J. gen. ViroL (I975), 28, 271-283
271
Printed in Great Britain
Arenavirus Inactivation on Contact with N-substituted Isatin
beta-thiosemicarbazones and Certain Cations
By J. C. L O G A N , M. P. F O X , J. H. M O R G A N ,
AND C. J. P F A U
A. M. M A K O H O N
Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181, U.S.A.
(Accepted 21 April I975)
SUMMARY
N-methyl and N-ethyl isatin beta-thiosemicarbazones inactivate cell-free Parana
and Pichinde viruses as well as three strains of lymphocytic choriomeningitis
virus. This antiviral activity is abolished in the presence of the chelating agent
EDTA. The rate of virus inactivation by N-methyl isatin beta-thiosemicarbazone
is greatly enhanced and contlolled by the addition of cupric sulphate. Divalent
cations of other first transition series metals are less effective. A difference exists in
the copper requirement for fast inactivation of the prototype arenavirus (lymphocytic choriomeningitis) and the Tacaribe Complex of viruses (Parana and Pichinde).
In the presence of 20/zi-N-methyl isatin beta-thiosemicarbazone, LCM and
Pichinde viruses can be inactivated at about the same rate if 2o/zi-CuSO4 is added
to the former and 16o #M-CuSO4 is added to the latter. Using 20/zM-N-methyl
isatin beta-thiosemicarbazone and CuSO4 the inactivation of LCM is reduced, but
not eliminated, in the presence of an equal amount of infectious Pichinde virus.
Crude and highly purified Pichinde virus are inactivated at the same rate when
exposed to identical concentrations of N-methyl isatin beta-thiosemicarbazone
and cupric sulphate. There is little detectable change in inactivation rates when
Pichinde or LCM viruses are grown in a variety of different cell lines.
INTRODUCTION
(S,S)-I,2-bis-(5-methoxy-2-benzimidazolyl)-i,2-ethandiol is a potent inhibitor of arenavirus synthesis in L cells (Stella et al. I974b). Most mice treated with the compound and
infected with a lethal dose of the prototype arenavirus, lymphocytic choriomeningitis
(LCM), live at least four times longer than the controls. However, no inhibition of virus
synthesis could be demonstrated in the mice receiving this bis-benzimidazole (Stella et al.
I974a). A search was then begun for a structurally related compound that might have both
in vitro and in vivo antiviral activity. There is some similarity in structure between isatin
beta-thiosemicarbazone (IBT) and the above compound (O'Sullivan et al. r97o). Both
consist of fused benzene and 5-membered nitrogen-containing rings with a single side chain
attached to the heterocyclic ring. The heterocyclic systems, however, are unlike in properties
and the characteristics of the side chains are quite different. An extensive literature exists
on the ability of IBT (and related compounds) to inhibit a late stage in the replication of
vaccinia virus in tissue culture, as well as to protect mice against intracerebral infection
with various pox viruses (Bauer, I972; Levinson, I973)o The IBTs have also been reported
to inhibit, in tissue culture, other D N A viruses in the adeno and herpes groups and certain
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J. C. L O G A N A N D O T H E R S
reo, arbo, myxo, paramyxo and picornaviruses containing RNA (Bauer, Apostolov &
Selway, I97O). In this respect IBT also shares a biological resemblance with the bis-benzimidazoles, since these have also been reported to inhibit the synthesis of picornaviruses
(Schleicher et al. I972). Indeed, it was found that IBT and N-methyl isatin beta-thiosemicarbazone (M-IBT) were effective in lowering the extracellular virus yields from HeLa and
L cells infected with LCM and Pichinde viruses (Young et al. I974). An unexpected finding
in these studies was that the later M-IBT was added to infected HeLa cells the more effective
it was in lowering the extracellular infectious virus harvests. Contact inactivation of cellfree virus was considered as a possible explanation for these results and, in fact, this has
been described as the mode of action of the IBTs against several viruses. These include
Rous sarcoma virus (Levinson, Woodson & Jackson, I97I); three viruses causing slow
infections of s h e e p - Visna, Maedi and progressive pneumonia (Haase & Levinson, I973);
herpes viruses (Levinson et al. I974); murine sarcoma and leukaemia viruses (Levinson
et al. I973a; and feline sarcoma virus (Levinson et al. I973b). In this communication we
show that the arenaviruses can now be added to the above category.
METHODS
Compounds. N-ethyl isatin beta-thiosemicarbazone (E-IBT) was a generous gift from
D. J. Bauer. M-IBT was from either Aldrich Chemical Co. or D. J. Bauer. Thiosemicarbazone (TSC) was an Eastman Organic Chemicals product. Stock solutions of the substituted IBTs were prepared, as recommended by D. J. Bauer, in the following manner:
23 mg of M-IBT, or 24"8 mg of E-IBT, were dissolved in o'5 ml of N,N-dimethyl formamide
(Fisher Certified Grade) and quickly added to 86o ml of double distilled water. The compounds immediately precipitated and were dissolved by autoclaving for Io min at Io lb
pressure. The solutions were stored at 37 °C in the dark and were kept for no longer than
two weeks, after which insoluble precipitates began to be visible. Immediately before use,
IOO ffM working stocks of the compounds were made by adding 7 ml of 8 x concentrated
(with glutamine and bicarbonate) Eagle's minimal essential medium (MEM) to 43 ml of one
of the above solutions. These were then diluted appropriately to give the working concentrations desired. TSC was completely soluble in MEM and was prepared as a Io mM stock
solution by adding 45"6 mg to 5o ml of medium. Inorganic compounds (either Fisher
Certified or Baker reagent grade) were added to sterile distilled water and stored, as I mM
solutions, at 4 °C. The disodium salt of (ethylenedinitrilo) tetra acetic acid (EDTA) was the
reagent grade product of Mallinckrodt Chemical Works.
Virus and assay techniques. The origin of the LCM strains and their plaque assay have
been described (Pulkkinen & Pfau, I97o). Details of the origin of Parana and Pichinde
viruses are given elsewhere (Pfau et al. I972; Stella et aL I974b). These Tacaribe complex
viruses were measured by our standard BHK2~/I3S suspension assay (Pulkkinen & Pfau,
I97O). Pichinde plaques were stained with neutral red and counted on the third day after
infection while the Parana plaques were stained and counted on day 5 (vs. day 4 in the LCM
assays). The standard error in these titrations was no greater than lO ~.
In some experiments, as noted in the results section, LCM plaques were counted through
use of the vital stain 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT)
according to the method of Cooper (I959).
Cells. BHK21/I3S, HeLa, L-929 and Vero cells were propagated as previously described
(Stella et al. I974b). Chinese hamster ovary (CHO) cells, from T. T. Puck, were grown in
FI2 medium (Ham, I965)+ IO ~ heat-inactivated foetal calf serum.
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Arenavirus inactivation
273
Table ~. Inactivation of L C M strain UBC at 37 °C
p.f.u./ml x 1o-4
Incubation
time
M-IBT
~
r
,
(h)
Control
4o/ZM
8O/ZM
o
4
I3O
6I
IIO
I8
230
7
8
I2
I'5
0"7
Virus growth and purification. All virus stocks were the 36 to 48 h post infection supernatant fluid harvests of monolayer cultures infected at an input multiplicity of o-~ to
o.2 p.f.u.fcell. The fluids were centrifuged at low speed to remove cell debris and stored at
- 7 o °C until use. These unpurified preparations were used in all experiments except as
noted. Pichinde virus was purified by a combination of polyethylene glycol precipitation
followed by centrifuging through discontinuous and continuous sucrose gradients (Ramos,
Courtney & Rawls, I972 ). The final 5 ml gradient was fractionated into about 2o samples
and the three containing over 95 ~ of the infectivity in the entire gradient were frozen
at - 70 °C.
Treatment of virus. One ml of virus at crushed-ice temperature was rapidly added to a
16 x 75 m m screw-cap test tube containing 8 ml of M E M ( + IO ~ heat-inactivated foetal
calf serum) pre-heated to 37 °C. This medium contained the appropriate dilutions of drug
and/or inorganic compound solutions. Incubation was continued at 37 +-o'25 ° C. The p H
of the reaction mixtures in a specific group of samples was the same, and remained constant
during the entire observation period, but varied in different experiments between 7"45 and
7"95 depending on the age of the medium used. Tenth ml samples were withdrawn at various
times and immediately diluted io-fold into M E M plus to ~ calf serum. Unless otherwise
stated these were then rapidly frozen at - 7 o °C until the time of plaque assay.
RESULTS
Contact inactivation of cell-free L C M virus
Slow reaction with M - I B T
The loss of infectivity of L C M strain UBC (grown in L ceils) was followed at 37 °C in
the presence or absence of M-IBT. After 8 h the control stock had lost over one log10 unit
of activity (Table I). However, after the same interval in the presence of 4o #M-M-IBT the
titre of the virus stock decreased by almost two log10 units. Even further inactivation was
noted with 8o #M drug.
Fast reaction with E-IBT plus either CuS04 or HgCI2
Because a strong synergistic effect was reported (Levinson et al. I973b) with CuSO4 and
M-IBT, at concentrations that gave little activity when used singly, the inactivation of
L C M - U B C was followed after mixing with equimolar concentrations of E-IBT and CuSO4
or HgClz. While the drug or inorganic compounds alone, at eo #M, had measurable antiviral
activity comparable to M - I B T (Table I) the inactivation rate of virus was precipitous when
an E-IBT-salt mixture was used (Fig. I). The E-IBT-CuSO4 combination was the most
effective, causing over a one log10 drop in titre within I5 min (the earliest interval at which
samples were taken). A comparable drop in infectivity required almost six times as long
with the E-IBT-HgC12 mixture.
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J. C. L O G A N
AND
OTHERS
-~ l0s
10 4
1031
I
I
I
I
1
I
I
I
I
2
Time at 37 °C (h)
!
3
4
Fig. I. L C M - U B C inactivation by N-ethyl isatin beta-thiosemicarbazone and divalent cations:
A - - A , no deliberate addition o f c o m p o u n d s ; [ ] - - [], zo #M-E-IBT; • - - • , 2o/~M-CuSO4; • - - • ,
2o #M-HgC12; ( 3 - - ( 3 , 2o #M-E-IBT plus 20 #M-CuSO4; I1--11, 2o #M-E-IBT plus 2o ~M-HgC12.
Comparison of the efficacy of E-IBT and M - I B T
E-IBT and M-IBT, with no deliberate metal addition, were found to have similar activities
against herpes simplex (Levinson et al. t974), and Rous sarcoma virus (Levinson et al. I970.
An identical experiment to that presented in Fig. I was repeated using 2o #M-CuSO4 with
either 2o #M-E-IBT or M-IBT. After 3o min the infectivity loss of the M-IBT-treated virus
was 94 ~ while that of E-IBT-treated virus was 96 ~ . After 2 h the infectivity loss of M-IBTtreated virus was 99"75 ~ while that of E-IBT-treated virus was 99"85 ~.
Effect of host celt origin on reaction with M-IBT-CuSO 4
Confluent monolayers of HeLa or Vero cells were infected with LCM-UBC (L cell origin)
at an input multiplicity ofo. I p.f.u./cell. After a standard I h adsorption period the inoculum
was removed and M E M was replaced to the original volume. Twenty-four h post infection
the supernatant fluids were harvested and stored at - 7 o °C until use. Both virus stocks
were exposed to CuSO4 and M-IBT under the conditions used for the experiment presented
in Fig. I except that virus infectivity was measured only at the o, 3o and 6o min intervals. These titres were, respectively, for Vero- and HeLa-grown virus: o time, 4"8 and
3"8 × Io 4 p.f.u./ml; 3o rain, I. 7 and 3"4 × Io3 p.f.u./ml; 6o min, 4o and 60 p.f.u./ml.
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Arenavirus inactivation
Table 2. L C M - U B C inactivation as a function o f M-1BT concentration
Time
(h)
M-IBT
(#M)
CuSO4
(~M)
p.f.u./ml
x 1o-4
o
0
O
78
2
0
0
52
2
0"2
2O'0
24
2
I'0
20"0
I8
2
2
5'0
20.0
2o.o
20.0
3"2
0"4
The protocol followed the pattern used for obtaining the results presented in Fig. I.
Effect o f L C M strain on reaction with M - I B T and CuSO 4
The Traub and UBC strains of LCM "were inactivated, side by side, in the presence of
20/zM-M-IBT and 2 0 / z M - C u S O 4. The inactivation rates, measured 3o and 12o min later,
were almost the same. However, an identical type of experiment with the CA~37~ strain
(grown in L cells, as was the Traub strain) showed only a 28 ~ loss in infectivity after ~ h
exposure to the above combination, whereas the control UBC infectivity loss was in the
expected range (88 ~ ) after only 3o min. The infectivity of another sample of CA~37I
exposed to 2o/zM-M-IBT and twice the usual concentration of CuSO~ (4o/ZM) dropped by
96 ~ after 3o min and this increased to 99"3 ~o after an additional 3o min.
Since an initial experiment with LCM-CAI37I using only the 2o#M-CuSO~-M-IBT
combination showed very little inactivation (compared to the UBC strain), the possibility
was considered that this was due to the fact that samples were diluted and assayed immediately, without the usual storage at - 7o °C. Thus an LCM-UBC virus stock was inactivated
in the presence of 2o #M each of M-IBT and CuSO4. At various times samples were withdrawn
and diluted m-fold in MEM. Part of this dilution was immediately frozen at - 7 o °C,
while the other part was diluted further and placed on standard B H K suspension assay
plates. The resulting plaque counts were then compared with those from plating of the
same sample which had been frozen for several days. In this way it was found that there
could be as much as a threefold difference in the titre of a single sample. However, the titre
of neither the freshly assayed nor the frozen and thawed sample was consistently lower than
the other (i.e. no pattern emerged indicating further inactivation caused by freezing a n d
thawing).
Effect o f M - I B T concentration on reaction velocity
The extent of the inactivation of LCM-UBC (grown in L cells) was determined after 2 h
exposure to 20 #M-CuSO 4 plus varying amounts of M-IBT. As shown in Table 2, the lower
the concentration of drug the lower was the loss in titre. Even a Ioo-fold dilution of M-IBT
(o.2 #M) below that used in previous experiments caused over a 5o ~ decrease in titre when
compared to the 2 h control figure.
Reaction with divalent cations o f various first transition series metals
Since thiosemicarbazones are well known for their ability to form coordination compounds (Levinson, I973) with metals of the first transition series (Mn, Fe, Co, Ni, Cu, Zn),
FeSO4, NiC12 and ZnCI~ were substituted for CuSO4 in the reaction with M-IBT and LCMUBC (Fig. 2). Copper was the most effective, followed by zinc, iron and nickel. All these
divalent cations were at twice the concentration (4o/zM) used in the previous experiments
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J. C. L O G A N
T a b l e 3.
AND OTHERS
Effect of EDTA on the interaction between M-IBT and LCM-UBC
loss of original
infectivity after
g-
Treatment
Concentration
4h
8h
M-IBT
None
M-IBT+EDTA
DMF
DMF+CuSO,
EDTA
20/ZM
90
77
79
82
8I
58
98"8
92'3
94"O
94'8
94"7
75
zo #M+ tmM
o'o2 ~ *
o'o2 ~ + 2 o #M
/ mM
* The concentration of dimethylformamide in a 40/ZM solution of M-IBT.
I
I
105
"~. ]0 4
103
10z
I
0
I
1
Time at 37 °C (h)
Fig. 2. LCM-UBC inactivation by N-methyl isatin beta-thiosemicarbazone and cations of first
transition series metals: O - - O , no deliberate addition of compounds; A - - A , 4o #M-ZnCIz;
®--®, 4O#M-FeSO4; • - - • ,
4O#M-NiC12; 0 - - 0 , 2O#M-M-IBT plus 4O#M-ZnCI~; I1-~11,
20 #M-M-IBT plus 4o #M-FeSO4 ; D - - [ ~ , 2o #M-M-IBT plus 4o #M-NiCI2; A - - A , 2o #M-M-IBT
plus 40 #M-CuSO~.
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Arenavirus inactivation
277
106i
105
104
103I0 I L I 1I
t
2
Time at 37 °C (h)
Fig. 3. Pichinde virus inactivation by N-methyl isatin beta-thiosemicarbazone and various concentrations of CuSO4: O--O, no deliberate addition of compounds; ~ - - A , 80/zM-CuSO4;
@--@, I60#M-CuSO4; I1--11, 20#•-M-IBT plus 20#M-CuSO~; [2]--[], 2O/zM-M-IBT plus
4o/zM-CuSO4 ; &--&, 20/tM-M-IBT plus 80 #M-CuSOa; Q--@, 2O/ZM-M-IBTplus 16o #M-CuSO4.
with C u S Q . Under these conditions there was an almost Io-fold greater loss in titre after
the first 30 min of incubation than observed with 20/zM-CuSO4 and M-IBT.
Activity of M - I B T + EDTA or TSC + CuS04
Because the divalent first transition series cations, especially copper, seemed so important
for enhancing the activity of M-IBT, the possibility was considered that M - I B T intrinsically
had no antiviral activity without forming coordination compounds with trace amounts of
metals found in the tissue culture medium or glass tube. Furthermore, it has been reported
that a I mM concentration of the chelating agent E D T A abolishes the ability of M-IBT to
inactivate Rous sarcoma virus (Levinson et aL I973b ). Thus, under conditions of no
deliberate addition of metals with the concomitant weak inactivation of L C M by M-IBT
(as in Table I) it was found that addition of E D T A to the system eliminated the activity
of M-IBT (Table 3). These data, firmly reproduced in a second experiment, showed that:
(I) the inactivation of L C M by M-IBT was consistently greater than that seen with M - I B T +
E D T A ; (2) the inactivation caused by M-IBT + E D T A was no greater than that caused by
exposing the virus to the amount of drug solvent, dimethylformamide (DMF), found in
working concentrations of M-IBT; (3) the D M F alone gave a slightly higher rate of inactivation than the L C M control but was not enhanced by copper; (4) E D T A alone distinctly
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J.C. LOGAN
AND
OTHERS
Table 4. Independence of Pichinde virus purity on contact inactivation rate
with M-IBT and CuS04
Virus
stock
Cruder
Purified:~
Crude
Purified
Time o,
p.f.u./ml x io -4
M-IBT +
CuSO4*
250
22
25o
26
+
-~None
None
~o 10ss o f infectivity after
,
* - - ~
Ih
4h
89
95
8
-- 8
99"2
99"3
32
42
* 20 # M - M - I B T + 4 o #M-CuSO4.
I Supernatant harvests from infected L cells.
Purified virus (grown in B H K cells) from a highly infectious fraction o f the final continuous sucrose
density gradient. The sucrose had been dissolved in T N E buffer (Ramos et al. I972) containing I mM-EDTA.
The virus was diluted Ioo-fold in the final reaction mixture.
enhanced virus thermostability. L C M virus falls into a class of viruses that are more thermostable in the absence than in the presence of high concentrations of MgC12 and CaC12 (Pfau,
I965). These divalent cations are normal constituents of the tissue culture medium and
also must interact with the EDTA.
TSC appears to have almost as much activity against Visna and herpes viruses as M - I B T
(Haase & Levinson, ~973; Levinson et al. ~974). However, between ~o- and Ioo-fold more
TSC than M-IBT was required to achieve comparable inactivation of Rous sarcoma virus
(Levinson et al. I973b). Thus an attempt was made in our system to determine the active
part of the M-IBT molecule. Compared to M-IBT, TSC was relatively ineffective against
LCM-UBC. After a 2 h incubation period with 2o #M each of TSC and CuSO4 virus titre
was reduced by 42 % while the untreated control was down 27 % and a virus solution where
M - I B T had been substituted for TSC contained less than o" 1 % of the original infectivity.
Under the above conditions 20/zM-TSC+ I6O/zM-CuSO 4 reduced virus titre by 75 %, but
I6o/zM-CuSO4 by itself caused a 57 % drop in titre; 4oo/zN-TSC+2o/zM-CuSO4 led to a
96 % loss in infectivity, with 4oo/zM-TSC alone resulting in a 59 % loss.
Contact inactivation of cell-free Pichinde virus
Fast reaction with M-IBT and high concentrations of CuS04
Pichinde virus (CHO cell origin) was used in an experiment identical to that presented
in Fig. t. No fast inactivation was observed at a 2o/~M-concentration of E-IBT and CuSO4.
At the end of 4 h the titre of the treated virus was still 4o % of the control. However, it was
possible to increase the inactivation rate of Pichinde (Fig. 3) by increasing the C u S Q
concentration while keeping M-IBT at a constant 2o/ZM. Using 16o #M-CuSO4 an inactivation rate similar to that of L C M was achieved. This need for I6O/ZM-copper was shown by
Pichinde grown in either L, C H O or B H K cells.
Comparison of the inactivation rate of crude and purified virus
In an attempt to determine whether the high CuSO4 concentration was an intrinsic need
for the rapid inactivation of Pichinde, or whether some component in the system, beside
the virus or M-IBT, was competing for copper (a by-product from virus-infected cells, for
instance) the inactivation rates of crude and purified virus were compared. For ease of
measurement, a concentration of CuSO4 (4o/ZM) was chosen that gave an intermediate
inactivation rate with crude virus. As shown in Table 4, both virus stocks (over the course
of 4 h) were inactivated at about the same rate. Since the purified Pichinde virus contained
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Arenavirus inactivation
279
T a b l e 5. Effect of M-IBT and CuS04 on an LCM-Pichinde virus mixture
p.f.u./ml x lo -5
incubation time (min)
(
Virus*
Treatmentt
Stain$
LCM
-
NR
13"o
13'o
-
INT
IZ.O
14"o
+
+
+
-
NR
INT
NR
NR
I3'4
13"0
I I'O
26.0
LCM
Pi
LCM + Pi
LCM + Pi
o
-
INT
t4'o
+
+
NR
INT
27"0
I3.o
15
30
45
6.6
6"4
5"5
5"I
2.1
2.2
I3'O
22.o
20"0
7"2
15'o
6'7
I I "o
4'6
12"o
* LCM, strain UBC: Pi, Pichinde virus.
t 2O/zM-M-IBT+20#M CuSO4 (+ =added, - = n o t added). The standard inactivation conditions of
the virus were followed (see Methods) except that I ml addition of virus to the treatment medium (MEM
+ [ - M-IBT-CuSOa) contained either o'5 mI LCM stock+o-5mI MEM; 0"5 ml LCM stock+o'5 ml
Pichinde stock; or o'5 ml MEM+o'5 ml Pichinde stock.
.+ NR=neutral red, INT=2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride. All assay
plates were stained on the third day after infection.
a small a m o u n t o f E D T A (0.0I m i o r less) it could be argued t h a t the virus r e q u i r e m e n t
f o r CuSO4 was indeed lowered, b u t chelation b y E D T A fortuitously m a d e it a p p e a r t h a t
the original r e q u i r e m e n t h a d n o t changed. However, the inactivation rates o f crude Pichinde
virus m e a s u r e d at one a n d f o u r hours after the a d d i t i o n o f 20/zM-M-IBT a n d 40/*M-CuSO4
were identical in the presence a n d absence o f 0.0I m M - E D T A .
Co-reaction of Pichinde and L C M viruses with M-IBT and CuS04
The a b o v e experiment i n d i c a t e d t h a t Pichinde virus itself did indeed require t h e p r e s e n c e
o f 160 #M-CuSO4 to be inactivated at a b o u t the same rate as n o t e d with L C M a t 20 #MCuSO4. I n an a t t e m p t to d e t e r m i n e if Pichinde w o u l d interfere with the i n a c t i v a t i o n rate o f
L C M , a mixture o f b o t h viruses was exposed to 20 # i c o n c e n t r a t i o n s o f M - i B T a n d CuSO4.
By m a k i n g use o f the fact that I N T - c o n t a i n i n g overlays on p l a q u e assay plates failed to
stain L C M - i n f e c t e d cells b u t w o u l d stain cells infected with Pichinde only, as well as
uninfected cells (J. H. M o r g a n & C. J. Pfau, u n p u b l i s h e d observations), it was possible to
selectively assay L C M virus in such a mixture. Thus the i n a c t i v a t i o n rates o f the virus
mixture a n d L C M alone were followed b y m a k i n g d u p l i c a t e t i t r a t i o n plates f o r each dilution
o f virus, i.e. one set o f dilution plates being stained with I N T a n d the o t h e r with n e u t r a l
red. A s shown in T a b l e 5, L C M virus in the presence o f Pichinde was inactivated at a
progressively slower rate t h a n the L C M virus alone. A f t e r i n c u b a t i o n for 45 m i n there was
over twice as m u c h infectious virus r e m a i n i n g in the mixture as in the solution c o n t a i n i n g
only L C M .
Effect of M-IBT concentration on reaction velocity
The extent o f the i n a c t i v a t i o n o f Pichinde (grown in L cells) was d e t e r m i n e d after 2 h
exposure to I60/zM-CuSO4 plus v a r y i n g a m o u n t s o f M - I B T . As shown in T a b l e 6 the lower
the c o n c e n t r a t i o n o f d r u g the lower was the loss in titre. A s with L C M (Table 2) the M - I B T
c o n c e n t r a t i o n Ioo-fold b e l o w t h a t used n o r m a l l y ( o - 2 / z i ) still caused significant i n a c t i v a t i o n
o f virus when c o m p a r e d to the 2 h c o n t r o l figure.
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Table 6. Piehinde inactivation as a function o f M - I B T concentration
Time
(h)
M-IBT
(~M)
CuSO4
(#M)
p.Lu./ml
X IO-4
0
2
2
2
2
2
0
O
0"2
I.O
5"0
20.o
0
O
~6o.0
I60-o
I6o.o
I6O.O
I80
I80
6o
I2
0.6
0'02
The protocol followed the pattern used for obtaining the results presented in Fig. L
Table 7. Parana virus inactivation by M - I B T and CuS04
Time
(min)
CuSO4
(~M)
M-IBT
(~M)
p.£u./ml
X IO-a
O
15
30
60
120
12o
O
I6o
16o
I6o
o
80
O
20
20
2o
o
20
I70
14
2.8
0'4
I8O
4"5
T h e protocol followed the pattern used for obtaining the results presented in Fig. I.
Contact inactivation o f cell-free Parana virus
Fast reaction with M - I B T and high concentrations o f CuS04
Parana virus (L cell origin) was used in an experiment identical to that presented in Fig. I.
No fast inactivation was observed at 2o #M concentrations of E-IBT and CuSO4. At the
end of 4 h the titre of the treated virus was still 8o ~ of the control. However, as with
Pichinde (Fig. 3) use of 8o or I6o/zM-CuSO4 combined with 2o/zM-M-IBT led to rapid
inactivation of plaque-forming activity (Table 7).
DISCUSSION
There are several similarities and differences in the IBT reaction with the arenaviruses
and those groups previously studied. The arenaviruses share with Rous sarcoma virus
(RSV): insensitivity of the IBTs with concomitant E D T A treatment (Table 3, Levinson
et al. ~973b); a strong synergism with CuSO4 (Fig. I, Levinson et al. ~973b); decreasing
sensitivity (along with herpes virus, Levinson et al. ~974) to copper, zinc, iron and nickel
(Fig. 2, Levinson et al. I973 b); and relative insensitivity to TSC (Levinson et al. 1973 b).
The arenaviruses appear to be the first group studied in which both IBT and deliberately
added divalent cations must be present for rapid contact inactivation. This obvious need
for the cations might not have been apparent if the experiments had been carried out in
phosphate-buffered saline (PBS) instead of tissue culture medium. If herpes viruses (but
none of the others studied by Levinson and co-workers) were exposed to E-IBT in tissue
culture medium little if any inactivation was observed (Levinson et al. I97I), but rapid
inactivation occurred in PBS. The inhibitor of the reaction was traced to the essential amino
acid mixture used in the tissue culture medium (Levinson et aL ~974). This approach,
however, was not attempted since LCM rapidly loses activity when diluted into PBS (Pfau &
Camyre, I967). Not only must the IBTs and first transition series cations be present to cause
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Arenavirus inactivation
28
rapid inactivation of arenaviruses, but the amount of the latter needed varies with the type
of virus used. This unique difference may be a reflection of the stability (or permeability ?)
of the virus particle coats. Pichinde and Parana can be almost quantitatively recovered from
sucrose or caesium chloride density gradients whereas very large losses are incurred with
LCM strains (Mifune, Carter & Rawls, I97I; Pfau, I974; F. Dutko, L. Verone & C. Pfau,
I975, unpublished observations). Unlike the I6o #M-CuSO4 requirement for the Tacaribe
complex viruses the Traub and UBC strains of LCM virus required zo #M-CuSO4 to achieve
a comparable inactivation rate. However, the CA~37~ strain required 4o /ZM and this is
the most stable of three tested strains (the others being Traub and WCP) with respect to
recovery from potassium tartrate density gradients (Camyre & Pfau, I968). Pichinde and
Parana (Tables 5, 6) are also more thermostable than LCM-UBC (Table 2). Preliminary
evidence indicates that the CAI37 x is somewhat more thermostable than the UBC strain
(J. C. Logan, unpublished observations).
It is difficult to draw a specific conclusion about the mode of action of thiosemicarbazones
in the contact inactivation of viruses studied by Levinson's group. Using [ZH]-uridinelabelled RSV it was found that M-tBT inactivated virus did not lose its ability to absorb to
chick embryo cells (Levinson et al. I973a ). A striking correlation was found between the
ability of various thiosemicarbazones to inactivate the infectivity, and the virus particleassociated reverse transcriptases of RSV (Levinson et aL I973 b) and Visna virus (Haase &
Levinson, I973). For a while it was considered that the contact inactivation of the viruses
was a reflection of the ability of the thiosemicarbazones and copper to inactivate these
enzymes. Because of this, a virus particle D N A or RNA polymerase was sought from
purified herpes type ~, yet none was found (Levinson et al. r974). Further work showed that
the Rous sarcoma virus particle-associated enzymes not requiring nucleic acid template,
lactic dehydrogenase and t-RNA nucleotidyl transferase, were not sensitive to M-IBT
(Levinson et al. I973a). A variety of other enzymes not utilizing nucleic acids were found to
be insensitive to M-IBT (Levinson et al. I973a ). Recent unpublished evidence indicates
that M-IBT-eopper complexes bind to and precipitate both single- and double-stranded
D N A and RNA (quoted in Levinson et al. I974). Thus, direct interaction with nucleic acids
and interference with genome function may be involved in the general mechanism of action
of the drug, which is perhaps serving as a scavenger and vehicle to transport first transition
series metals to the appropriate site(s). However, polio virus infectious RNA has been
reported to be insensitive to E-IBT (Levinson et al. I97I). It should also be noted that data
have been presented which have been interpreted as indicating the presence of an RNAdependent R N A polymerase in Pichinde virus (Carter, Biswal & Rawls, i974).
The contact inactivation of viruses by M-IBT has not been found to be a general phenomenon: 4 ° #M-E- or M-IBT has been reported to have no effect on Newcastle disease,
polio or vaccinia viruses (Levinson et al. I97I); vesicular stomatitis virus (Levinson et aI.
I973b), or polyoma virus (Levinson et al. I973a). Since Pichinde virus required a relatively
high dose of C u S Q to be contact inactivated by M-IBT (Fig. 3), the possibility was considered that the above viruses would become susceptible to M-IBT if exposed to the proper
concentration of CuSOa. Under conditions similar to those presented in Fig. 3 the first
virus investigated, vesicular stomatitis, lost two log10 units of infectivity after z h exposure
to 2o #M-M-IBT and ~6o/zM-CuSQ (M. P. Fox & C. J. Pfau, unpublished observations).
Further work is under way to expand this possible generality.
In view of the profound effect exerted by IBT on the course of infection with vaccinia
virus in mice, the efficacy of this compound was explored in other murine virus infections
(Bauer, I955). IBT was reported to have no effect on LCM infection (Bauer, ~955), but
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what was actually used was pseudo lymphocytic choriomeningitis (ectromelia) virus. Later
an authentic strain was used, still with negative results (D. J. Bauer, I974, personal communication). The synergism between CuSO4 and M- or E-IBT seen in vitro would seem to
warrant further in vivo investigation.
We thank Frank Dutko for the purified Pichinde virus and his interest and many helpful
suggestions in this work. This study was supported by U.S. Public Health Service Grant
AI-Io6t2 from the National Institute of Allergy and Infectious Diseases.
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