Effect of metabolic inhibitors on interferon activity

LIFE SCIENCES Vol. 6, pp. Printed in Great Britain.

EFFECT OF METABOLIC A. K. Field,

1483-1491,

INHIBITORS

1967.

Pergamon Press

Ltd.

ON INTEIKFERON ACTIVITY

G. P. Lampson and A. A. Tytell

Division of Virus and Cell Biology Research Merck Institute for Therapeutic Research West Point, Pennsylvania

(Received 6 March 1967; in final form IO April 1967) Although nearly ten years have elapsed

since the discovery of interferon

(1), until recently little progress has been made toward understanding mechanism by which interferon Glasky et al.

(2) reported

of a RNA synthetase have presented

mediated

through

evidence

cells from virus infections.

that interferon directly

from virus infected

evidence

specific blockade

protects

cells.

that the antiviral

of viral RNA translation induction

action was based primarily

DNA-dependent

inhibitors

activity

(8).

as actinomycin

antimetabolites

periodically

embryonated

eggs.

for interferon

action was inhibited by Actinomycin

medium containing

(5,6).

was to broaden the spectrum of the

action.

in continuous

culture

lO~ agamma calf serum.

in Eagle's

These cultures were

free of PPL0 contamination.

Disease Virus

Allantoic

D inhibits

and Methods

cells were carried

Newcastle

is required

activity to better understand

in interferon

checked and maintained

Virus.

from

(7) and also blocks at least one enzymatic

Materials

minimal essential

synthesis

D and puromycin.

tested for anti-interferon

L(MCN)

resulted

and suggested that this block was

of this investigation

role of cellular macromolecules

Cells.

the action

Marcus and Salb (3)

(4), but this may not be its sole activity

synthesis

The purpose

inhibited

activity of interferon

on the fact that interferon

RNA synthesis

Puromycin blocks protein

Recently,

of a new protein by interferon.

The concept that new RNA and protein

such metabolic

However,

the

(NDV) stock was prepared

fluids containing

1483

from infected

NDV were stored as i mi aliquots

1484

INTERFERON ACTIVITY

Vol. 6, No. 14

in sealed ampules at -70°C. Interferon.

Interferon was prepared from the maintenance medium of mouse

spleen suspension cultures 18-20 hours after infection with NDV.

Crude inter-

feron was treated with O.IN HCIO 4 overnight at 4°C and then chromatographed on CM-gephadex.

This partially-purified mouse spleen interferon

in a fraction eluted at pH 6.6.

(MSIF) was obtained

The interferon prepared in this manner exhibited

no cell toxicity and contained no infectious NDV. Interferon Assay by Hemadsor~tion Inhibition.

In 1964, Finter

duced the use of hemadsorption inhibition for interferon assay.

(9) intro-

The following

modified procedure was found convenient for purposes of this study. Growth medium drained from Leighton tube cultures containing approximately 105 cells was replaced by addition of i ml samples of MSIF dilutions. Normal and NDV control cultures received i ml of growth medium. incubated overnight at 35°C and then inoculated with 0.i ~

Cultures were

of sufficient NDV

to infect approximately 50% of the cells after an additional overnight incubation period.

Normal control cultures received rlo inoculum.

The percent of

cells infected was determined by replacing the overlay fluids with 2 ml of a 0.5% guinea pig RBC suspension,

incubating at 4°C for 15 minutes,

and counting

both cells that had adsorbed RBC (HAd+) and those that had not (}~d-). infected = i00 x

% cells

# cells HAd+ cells HAd+ plus # cells HAd-

Detection of Anti-interferon A~ents.

Chemicals tested for their effect

on interferon action were added 0-3 hours before addition of interferon and maintained at the stated concentrations for the duration of the experiment. those cases where the chemical was inhibitory to NDV infection,

In

cultures were

thoroughly washed to remove the chemical and excess inter2eron before infection with NDV.

Such a procedure had no effect on the ability of interferon-treated

cells to resist NDV infection. Chemicals. in Table II.

Concentrations used in the experiments described are indi-

Vol.

6,

No.

14

INTERFERON ACTIVITY

1485

Results Serial dilutions NDV replication,

of interferon have been titrated for ability to inhibit

as reflected by the proportion of cells in an infected culture

capable of adsorbing guinea pig RBC (HAd+ cells) cells was inversely proportional

(lO).

to the interferon concentration.

untreated MCN cells were HAd-, whereas NDV infected overnight

The proportion of HAd+

incubation but not immediately

Normal

cells became HAd+ after

after virus adsorption

(Fig. 1).

70 60 I

50 40 I--I.u ~J

30 20 I0 x

20

x

x

x

i

i

I

40

I

I

I

I

i

T

80 160 320 640 1280 2560 5120 10,240 RECIPROCAL INTERFERON DILUTION

FIG. i Interferon t~tration by hemadsorption inhibition. X = percent HAd+ cells after treatment with the indicated IF dilution and NDV infected; 0 = percent HAd+ cells in NDV infected culture; • = percent HAd+ cells in uninfected culture.

1486

INTERFERON ACTIVITY

The action

effects

of' m e t a b o l i c

are presented

summarized

in T a b l e

in T a b l e

Addition protective

with

cell

MSIF

capacity

of actinot~ycin

indicating

The presen
interferon

NDV

with

D to

free

dilutions

inl,~ri'eron :fh¢.sc r s u i t s

~.r~

the

subsequent

<:hall age.

Preincu0ation

and

NDV

NDV

actinomycin

D if

. r±'c't wa:

to

~u.lm pro-

ii;tinq

/rom

~h<

imr( :iSot . i~thibiL, e.~ 'k'V i J l ~ < t i o n .

by c~'cAok~}xi::ii<

Cu±tur<

before

inhibited

susc~ptibility

of N D V

or
of H S I F incr
the a n t i - i n t ~ r ~ e r o n

infecJion.

: treabo~

2[DV e h a l l e n g <

an<

}{ow-

i ~ ~, :~.ro.~d ~y

w_1:~ ± h ~ r:< ±'
,xiS:.'t;(,i ~A{'k

effect

C,

were

each

infection.

The

interferon

actio~

O-mercaptopurim<

interferon

before

P]itomycin

action,

on

NDV

chaikeng(

5-mercaptomcthy±

inhibitory rovcrs9

~ i~Lh~bi~:ion O~

wai: c a u s e d


in ,:<:iX:/ pr{ tr<<<<. [ wi~n

~rc:/,:i.i

(~)-SilCH..-i)

an:

;%imul~N;ion

by

:i fix<

]xh z::<:~i ~])V inf
<-SII-[)U


.

%o i n t e r f e r o . u cSl'cct,

(<:-S[{-PU)

ilowu/cr,

[fl)Y inre<:tior:

5-fluoro-2'-deoxyuridin< NDV

14

simultaneously

;;~imulate

cycloheximide,

inhibited

washed

(idU)

~hat

washed

ha(l ~'io s t i m u l a t i n g then

No.

protection.

As an~l a l s o

~o

addition

eL c e l i s

au~(] t h e n

a~x

respectiwly.

o! c y c l o h e x i m i d ~ ~ COil!p±tt~ly

pretreatmenL

heximide

2 A-H,

serial

while

population

before

thus

enhanced

D with

of i n t e r f e r o n

tection,

washing

Fig.

on ~'YDV i m f ¢ c t i o n

6,

II.

of t h e u n p r o t e c t e d

evexb

I and

of a c t i n o m y c i n

action

overnight

inhibitors

Vol.

on withodt

al::

io, io ~ < c / j r r i , ! i n <

afpar~t

ff< :~ ,~n riD'.'

o k l'Jl) i~]k .'tio:~ ri::iot~

a!.i~onuci~o;::i,~: (l't!dR) u l t < r c ]

I


{~u_"ot~%-.!; ~)
in~.'

/yo:~ :<~:![c,~ ::or

infection. D i s c u s { io:l Harcus

of a new This

cellular

mechanism

apparatus iation

and

for

to t h e

for

Salb

have

protein

suggested

which

expression

cellular active

DNA

then

Lllilt iliL(rioro:~ block'

of a m % i v i r a l

transcription

protein.

However,

~:ck

ti'~nli:N~iol~ activitj

wo~tli

to m e s s e n g e r inhibition

o.

RNA

o[' D N A

:;~. iI ~%tc_"nd 'syKth(}: 2;2 [~_'a/i m
th,

h tact

;k~osequent

{f~th
RNA.

trap_:>

a'. 're:i± :~,;

Vol.

6,

No.

14

INTERFERON ACTIVITY

1487

TABLE I Effect of Chemical Treatment on NDV Infection

1 Relative proportion of cells infected

Chemical treatment of cells prior to NDV infection 2 3 4 5 6 7 8 9 i0 ii

1.O

1.8

0.0

1.6

1.O

i.i

i.i

0.9

0.2

i.i

1.6

i. no pretreatment, 2. actinomycin D (0.5 ~g/ml), 3- cycloheximide (i0 ~g/ml), 4. cycloheximide (i0 ~g/ml but removed by washing before addition of NDV), 5. mitomycin C (5 ~g/ml), 6. IdU (20 ~g/ml), 7. FUdR (20 ~g/ml), 8. 5-SHCH2-U (i0 ~g/ml), 9. 6-SH-PU (i0 ~g/ml), i0. 6-SH-PU (i0 ~g/mlbut removed by washing before addition of NDV), and ii. aminonucleoside (30 ~g/ml).

TABLE II Summary of Alterations in Interferon Action and NDV Infection by Chemical Pretreatment of Cell Cultures

Concentration tested

Effect on interferon

Effect on NDV infection

Actinomycin D

0.5 ~g/ml

inhibited

stimulated

Cycloheximide

i0 ~g/ml

inhibited

inhibited* stimulated

5 ~g/ml

inhibited

none

5-mercaptomethyl uracil

lO ~g/ml

inhibited

none

6-mercaptopurine

i0 ~g/ml

inhibited

inhibited

lododeoxyuridine

20 ~g/ml

inhibited

none

5-fluoro-2'-deoxyuridine

20 ~g/ml

none

none

Aminonucleoside (puromycin)

30 ~g/ml

none

stimulated

Mitomycin C

*NDV replication was inhibited by the presence of cycloheximide but enhanced in cells pretreated with and then washed free of cycloheximide.

1488

INTERFERON A C T I V I T Y

Vol.

6, N o .

i.O

A

0.8

K

0.6 0.4" A T

0.2 (/) J J W L) 0 bJ l0 bJ b. Z

i,

I

0

0

1.0

~.0"

0.8

0.8

0.6,

06

MC $ IF

0.4,

0.4

0.2.

0.2

0

b. O

0

1.0"

Z O FE O Q. O E n W >

1.0

0.8,

08

0.6

06

0.4

04

0.2

02

0

0

=°~ " ~ e ~

.J hi E

04t

0.2

°81

o.6t

G

~.N.,F o

l0 08"

~

H

0.6

0.4 0.4 t 0.2

0.2

0 2O

0 Fig.

2.

RECIPROCAL

INTERFERON

DILUTION

Effect of chemical treatment on interferon activity. IF= interferon, Act D=ectinomycin D, Cy =cycloheximide, MC = mitomycin C, IdU =iododeoxyuridine, 5-SHCH z- U= 5- mercaptomethylurocil, 6- SHPU= 6- mercaptopurine, AN = aminonucleoside (puromycin), FUdR = 5- fluoro- 2'- deoxyuridine.

14

Vol.

6, No. 14

INTERFERON ACTIVITY

1489

ribosomal RNA synthesis, would not be expected to interfere with interferon function. FUdR blocks thymidine synthetase activity, resulting in the inhibition of DNA synthesis (ll).

Lack of anti-interferon activity by FUdR, present in a

concentration exceeding that required to inhibit DNA synthesis, indicated that DNA synthesis was not necessary for expression of interferon activity. Mitomycin C has been shown to cause interstrand cross-linking in DNA molecules, inhibition of DNA synthesis, and degradation of DNA during posttreatment incubation (12).

IdU (13) and perhaps 5-mercaptomethyl uracil are

thymidine analogs, while 6-mercaptopurine is a purine antimetabolite.

The anti-

interferon activity demonstrated by these compounds emphasized the requirement for functional DNA as well as functional RNA for interferon action. As shown above and elsewhere (14), actinomycin D blocks interferon action, further suggesting that transcription from DNA to RNA is important for interferon expression.

However, since aminonucleoside puromyein has been shown to pri-

marily interfere with ribosomal RNA synthesis while leaving messenger RNA and transfer RNA synthesis intact (15) and was not inhibitory to interferon action, the required transcription probably involves messenger RNA and/or transfer RNA. The need for protein synthesis in interferon action was substantiated by the inhibitory action of cycloheximide, an inhibitor of protein synthesis (16). However, whether this new protein acts directly as the antiviral substance, is involved in altering the interferon molecules to become the antiviral substance, or alters the cell so interferon may act, remains pure speculation. Another point which has become clear from the above studies is that the stimulation of NDV replication did not necessarily result from inhibition of interferon activity.

Miton~cin C and 5-mercaptomethyl uracil inhibited inter-

feron action without stimulating NDV replication, while aminonucleoside stimulated NDV replication without affecting interferon action. Finally, these studies emphasize the need for caution in interpreting the effects of antimetabolites on interferon induction if rigorous measures have

INTERFERON A ~ I ' I V I T Y

1490

not been

taken

a recent

demonstration

duction viral

may

to e l i m i n a t e

actually

inhibitory

by F,urk< refl(ct

(£()

eff
o11 ]n!
~ X.LU'~:s~,Lol~.

mito~ro-< ]:~ i ~ h i b ] :

an i n h i b i t i o n

(i~ N o .

Ve, l.

oY oxpr<:,sion

]~

r[

1.1

[~hi~ ,

I~NL ])ro-

,oY ]nt, r!, to:,': a n t ] -

activity. C o ~ c l u s ion A rapid

Diseas( The

Virus

has bFen

action.

uracil~ of

to a d s o r b

technique

feron

t
bas(d red

6-mercaptopurin~.~

deoxyuridine

but ~nd

not

~be a b i l i t y

biood

ceils

D~

These

requires

functional

i( t.Lopid

o[' : ' o m p o ~ n d


~:: :J.ht~So:~J ;t : !,o itlt(,r-

iodo,[I t)i£/i~ri< [i!,

~uH] mito~l%;cin C~ w(r(~ i n h i t i L o r j

hich

studi(~:

of c~3 [: in!(~ (, i ~',7 i],wc;
h(~n

<'J,:?lo~Lek]H:Jl]< ;

conc<~!b~'&~ioii;i

~iilinoiluc].(:osidt

t%ction.

has

l{sed f o r e v % ± u a t i o n

Actinor:%'cJn

interferon

on

iN[~(:r'J£'~'!)~

to

]t:,w . . e ~ , : ~ L t . r a t : [ , o n

Wi i.i~

:-i ] iot.q-2k'-

pNroH~'eili w(]~( liOl ]nil] i;[ i~)r',' : o

i{,~<] su[,[~orl

(
Oi

i -~!!(r<'~i~ [_o!K?[h2/]

Io the i~ypot}~ : i: ~h:~

])li2\~ transcrip!

k] (]~ [

]i[ ~ i'_t'(FOL~

:~ f :':, roll :<,'%[o!~

]/{ "l [[ (; [ : : { [ ] [{ ~ " ~ : [ J/~ ,

< ] ~ [:

[ [ I: : [ : '~[ [ ~

[

to p r o t e i n . Acknowie4o{ i < n t T-ha a u t h o r s invaluabi(-

wish

techni<'ai

to % h u ~ k

Hr:.

i. Li{~I k~, i ~

~,'::. ~ . k , n ~ '

I 9r :,k~Jr

:i: ::]::!,an<, . ]<(:l'<~r'Oll''<

l°

2.

A. J .

Glasky~

3.

P.

H a r e m s an~ J .

4.

E. R e i c h ,

I.

L. S i m o n ~uld J .

C. Ho.]p<~r~ Sci
Pl. S&!b~ V i r o l o g 3 , 30~

R. H. 9'r
A. d.

Sh~tkin

.N~'t~ ~ .i (i }()ii).

£,.02 (.~ < £ ) .


i'~'l
.aJ~

>;!

(1961). ~.

V. H. Zhdazlov and I". i. Y~'rshov~ d. C
6.

J.

7-

M.

$.

H. H.

9"

N. B. Finter, Viroio6~/ 2~, t~+:) (lp6~).

Laszlo~

(~).

D. S. M i l l e r ~

]3, Y a r m o l i n s k y Al0pleman

K. S. H~:Curby ~nd ].). t[<)<£~:t
a n d G. L.

and R.

bc La }laba~

C. K ( m p .

]3ioehei~.

10. P. I . Marcus, B a c t . Proe. ±$1 ( i Q 6 2 ) .

Pro~'.

Nat.

Aea:].

1{io£)h@r~]. [{
:~ : ('~,~).

S~i.h,<

N
.L>i~ .ki)C,

~, , i l:IL (i V/',0

Co:u~. 2 4 ~

b~:~

(i)0<).

Vol.

6,

No.

14

INTERFERON ACTIVITY

1491

11. S. S. Cohen, J. G. Flaks, H. D. Barner, M. R. Loeb and J. Lichtenstein, Proc. Nat. Acad. Sci. 44, 1004 (1958). 12. A. Terawaki and J. Greenberg, Bi_Ochem. Biophys. Acta 119, 540 (1966). 13. Y. Centifanto and H. E. Kaufman, Proc. Soc. Exp. Biol. and Med. 12__~0,23 (1965) 14. J. Taylor, Biochem. Bio~h~s. Res. Comm. 14, 447 (1964). 15. A. E. Farnham and D. T. Dubin, J. Mol. Biol. 14, 55 (1965). 16. L. Felicetti, B. Colombo and C. Baglioni, Biochem. Biophys. Acta 119, 120

(1966). 17. D. C. B u r k e , Bioehem. J . P r o c .

9~, 2p ( 1 9 6 5 ) -