Journal of General Virology (1990), 71, 2989-2997. Printed in Great Britain
2989
The mechanism of the antiherpetic activity of zinc sulphate
Gunther Kiimel, 1,2. Stefan Schrader, 1 Hanswalter Zentgraf, 3 Heiner Daus 4 and Martin BrendeP
l lnstitut far Mikrobiologie (Virusforschung), 2Z. Hygiene, J. IV. Goethe-Universiti~t, Mayerstr. 9, D-6000 Frankfurt am
Main 3institut f a r Virusforschung, Deutsches Krebsforschungszentrum, Heidelberg and 4L Med. Klinik, Homburg,
Germany
The molecular mechanism of the effects of zinc ions
against herpes simples virus (HSV) infection was
investigated. Zinc sulphate (100 ~tM) in the culture
medium of an HSV-infected African green monkey
kidney cell line did not block viral DNA synthesis and,
at this concentration, only moderate cytotoxic effects
were observed in uninfected cells. Nevertheless, virus
yields were reduced to less than 1%0 of the control.
Thus the long standing hypothesis that zinc might
block multiplication of HSV by selective intranuclear
inhibition of the viral DNA polymerase apparently has
lost its validity. Inhibition of virus growth in the
absence of severe cytotoxicity must therefore result
from other effects of ZnSO4. Free virus is inactivated
by 15 mM-ZnSO4 within a few hours of its addition.
The inactivated virus is defective in the glycoproteindependent functions of penetration and, to some
extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a
virion, transmembrane transport of zinc ions is not
expected and the established antiviral effect is therefore
explained by an inhibition of virion glycoprotein
function after non-specific accumulation of zinc into
many virion membrane components.
Introduction
In order to explain the antiviral effect of zinc, we reevaluated the effects of ZnSO4 on HSV replication,
adsorption, penetration, viral DNA synthesis and virus
egress, and determined the cytotoxicity of zinc ions. We
also studied in detail the inactivation of free HSV
particles by Zn 2÷. Surprisingly, control experiments of
this sort were not part of any of the previously quoted
studies on zinc inhibition of HSV. Investigators performing experimental therapy with topical or systemic
application of zinc to HSV-infected mice also failed to
take into consideration the possibility of direct inactivation of HSV by zinc salts (Tennican et al., 1979).
The need for drug safety and the avoidance of
development and spread of virus strains resistant to
nucleoside analogues (Eggers, 1989) has increased the
importance of physiologically tolerable drugs for the
treatment of non-severe herpes simplex virus (HSV)
infections. Recently, a study on the clinical efficacy of
harmless combinations of heparin and ZnSO4 has been
published (Holzmann et al., 1988) but the mechanism of
the alleged antiherpetic activity has not been elucidated.
Inhibitory effects of zinc salts on the replication of
HSV have been studied in various systems since 1967
(Falke, 1967; Gordon et al., 1975; Shlomai et al., 1975).
In cell homogenates, Fridlender et al. (1978) found HSV
DNA polymerase to be inhibited by zinc ions at a lower
concentration than the cellular DNA polymerases 0t and
ft. Despite the conflicting results of other investigators
(Gupta & Rapp, 1976), the interpretation of Fridlender
et al. (1978) that addition of zinc salts to the culture
medium led to a selective inhibition of the HSV DNA
polymerase in the nucleus of the infected cell, and thus to
a block in HSV replication, found general acceptance.
However, accumulation of zinc within the cell is strictly
controlled by the homeostatic mechanism and it has
recently been demonstrated that zinc uptake into various
culture cells is indeed minimal (Meshitsuka et al., 1987).
0000-9575 © 1990 SGM
Methods
Cells and virus. All experimentshavebeenperformedon the African
green monkeykidney(AGMK)cellline RC 37 Rita (Italdiagnostics)
with HSV-1 strains KOS, WAL and ANG and HSV-2 HG52. Cell
culture conditions,origin of virus strains, the methodsof stock virus
preparationat lowm.o.i,and the techniqueof virustitrationhavebeen
described previously(Gloriosoet al., 1984;K~melet aL, 1985;Bishop
et al., 1986).The cellculturemediumwas Eagle'sMEM supplemented
with 7% foetal calf serum, non-essentialamino acids, penicillinand
streptomycin.Radioactivelylabelledviruswaspreparedby infectionof
a cell culture(m.o.i. 3) and additionof 20 ~tCi/ml[5-3H]thymidineto
the mediumafterthe adsorptionperiod. After24 h, cellswerelysedby
repeatedfreezingat - 70 °C. The virussuspensionwas subjectedto low
speedcentrifugation(10min, 1500g, 4 °C)and dialysedagainst 10mMHEPES, 100 mM-MgSO4pH 7.3.
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2990
G. Kiimel and others
Colony formation. Cells were seeded in appropriate dilutions onto 6
cm plastic dishes. The cell culture fluid was replaced 12 h later by
medium containing ZnSO4. After 48 h the dishes were stained with
crystal violet and photographed. The number of single cells, and of
colonies containing two, three, four or more cells per colony, was
determined.
a tight fitting plunger. The nuclei were collected and cell debris was
removed by three cyclesof centrifugation (t000 g, 2000 g, 2000 g, 5 rain
each, 4 °C) and resuspension in RSB/sucrose/Triton X-100. All steps
were monitored by phase-contrast microscopy. Radioactivity in acidprecipitable material from the cell nuclei was taken as a measure of
viral DNA transported into the cell nucleus after penetration.
DNA synthesis in uninfected cells. Culture medium containing ZnSO4
in various concentrations was added to cell monolayers. At appropriate
times the cultures were labelled for 20 min by addition of [53H]thymidine to a concentration of 0.75 ~tCi/ml.After the pulse, cells
were washed three times with phosphate-buffered saline (PBS) and
homogenized by freezing. Radioactivity of acid-precipitable material
was determined.
Results
DNA synthesis in infected cells. Cell monolayers were infected with
HSV-1 KOS (m.o.i 1). After 1 h, free ir~put virus was removed by
washing with PBS and the culture medium replaced by medium
containing ZnSO4. Before the onset of viral DNA synthesis at 4 h postinfection (p.i.), [5-3H]thymidine was added to a concentration of 2
~tCi/ml.DNA was extracted 24 h p.i. by sarkosyl lysis and proteinase K
digestion, separated from cellular DNA by CsCI centrifugation and
characterized by restriction enzyme analysis, as described previously
(Kiimel et aL, 1982).
Effect ofZnSO 4 on virus yield. Cells were infected by HSV-1 at a
m.o.i, of 3 p.f.u./cell. At the end of the adsorption period (1 h), nonadsorbed input virus was removed by three cycles of washing with PBS
and the medium replaced by a medium containing the appropriate
concentration of ZnSO4. Virus was harvested from the cultures 24 h p.i.
by repeated freezing at - 7 0 °C, and titrated.
Virus adsorption kinetics. Virus was radioactively labelled with
[3H]thymidine and used to infect cell monolayers in culture dishes
(m.o.i. 10) in the presence or absence of ZnSO4. At different times
thereafter non-adsorbed virus was removed by three steps of washing
with PBS. The cells were lysed by repeated freezing at - 2 0 °C and
radioactivity was determined in TCA-precipitable material.
Effect of ZnS0 4 on virus penetration. Cell monolayers in culture dishes
were pre-washed with ice-cold PBS and either mock-infected or
infected at an m.o.i, of 0-3 with HSV-1 KOS at 4°C. After an
adsorption period of 2 h, non-adsorbed virus was removed by repeated
washing with cold PBS. Medium containing various concentrations of
ZnSO4 was added and the temperature shifted to 37 °C. After 4 h the
medium containing ZnSO4 was replaced by culture medium. Virus
yields were determined by titration of cell lysates prepared by freezing
at - 7 0 °C 16 h p.i. One control dish left without zinc treatment was
harvested 12 h p.i. and another 16 h p.i. If ZnSO4 prevented
penetration, virus yields in zinc-treated samples should have resembled
that from the 12 h controls because penetration would have occurred
only after removal of ZnSO~. If virus penetration occurred in the
presence of ZnSO~, the respective virus yields should correspond.with
the 16 h control. Mock-infected cultures were infected after temperature shift using the same protocols to demonstrate that zinc treatment
had not abolished the ability of cells to adsorb and replicate virus.
Penetration of inactivated virus. Cell cultures in plastic dishes were
infected at 4 °C with zinc-inactivated radioactively labelled HSV-1
KOS at an m.o.i, of 10. After 2 h adsorption free input virus was
removed by three cycles of washing with ice-cold PBS and the cells
were covered with culture medium and shifted to 37 °C. At appropriate
times p.i., sample cultures were washed with hypotonic buffer (RSB:
0.01 M-Tris-C1, 0-001 M-NaCI, 0.0015 M-MgC12) and the cells were
scraped offthe plastic surface and put in Eppendorf tubes. After 10 rain
swellingon ice the cells were collected by centrifugation (5 min, 500 g,
4 °C) and suspended in RSB containing 2 M-sucroseand 0.5 % Triton X100. Ceils were broken at 4 °C by 15 strokes in a glass homogenizerwith
Cytotoxieity o f Z n S 0 4
T o establish a m e a n i n g f u l system it was of p r i m a r y
i m p o r t a n c e to d e t e r m i n e the threshold c o n c e n t r a t i o n of
ZnSO4 toxic to u n i n f e c t e d cells. Besides microscopic
observation, we m o n i t o r e d the kinetics of D N A synthesis a n d performed colony f o r m a t i o n tests in the presence of i n c r e a s i n g c o n c e n t r a t i o n s of Z n S O 4 in the culture m e d i u m (Fig. 1 a to e). C o l o n y f o r m a t i o n c a n be
considered a sensitive a n d c o m p r e h e n s i v e i n d i c a t o r for
all m e t a b o l i c p a r a m e t e r s i n f l u e n c i n g cell growth, D N A
synthesis a n d mitosis. T h e results o b t a i n e d were confirmed by a s t a n d a r d dye exclusion test ( t r y p a n b l u e ;
L i n d l & Bauer, 1987) a n d a m i n o acid i n c o r p o r a t i o n (data
not shown). T h e applied m e t h o d s defined a c o n c e n t r a tion range of c o n t i n u o u s l y i n c r e a s i n g cytotoxicity. Z i n c
at a c o n c e n t r a t i o n of 200 ~tM had a strong influence o n
cell morphology, D N A synthesis, cell growth a n d colony
formation. A t 100 ~tM, some i n d i c a t i o n s of cytotoxicity
could be detected by visual i n s p e c t i o n of cell m o n o l a y e r s
after 24 h t r e a t m e n t a n d D N A synthesis a n d colony
f o r m a t i o n were i m p a i r e d to a m o d e r a t e extent only. A t
50 ~tM, only p r o t e i n synthesis showed some i m p a i r m e n t
(confirming the results of M e s h i t s u k a et al., 1987), the
other p a r a m e t e r s of cytotoxicity r e m a i n i n g virtually
u n c h a n g e d . T h u s the u p p e r limit of c o n c e n t r a t i o n for
d e t e r m i n i n g the influence of ZnSO4 o n H S V replication
in the absence of severe cytotoxicity lies b e t w e e n 50 ~tM
a n d 200 ~tM.
Effect o f Z n S 0 4 on H S V replication
This issue was addressed by d e t e r m i n i n g virus yields in
the presence of ZnSO4 (Fig. 2). W i t h a c o n c e n t r a t i o n of
50 ~tM-ZnSO4 the virus yield after i n f e c t i o n with HSV-1
K O S was only slightly reduced, whereas at 100 ~tM the
yield fell sharply to values below 1%o of the control. A t
200 ~tM, virus yield was reduced 50-fold. It is r e m a r k a b l e
however that at a c o n c e n t r a t i o n of Z n S O 4 in w h i c h
severe cytotoxicity was observed by all the m e t h o d s
applied, cells were still able to m a i n t a i n some virus
synthesis, the virus yield at 300 ~tM-ZnSO4 b e i n g 8 x 102
p.f.u./ml. T h e a m o u n t of infectious virus in the eclipse
phase (2 h p.i.) was 10 p.f.u./ml whereas the final yield in
u n t r e a t e d controls was 3 x 108 p.f.u./ml. This e x p e r i m e n t
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Zinc ions inactivate the virion of H S V
2991
was repeated with several other virus strains (HSV-1
ANG, HSV-2 HG52) and similar results were obtained
(Fig. 2).
Effects of ZnSO~ on cellular and viral DNA synthesis
Because of the earlier suggestion that the primary cause
of impairment of virus synthesis by ZnSO~ was
intranuclear inhibition of HSV D N A polymerase, viral
and cellular D N A synthesis was studied in the presence
of ZnSO4. Fig. 3 shows the CsCI density gradient profiles
for two ZnSO4 concentrations. The amount of viral
DNA synthesis with 100 ~tM-ZnSO4 in the culture
medium was not distinguishable from the synthesis in the
absence of zinc. According to the data on cytotoxicity
and virus yield, this concentration of ZnSO4 is apparently the highest that is compatible with effective
synthetic processes in the infected cell. Viral and cellular
D N A were unaffected, although formation of infectious
virus was very strongly impaired. If the concentration of
ZnSO~ was increased beyond 100 #M, synthesis of both
viral and cellular D N A dropped to very low values.
(d)
!
i
~ ~
I00
80-
t,
60
40
<
Z
20
0
i
0
4
8
12
16
Incubation time (h)
24
20
Effects of ZnSO , on virus adsorption, penetration and
egress
In experiments designed to determine virus yield in the
presence of ZnSO4, the compound was added only after
virus adsorption. Therefore, the observed reduction in
virus yield does not reflect an effect on the adsorption
process. A possible influence of ZnSO4 on HSV
adsorption was investigated using [3H]thymidinelabelled HSV-1 ANG. No difference in the absorption
kinetics was found whether ZnSO4 was present or absent
(Fig. 4).
Penetration of HSV is a fast process, directly
connected to virus adsorption. Hence the influence of
ZnSO4 on penetration was tested by separating the
processes of adsorption and penetration by appropriate
(e)
I
I
I
!
2OO
150
e~
100
Fig. 1. Cytoxicity of ZnSO, for the AGMK cell line RC 37 Rita. Light
microscopy of cell cultures held in culture medium alone (a) or in
medium containing ZnSO, at either 100 ~tM(b) or 200 p.M (c) for 24 h
and examined for morphological changes by phase-contrast light
microscopy. Bar markers represent 35 ~tm. (d) D N A synthesis in
uninfected cell. Cultures held in medium containing ZnSO4 were
subjected to a pulse with [3H]thymidine at the times indicated in the
figure, washed and the radioactivity was determined in acidprecipitated aliquots of cell homogenates. Without zinc, • ; / % 50 #M-
o
o
50
2
3
4
Cells per colony
>4
ZnSO,; O, 100 ~tM-ZnSO,; [3,200 l.tM-ZnSO4; V, 300 p.M-ZnSO,. (e)
Colony formation in the presence of ZnSO,. Cells were seeded onto
culture dishes in appropriate dilution. After 10 h, medium was replaced
by unmodified medium, [] ; or medium containing ZnSOa at 50 ~tM k~;
100 p.M, B; or 200 ~tM, II. Two days after seeding, cells were stained
with crystal violet and colonies were counted.
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G. Kfimel and others
2992
I
I
I
I
108
~1o
.=
107
]5
<
106
>
104
10 3
10:
I
0
I
I
I
I
I
100
150
200
250
300
Zinc concentration (~tM)
Fig. 2. Virus yield in the presence of ZnSO4. Cell monolayers were
infected with HSV-1 KOS, • ; HSV-1 ANG, O; or HSV-2 HG52 A.
The culture medium was changed to media containing ZnSO~. After
24 h virus was harvested and the yield determined.
50
i
(a)
(b)
(c)
15
x
6. 10
< 5
Z
c~
0
0
I
0
20
I
40
60
80
Adsorption time (rain)
I
I
100
120
Fig. 4. Adsorption of native virus in the presence of ZnSO4.
Radioactively labelled HSV-1 KOS was allowed to adsorb to cell
monolayers in the presence of various concentrations of ZnSO4 for the
times indicated. Virus adsorption was monitored after removal of
unadsorbed virus as described in Methods by determination of acidprecipitable material. Adsorption was in the presence of unmodified
culture medium, • ; or medium containing ZnSO4 50 ~tM,r-q; 100 IxM,
A; 200 gM, M; 300 ~tM, C), respectively.
.~ 105
20
0
adsorption period. Table 1 shows the virus yield in cell
cultures when Z n S O 4 had been r e m o v e d after the 4 h
period allowed for penetration. Virus yields 16 h after the
end o f the adsorption period demonstrate that ZnSO4
does not prevent either penetration or the early events o f
virus replication.
Since ZnSO4 is hardly transported into cells (Meshitsuka et al., 1987), one would expect it to exert cytotoxicity
by reacting with surface structures o f either the cell or the
virion itself. Zinc ions m i g h t in this w a y affect the last
stage in H S V replication, egress o f the virus from the
m e m b r a n e systems o f the cell. This possibility was tested
by separate determination (by titration) of the ratio of
virus remaining within cells to that transported into the
supernatant. W e c o m p a r e d cell cultures in w h i c h HSV-1
had been p r o p a g a t e d in the absence or presence o f
ZnSO4; the ratio o f intracellular to released virus
remained the same (data not shown).
_J
J
10
20
30
10
20
30
10
20
30
Fractions
Fig. 3. DNA synthesis in infected cells in the presence of ZnSO4. Cell
monolayers were infected with HSV. After adsorption, the medium
was replaced by medium containing no ZnSO4 (a), 100 ~tM-ZnSO4(b) or
200 ~tM-ZnSO4(c). Synthesis of viral and cellular DNA was allowed to
proceed in the presence of [3H]thymidine. Each of the three panels
shows acid-precipitable material in fractions of the CsCI gradients
used to separate viral from cellular DNA. The arrow shows the position
of 14C-labelled viral marker DNA.
temperature shifts (Fuller & Spear, 1985), ZnSO4 being
added during the period o f virus penetration. I f ZnSO4
could prevent virus penetration, this would have led to
very low titres o f virus 16 h after the end o f the
Inactivation o f free virus by Z n S 0 4
The true m e c h a n i s m underlying the d o c u m e n t e d antiherpetic effect o f ZnSO4 ( H o l z m a n n et al., 1988) was
revealed by experiments where the interaction o f ZnSO4
with free H S V was tested in the absence o f cells. E a c h o f
three HSV-1 strains ( A N G , KOS, W A L ) or HSV-2
H G 5 2 was used either in the form o f an unpurified stock
(Kfimel et al., 1982) or a virus purified by sedimentation
in a sucrose gradient (Spear & R o i z m a n , 1972). Virus
was incubated for various periods of time with ZnSO4
dissolved in PBS without C a 2+ and M g z+ in concentrations ranging from 150 ~tM to 15 mM. After dialysis or
dilution to reduce the zinc concentration the a m o u n t o f
residual virus was determined by titration. Fig. 5 shows
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2993
Zinc ions inactivate the virion o f H S V
Table 1. Influence of ZnS04 on H S V penetration
Infection
HSV-1 KOS
Mock
ZnSO4
concentration ~M)
during virus
penetration
Interval
allowed
for virus
replication (h)
0
0
50
100
150
200
0
200
12
16
16
16
16
16
16
16
108
Virus yield
2-2
1.0
2.2
1-8
1'9
1.5
2-5
2.8
×
x
x
x
×
×
×
×
103
107
10r
107
107
107
103
lO ~
,
l0 6
J
,
~
~
i
,
,
"IL_
6. lOs
104
i
o
103
10:
the kinetics of inactivation of HSV-1 KOS by ZnSO4.
The infectivity of free HSV is effectively destroyed by
the presence of ZnSO4.
Zinc inactivation can be demonstrated with purified
HSV and the effect is not simulated by a possible pH
shift. Incubation with Na2SO4 or MgSO4 does not
inactivate HSV, whereas zinc salts other than
the sulphate show an effect similar to that of ZnSO4
(Table 2).
Inactivation is strongly temperature-dependent (Fig.
5). Even at high concentration (15 mM), ZnSO4 had no
significant effect on HSV infectivity in the temperature
range 4 °C to 18 °C. With elevation of the temperature
above 20 to 25 °C, inactivation increased strongly. We
observed considerable differences in the dose dependence of ZnSO4 inactivation between individual HSV
strains (Fig. 6).
The consequence of inactivation might be impairment
of the main glycoprotein functions, adsorption and
penetration. Alternatively, virus might be precipitated,
aggregated or have its structure destroyed by the divalent
cation. We used [3H]thymidine-labelled HSV-1 KOS to
study glycoprotein functions of zinc-inactivated virus.
The kinetics of virus adsorption was determined for
native or inactivated virus (Fig. 7). In a parallel
experiment, each of the two virus preparations was
adsorbed to ceils and the appearance of acid-precipitable
radioactive material in cell nuclei was followed (Fig. 8).
We found that DNA of native virus accumulated in
nuclei in a linear fashion, whereas no DNA from
inactivated virus could be detected from 0 to 4 h p.i.
These results suggest that zinc-inactivated virus is not
blocked in adsorption but penetration or transport to cell
nuclei is apparently totally prevented.
In order to test for morphological alterations or
aggregation of the virus we used transmission electron
microscopy (Fig. 9). In electron microscopy, biological
structures are visualized by the adsorption of an electrondense heavy metal so zinc adsorption into the virion can
be monitored if uranyl acetate contrasting is avoided.
10 ~
I
01
10
20
30
Incubation time (h)
40
Fig. 5. Inactivation of free HSV-1 KOS by ZnSO4; kinetics and
temperature dependence. Free virions of HSV-1 KOS were incubated
in the absence of cells with various concentrations of ZnSO4. The
residual virus concentration was determined by titration. II, Incubation at 30 °C, 15 mM-ZnSO4; O, incubation at 20 °C, 15 mM-ZnSO4.
Incubation at 37 °C: A, no zinc; (3, 0.5 m~,i-ZnSO4; N, 1.5 mM-ZnSO4;
V, 5 mM-ZnSO4; A, 15 mM-ZnSO4).
Table 2. Influence of counter ions and p H on the zinc
inactivation of the HSV-1 virion
Virus preparation*
Purified virus t
Stock virus:~
Stock virus§
Inactivation
PBS pH 4-5
PBS pH 5.5
PBS pH 6.5
PBS pH 7.3
PBS (pH 7.3)
5 mM-ZnSO4
5 mM-Zn(CH3COO)2
5 mM-Zn(NO3) 2
5 mM-ZnC12
5 msl-ZnI2
5 mM-Na2SO 4
PBS pH 7.3
5 mM-ZnSO4
Residual titre
1.5 x
2-0 x
3.5 x
3.7 x
4.2 ×
5-1 x
4"1 ×
3.8 x
2-5 x
2.5 x
3.7 ×
1-4 x
2.0 x
108
108
108
108
107
104
lO4
104
104
104
107
108
104
* Various preparations of HSV-1 KOS were incubated with equal
amounts of PBS or salts dissolved in PBS pH 7.3 for 24 h at 37 °C and
the residual virus concentration was determined by titration.
t Dialysed against 0.9~ NaCI, 10 mM-Tris-HC1 pH 7.3.
:~ Grown in medium without protein supplements, dialysed against
100 mM-MgSO4, 10 mM-HEPES pH 7-3.
§ Virus suspension in unmodified culture medium.
Purified virus (HSV-1 ANG) was inactivated (15 mMZnSO4, 24 h, 37 °C) or, as a control, incubated under the
same conditions with buffer only. Both samples were
subjected to preparation for electron microscopy; the
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2994
G. Kiimel and others
lOs
I
I
I
I
~
~500f
600 |
I
lO 7
,
~
~
~
~
,
~400 I
106
"~300 I
~200~
¢.*.2
6,
~ 105
.~
100
"~ 104
0t
0
J
I
0.5
1
~
t
t
1.5
2
2.5
Incubation time (h)
I
3
I
3.5
4
e~ 1 0 3
102
1 0 II
I
0
I
1
I
I
2
3
4
Zinc concentration (mM)
I
5
~I
6
Fig. 6. Inactivation of free HSV-1 KOS virions by ZnSO4; dose
dependence and strain differences. Suspensions of HSV-1 KOS (D),
HSV-1 A N G (O), HSV-1 W A L ( 0 ) and HSV-2 HG52 (z~) were
incubated for 24 h with ZnSO4 at the concentrations indicated and the
residual virus concentration was determined.
20
~
16
x
d
"~
8
0
0
20
40
60
Fig. 8. Kinetics of penetration and transport to the nucleus of native
and ZnSO4-inactivated HSV. Culture cells were infected at an m.o.i, of
10 with native or inactivated HSV-1 KOS radioactively labelled with
[3H]thymidine. At the times indicated radioactivity of acid-precipitable material in the nuclei was determined as detailed in Methods.
Native virus, A ; inactivated virus, O.
80 100 120 140 160 180 200 220 240
Adsorption time (min)
Fig. 7. Adsorption kinetics of ZnSO4-inactivated virus. Radioactively
labelled HSV-! KOS was inactivated in 15 mM-ZnSO, for 24 h or
incubated with PBS as a control. Virus adsorption was monitored by
removal of unadsorbed virus and determination of radioactivity in
acid-precipitable material at the times indicated (for details see
Methods). Native virus, A ; inactivated virus, 0 .
step of negative staining with uranyl acetate was
however omitted in two of the samples (insets in Fig. 9 a).
The micrographs show clearly that zinc is deposited in
considerable amounts onto virion structures, and that
disintegration, aggregation or precipitation does not
occur.
Discussion
The mechanism of inhibition of HSV infection by
ZnSO4 has had a chequered history in the literature. An
early study (Falke, 1967) described alteration of giant cell
formation, a glycoprotein-mediated phenomenon. The
laboratory of Y. Becker reported inhibition of viral D NA
polymerase in vitro (Fridlender et al., 1978) and this effect
was taken to explain in vivo HSV inhibition as observed
in their virus-cell system (Gordon et al., 1975; Shlomai et
al., 1975). Gupta & Rapp (1976) interpreted their data on
the allegedly specific failure of zinc-treated infected cells
to synthezise certain viral proteins as an additional
explanation for the inhibitory effect of ZnSO4. Zinc
uptake into cells is, however, subject to strict homeostatic control and it is for this reason that zinc, in contrast
to cadmium, is well tolerated and even beneficial to the
organism. Therefore it is unlikely that the inhibitory
concentration for viral D N A polymerase could be
reached in the nucleus of the infected cell.
In accordance with the work of Meshitsuka et al.
(1987) we found that ZnSO4 increasingly affects the
culture cells when used in a concentration range from 50
to 200 ~t~. No inhibition of HSV replication could be
observed below these limits. Only at 100 [AMwas virus
synthesis considerably decreased, although viral as well
as cellular D N A synthesis was sustained. At higher
concentrations, all D N A synthesis in the infected cell as
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Zinc ions inactivate the virion of H S V
2995
®
•
•
(b)
•~• ~:i~ • ii
••
•••i~,~•~•~=~•~
••~•
.....
2
¢
Fig. 9. Electron microscopy of zinc-inactivated purified HSV-1 KOS. (a) Mixture of untreated and zinc-inactivated virus particles
without uranyl acetate contrasting. The insets show untreated virions alone (left) and zinc-inactivated virions alone (right), without
contrasting. (b) Zinc-inactivated virus was contrasted with uranyl acetate. (c) Untreated virus contrasted with uranyl acetate. The bars
markers represent (a and b), 0.5 ~tm (c), 0-25 ~tm.
well as cell morphology and presumably metabolic
activity in general is severely affected.
Thus the early interpretation (Gordon et al., 1975;
Shlomai et al., 1975) must be revised. Inhibition of HSV
replication in culture cells is due to the cytotoxicity of the
effective zinc concentration. The inhibitory effect of zinc
ions on the formation of HSV D N A observed by
Becker's group might be explained as a secondary effect
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2996
G. Kiimel and others
in the particular virus-cell system used, possibly due to
non-specific inhibition of protein synthesis.
The concept that intracellular inhibition is the
consequence of the cytotoxicity of ZnSO4 rather than a
specific interaction with viral synthetic processes is
corroborated by results showing that zinc does not
interfere with adsorption, penetration or egress of native
virus, and that replication of different virus strains is
inhibited to the same extent by the presence of ZnSO4 in
the culture medium. Free virus, however, is very
effectively inactivated at the concentrations used in
topical therapy (Holzmann et al., 1988). In control
experiments, we clarified that this effect is indeed
mediated by the zinc ions in the virus suspension.
Virion inactivation is independent of the counterion of
the zinc salt and a possible pH shift due to the addition of
the salt solution is without effect. Electron micrographs
show that zinc does not simply precipitate or aggregate
the HSV particles but the presence of the zinc ions leads
to a considerable deposition of zinc ions in virion
components. Since the majority of virus particles are
non-infectious in a given suspension, information concerning the target component of zinc inactivation cannot
be derived from the micrographs. Transport systems in
viral membranes have not been described and are not to
be expected; therefore interior structures should not be
accessible to metal ions in intact virions. If only the
virion surface interacts with zinc, membrane-bound
surface glycoproteins are likely to be the target because
membrane lipids are identical between virus strains,
whereas glycoproteins are different. This could also
explain differences in inactivation between viral strains.
ZnSO, interacts with the virion only above the temperature range of transition of membranes from a rigid to a
fluid state (Powers & Pershan, 1977; Chapman et al.,
1974). This temperature effect is consistent with the idea
that sensitive domains of the target glycoprotein have to
be exposed by a conformational change. Indeed, ZnSO4
inactivation of the herpes virion interferes with glycoprotein functions; virus penetration is severely affected
whereas effects on adsorption are slight. These results
lend themselves to the following interpretation. Membrane glycoproteins accumulate zinc non-specifically
depending on zinc concentration and incubation time,
possibly by binding to sulphydryl groups. The deposition
of zinc interferes eventually with the function of HSV
glycoprotein B or D, thus preventing virus penetration.
This would be in line with conceptions of the mechanism
of adsorption and penetration by membrane fusion. Adsorption can be considered to be a simple and insensitive
process, mediated by several glycoproteins and independent of temperature and hence of active contributions
from the cell metabolism. Penetration by membrane
fusion in acidified vesicles, however, is thought to
involve multiple interactions between viral glycoproteins
and cell surface components (Fuller & Spear, 1985;
Kiihn et al., 1990). It appears to depend on energy
transfer, physiological temperature and membrane
fluidity. Thus, our finding that zinc inactivation affects
penetration rather than adsorption, corroborates the
interpretation that glycoproteins accumulate the metal
until the zinc content becomes incompatible with the
glycoprotein functions.
We conclude that the molecular mechanism of the
therapeutic effect of ZnSO, in the treatment of herpetic
lesions is not due to a cytoinvasive intracellular
inhibition of virus replication, but to the drastic
inactivation of free virus in skin tissues, intercellular
vesicles and blisters.
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