replication

polyamine

polyamines

the production

This

cells.

cells

of cells

markedly

synthesis

that

simplex

of those

infection

MGBG reduces

for

synthesis

replication

synthesis

or after

required

the

only

in vaccinia-infected

the

addition

that

virions

in herpes

affect

in vaccinia-infected

of vaccinia

biosynthesis

continues

cells. RNA and protein of lymphocytes

levels

in

ornithine

in herpes

showed

increases

of MGBG on polyamine

by the

in vaccinia-infected

activity

represent-

Exogenous

present

MGBG does not

when added

effect

is

of virions

viruses.

studies

as a precursor

study

progeny

Other

putrescine

consequently,

The present

from

from

can serve

found

(8).

decarboxylase

and ornithine

animal

parts

to the polyamines

and spermine

infected

results

DNA-contatiing

as a precursor

of spermidine

(1)

shown to be constituent

(10).

These

synthesis is

resulting

accompanied

increases

from

by marked

in macromolecular

124

concanavalin elevation synthesis

A-induced

of cellular continue

BIOCHEMICAL

Vol. 73, No. 1, 1976

unaltered

following

Under

these

ation

of thymidine

inhibition

conditions,

In the presence

(11). production

DNA, RNA or protein

in frequency synthesis,

association

of virus

been

compared

from bacterial

cells

and production

of progeny

in vaccinia present

virus

Since of virus cells

to the

residual

virus

higher

growth

virus

cores

inhibitor

virus

virus

both

replication

accumulation

of putrescine

inhibitor

(10).

residual

ornithine

of rat

spermidine virus ornithine.

fell levels

in HeLa cells Although

and after

Although

virus

hepatoma

cells

activity rapidly

dropped is

there

growth

virus-specific

nucleoids

a similar

role

shown to be

was

it

no

spermidine

virus

following ornithine.

that

of MGBG suggests replication.

inhibition

'The of putrescine

of DNA synthesis The replication

DNA synthesis

is

of the

Although

an oxyamino

of

pools

synthesis

in the presence

(13).

125

unlikely

in the presence

inhibition

by canaline,

is

exposure

of intracellular

is blocked

byoc-methyl

reductions

following

in vaccinia

significantly

inhibited

has

formation

further

infection,

continues

of putrescine

decarboxylase

concentrations

obtained

were

effect

from utilization

affected,

proliferation

in isolated

has been

yields

of infection.

participation

of

polyamine

of MGBG suggest

spermidine

a 60%

of the

in inclusion

not

results

at the

Thus,

This

of MGBG did

before

available

the possible

time

showed

suppression

reductions

formation

(2).

concentrations and similar

on HeLa

the

cells

DNA conformations

Indeed,

the

effect

Inhibition

inclusions.

in the presence

replication.

in vaccinia

in a marked

The parallel

(12).

maximally

was unaffected,

controls.

condensed

mitosis

or vaccinia-infected

untreated

cytoplasmic

the

reduces

of infected

results

incorpor-

into

was no measurable

uninfected

synthesis.

in the

of cells

cytoplasm

with

therefore,

shown to stabilize

there

of thymidine

in the

DNA with

of entry of MGBG that

in either

inclusions

spermidine

rate

virus

the incorporation

of DNA-containing reduction

, progeny

and spermine

was a 60% reduction

of a concentration

synthesis

Although

cells.

there

DNA and in the

of infectious

RESEARCH COMMUNICATIONS

by MGBG of spermidine

however,

into

AND BIOPHYSICAL

until

of vaccinia analogue

was reduced

of

by 40% only

(5>,

Vol. 73, No. 1, 1976

the

formation

compared

of DNA-containing,

with

inhibitor

non-inhibited,

of pyridoxal

decarboxylase the

BIOCHEMICAL

present

inclusions ization

(14). study, observed

of ornithine

MGBG that

leads

AND BIOPHYSICAL

cytoplasmic infected

These

effects

suggest

that

in the for

inclusions

cultures

phosphate-dependent

(6).

the association

polyamine

of the

of infectious

was supported

by grants

a 60% reduction is

including with

of virus

DNA with

the

can occur The nature

, progeny

virus

a potent

ornithine

together

inhibitors

biosynthesis.

to production

showed Canaline

enzymes,

of canaline,

presence

RESEARCH COMMUNICATIONS

results

of

cytoplasmic without

of resistance is currently

utilto under

investigation. This 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

work

from

the Medical

Research

Council.

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