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J. gen. Virol. (I97I), Io, 273-277
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The Inhibitory Effect of Compound 48/80 on the Formation of
Giant Cells Induced by Herpesvirus Hominis
(Accepted I9 November I97o)
Formation of giant cells is characteristic of the cytopathic effect of herpesvirus hominis on
cultured rabbit kidney cells. Giant cells are the result of the fusion of many single cells
(Falke & Richter, I96o ). The stimulus for this phenomenon is linked to the early phases of
virus multiplication. It includes transcription (2 hr after infection) and translation (3 hr
after infection) of the virus genome (Falke, I967; Falke, Bitter-Suermann & Clauss, 1969).
No information on the biochemical processes leading to giant cell formation is available at
present. A possible approach to this problem is the examination of chemical substances for
their capacity to inhibit giant cell formation. We describe in the following communication
the effect of such a substance.
Compound 48/8o is known as a histamine-liberating substance (Paton, I95I); it is a
mixture of monomers, dimers, trimers and possibly tetramers of 4-methoxy-phenethyl-Nmethylamine obtained from the condensation with formaldehyde (Paton, I95I). This substance inhibits the penetration process of herpesvirus hominis into rabbit kidney cells
(Falke & Netter, I969; Falke, I97o ). The phase of penetration sensitive to compound 48/8o
begins immediately after infection and lasts for about 3o min. When compound 48/8o is
applied later than 3o min. after infection, the penetration of virus can no longer be inhibited
and the synthesis of infectious particles proceeds normally. Under these conditions, however,
there is no formation of giant cells; instead, the cytopathic effect in the presence of compound
48/8o is characterized by the phenomenon of cell rounding. The inhibition of giant cell
formation by compound 48/8o can be accomplished by applying the drug during a period
from 3o min. up to 4 hr after infection. In the absence of this drug the first cell fusions occur
4 hr after infection. Under our conditions the replication process of the virus DNA does not
begin until 3½ hr after infection, whereas the first complete virus particles cannot be detected
until 5 hr after infection (Falke, I967). We conclude, therefore, that the effect of compound
48/8o is not connected directly with the early transcription and translation phase of the
virus multiplication cycle. The phase sensitive to compound 48/8o seems rather to be correlated to the ultimate period of the metabolism which immediately precedes the fusion process
(3½ to 4 hr after infection). This phase seems to contain the triggering mechanism for giant cell
formation itself (Falke & Netter, i969).
Application of 2 5 / % of compound 48/8o per ml. of cell culture fluid (pH 7"o to 7"2) did
not alter markedly the incorporation of [aH]thymidine, [aH]uridine and Jail]amino acids
(Table I). The inhibitory effect of compound 48/8o on giant cell formation was thus not
caused by an alteration of the biosynthesis of nucleic acids and proteins. In contrast, the
incorporation of [all]choline into lipids was markedly diminished. Compound 48/8o (IO,
25, 5o, IOO #g./ml.) interrupted giant cell formation in a herpes-infected culture even when
giant cell formation had already started when the drug was applied (Fig. I). Rabbit kidney
cells were infected with about 4oo p.f.u./Leighton tube. After incubation at 320 for 18 hr,
two infected tubes were taken to determine the number of nuclei/giant cell at I8 hr. At this
time the test substances were added. After further incubation at 37 ° up to 22 or 26 hr after
infection, respectively, all cultures were fixed and stained with Giemsa solution. Finally, the
x8-2
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number of nuclei/giant cell was counted. Under these conditions the initially observed
giant cells remained small and did not show the enlargement which normally follows the
fusion process and which is typical for the development of the culture in the absence of
compound 48/80. Furthermore, cycloheximide (0.6, 1.2 and 2"4/zg./ml.), an inhibitor of
Table I. Incorporation of tritiated compounds into normal rabbit kidney cells
Compound
48/80 (/zg./ml.)
Control
[3H]thymidine
[3H]uridine
[aH]teucine
[3Hlcholine
Ioo ~o
Ioo ~o
IOO ~o
IOO ~o
(7500 counts/min.) (915o counts/min.) (z67o counts/min.) (I42IO counts/rain.)
5
io
25
50
Specific activity
(c/m-mole)
95 %
ioo%
89 %
70 %
5
I25 %
ioo%
Io5 %
78 %
6
98 %
98%
9I %
83 %
34
58 %
46%
24 %
13 %
I5"4
The incorporation of the tritated compounds into acid-insoluble material was studied 6 to 7 hr after addition
of compound 48/80, as done by Falke, Bitter-Suermann & Clauss (I969).
The values are the mean of three independent experiments.
40
I
I
I
•
A
0
D
30
.~ 20
Z
10
l
I
I
I
18
22
Hr after infection
26
Fig. I. The influence of compound 48/8o, cycloheximide or heparin upon the growth of herpesinduced giant cells. • , Virus control; II, cycloheximide (0.6, 1.2, 2"4/zg./ml.); A, heparin (75/zg./
ml.); compound 48/80 (IO, 25, 50, IOO #g./ml.) each added I8 hr after infection.
the protein synthesis, was unable to block giant cell formation when applied 4 hr after
infection or later (i.e. after the translation phase). The inhibition of protein synthesis was
reduced to 18 ~ , 9"5% and 6 ~ , respectively, of the controls. The protein synthesiswas studied
as follows. Cycloheximide was added to zero time, the radioactive amino acid I hr later. The
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experiment was terminated at 8 hr and the incorporation into acid-insoluble material was
tested.
The same concentrations were able to prevent giant cell formation completely when
given during the translation phase. Under similar conditions cycloheximide (I.2#g.)
reduced the D N A synthesis to 37 ~ . Taken together these results demonstrate that, once
started, giant cell formation itself and its biochemical mechanism, do not require simultaneous de novo synthesis of proteins.
In further experiments chloroquine, I M C B H (Nl-isonicotinoyl-N2-3-methyl-4-chlorcbenzoyl-hydrazine) and heparin, as well as the monomer (N-methyl-2-(4-methoxy-3-methylphenyl-ethylamine-hydrogen-maleate) and the dimer (3,3-methylen-bis-(N-methyl-/?-(4methoxy-phenyl)-ethylamine)-dihydrochloride) of compound 48/80 were tested. The
quaternary amine 2-(4-methoxy-phenyl) ethyl-trimethyl-ammonium iodide was also included
in the tests. Chloroquine is an activator o f lysosomes (Fedorko, Hirsch, & Cohn, 1968).
I M C B H blocks the release of vaccinia virus from the host cell (Kato, Eggers & Rolly, I969),
the heparin inhibits the penetration of herpesvirus into rabbit kidney cells (Hochberg &
Becker, 1968). In addition, cromoglycate was included in our studies. This drug inhibits
phospholipase A (Orr & Cox, I969).
Table 2. Comparison of membrane-active substances
Reduction of Inhibition of Activation Reduction
penetration giant-cell
of
of choline Histamine
of virus
formation lysosomes incorporation liberation
Compound
Cpd 48/80
Monomer of Cpd 48/80
60 %
+++
+
70 %
(25)
o ~o
(IO)
-
(25)
+ +
(25)
18 %
75 %
(I)
-
(200)
(100)
(25)
(25)
(250)
Dimer of Cpd 48/80
o%
(IO0)
(IO0)
+
(25)
8o %
(25)
(250)
2-(4-methoxyphenyl)-ethyltrimethyl-ammonium iodide
Chloroquine
o%
(lOO)
o%
(too)
-
(IOO)
++
40 %
(25)
17 %
(4oo)
-
(3 ° )
(5 ° )
(I)
(25)
(I00)
--
--
IMCBH
o %
(I00)
Heparin
Cromoglycate
(100)
(100)
8o %
++
-
(IO)
(10)
(IO0)
o%
(IOO)
-
-
(200)
(IO0)
o%
(25)
3%
(25)
o%
(25)
-(lOt))
(200)
(250)
Figures in parentheses are the applied concentrations in #g./ml. - , no effect; +, slight effect; + +,
clear effect; + + + , strong effect.
Penetration by virus was tested by addition of the substance simultaneously with the virus.
Two hr after infection the unabsorbed virus was washed out and herpetic immune serum
was added. The test was read 18 hr after infection (Falke & Netter, I969). To determine
effects on giant cell formation, the drugs were added 3 hr after infection. The test was read
18 hr after infection. The tubes had been infected with about zoo p.f.u, of giant-cell-forming
herpes strains (Falke et aL I969). Histamine liberation was measured by incubating rat
peritoneal macrophages for 30 min. at 37 ° (pH 7"o) in the presence of the respective substances. Histamine was measured in the supernatant fluid and the cellpellet by a fluorimetric
method (Shore, Burkhalter & Cohn, I959), as described previously (Falke & Kahl, I97O).
18-3
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Short communications
The degree of activation of the lysosomes was studied 6½ to 7 hr after addition of the respective substances by application of euchrysine (Allison, 1967 ).
Only compound 48/8o and heparin were able to block giant cell formation. Both substances also inhibited the penetration of the virus (Table 2). Heparin could not block giant
cell formation completely (Fig. I). In addition, the effect was reversible, i.e. heparin could
be washed out, whereas the effects of compound 48/8o are irreversible (Falke & Netter,
I969). These data suggest that the action of compound 48/8o differs from that of heparin.
Furthermore, heparin did not release histamine, whereas compound 48/80 was highly
effective in this respect in very low concentrations. The m o n o m e r and the dimer of compound
48/8o which did not prevent giant cell formation also had no histamine-releasing activities.
Compound 48/8o and the dimer were the most potent inhibitors of choline incorporation
(Table z).
Besides its well-known properties, compound 48/8o has now been shown to exert other
pharmacological actions. Thus it blocks the further enlargement of giant cells in rabbitkidney cell cultures and inhibits the incorporation of choline into lipids. The inhibitory
action of compound 48/8o upon giant cell formation seems to be correlated neither with denovo
protein synthesis nor with the lipid metabolism of infected cells, since the dimer inhibits
choline incorporation but does not block giant-cell formation. This agrees with the results of
earlier experiments, which showed that lysolecithin does not play an important role in
giant cell formation (Falke, Schiefer & Stoffel, I967). The well-documented action of
compound 48/8o - apparently of the higher polymers - upon giant cells induced by herpesvirus seems to depend on the action of some unknown protein or enzyme and/or on steric
hindrance of hypothetical membrane subunit rearrangements during the process of cell
fusion.
We wish to thank Mrs Ursa Cohn and Miss Inge Raab for technical assistance. The
investigations were supported by the Deutsche Forschungsgemeinschaft. We thank Dr K.
Schmitt of Farbwerke Hoechst A. G. for synthesizing the monomers and the dimer o f
compound 48/8o and Burroughs Wellcome Inc., Tuckahoe, N.Y., for a gift of compound
48/8o.
Institut fiir Medizinische Mikrobiologie und
Pharmakologisches Institut,
Abteilung fiir Chemische Pharmakologie der
Universitiit Mainz,
65 Mainz, W. Germany
D. FALKE
G . F . KAHL
REFERENCES
ALLISON,A. ¢. (1967). Lysosomes in virus-infected cells. In Perspectives in Virology 5, 29. New York and
London: Academic Press.
FALKE,D. (1967)- Syncytienbildung bei Herpesvirus hominis. ZentrMblattfiir BakteriologieIOriginale205,16o.
FALKE, D. (I970). Wechselseitige Dissoziierbarkeit von Virus-DNS-Syuthese und Riesenzellbildung bei
Herpesvirus hominis dutch Cytosin-Arabinosid und durch Compound 48/80. Zentralblattfiir Bakteriologic I Originale 212, 390.
rALKE, D., mV'rER-SUERMA~q,D. & CLAUSS,L 0969). Riesenzellbildung und virusspezifische Antigene bei
Herpesvirus hominis: Informationsfluss der Eltern-DNS. Archivfiir die gesamte Virusforschung27, 317.
FALICE,D. & KAHL,G.r. (1970). Die Hemmung der Riesenzellbildung durch Compound 48/80 nach der
Infektion mit dem Herpesvirus hominis. II. Die Aktivierung der Lysosomen. Archiv flit die gesamte
Virusforschung 30, 353rALKE, n. & NETTER,R. J. (I969). Die Hemmung der Riesenzellbildung dutch das sogenannte Compound
48/80 nach der Infektion mit dem Herpesvirus hominis. I. Die Wirkung auf die Virussynthese. Archivfiir
die gesamte Virusforschung 28, 3o8.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 13:52:36
Short communications
277
EALKE, D. & RICHTER, I. E. (1960). M i k r o k i n e m a t o g r a p h i s c h e Studien tiber die E n t s t e h u n g v o n Riesenzellen
d u r c h Herpes-B-Virus in Zellkulturen. Archivfiir die gesamte Virusforschung I2, 73.
FALKE, D., SCmEEER, H. 6. & STOrFEL, W. (1967). L i p o i d - A n a l y s e n bei Riesenzellbildung durcb Herpesvirus
hominis. Zeitehrift fiir Naturforsehung z 2 b , 2360.
FEDORKO, E., HIRSCH, J. 13. & COHN, Z. A. (2968). A u t o p h a g i c vacuoles p r o d u c e d in vitro. I. Studies o n cultural
m a c r o p h a g e s exposed to cbloroquine. Journal of Cell Biology 38, 377.
HOCHUER~, E. & nECKER, V. (1968). Adsorption, p e n e t r a t i o n a n d u n c o a t i n g o f herpes simplex virus. Journal of
General Virology 2, 23I
KATO, N., EGGERS, H. J. & ROLLY, ~. (2969). Inhibition o f release o f vaccinia virus by Nl-isonicotinoyl-N2-3methyl-4-chlorobenzoyl-hydrazine. Journal of Experimental Medicine x29, 795.
ORR, T. S. C. & COX, J. S. G. (I969). D i s o d i u m cromoglycate, a n inhibitor o f m a s t cell degranulation a n d
h i s t a m i n e release induced by p h o s p h o l i p a s e A. Nature, London 223, 297.
PATON, W. D. i . 0 9 5 0 . C o m p o u n d 48/80: a p o t e n t h i s t a m i n e liberator. British Journal of Pharmacology 6,
499.
SHORE, P. A., BURKHALTER, a. & COHN, V. H. (2959). A m e t h o d for t h e fluorometric assay o f h i s t a m i n e in
tissues. Journal of Pharmacology and Experimental Therapy x27, 182.
(Received 2 April 1 9 7 o )
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