Journal of General Virology (1993), 74, 1519-1525.
1519
Printed in Great Britain
The effects of lithium and potassium on macromolecular synthesis in
herpes simplex virus-infected cells
C. E. Hartley, 1. A. Buchan, ~ S. Randall, 1 G. R. B. Skinner, 1 M . Osborne 2 and L. M . Tomkins 2
1Department of Infection and 2Department of Physiology, The Medical School, University of Birmingham, Edgbaston,
Birmingham B15 2T J, U.K.
All herpes simplex virus (HSV) infected cell-specific
polypeptides (ICSPs) were synthesized in the presence
of lithium at a concentration (60 mM) inhibitory to the
production of infectious virus. Yields of certain ICSPs
were increased and others, in particular glycoprotein
C, decreased. HSV DNA synthesis was completely
inhibited; synthesis and in vitro activities of HSV DNA
polymerase and thymidine kinase were decreased but to a
degree insufficient to account for the complete inhibition
of HSV DNA synthesis. HSV DNA synthesis was
inhibited to an equivalent degree by either incubation
with 60 mM-lithium or by potassium starvation; both
procedures decreased intracellular potassium by an
equivalent amount as adjudged by X-ray microanalysis.
We conclude that lithium inhibits HSV DNA synthesis
by displacement of potassium from a potassiumdependent biochemical reaction or by other physiological changes brought about by the loss of cellular
potassium. The possibility that lithium also directly
inhibits a virus replicative event cannot be excluded.
Introduction
reported. However, decreased yields of infectious HSV
were reported, together with decreased yields of viral
DNA, from infected Vero cells cultured with 10 to
30 raM-lithium (Patou et al., 1986).
Lithium influx will inevitably alter the intracellular
concentration of other cations and inhibition of HSV
replication by lithium may be a consequence of that
alteration. Potassium, for example, has been implicated
in the control of macromolecular synthesis. In BHK
cells, for example, a decreased concentration of intracellular potassium inhibited protein synthesis, DNA
synthesis and cell replication (McDonald et al., 1972).
We have investigated the effects of lithium and other
cations on HSV macromolecular synthesis to determine
the mechanism of inhibition of virus replication by
lithium.
Lithium is known to affect the behaviour and replication
of bacteria and viruses. Hadley et al. (1931) reported
that incorporation of 12 mM-lithium chloride into beef
infusion broth encouraged the generation of filterable Gforms of the Shiga bacillus, and Morishita & Tokada
(1976) reported that although lithium had a sodiumsparing effect for the growth of Vibrio parahaemolyticus,
higher concentrations, in excess of 300 mM, inhibited
growth of the organism. Leighton (1979) found that the
growth of a number of Gram-negative bacteria was
inhibited by comparatively low concentrations of lithium
chloride (12 m~), and recently we found that higher
concentrations (80 mta) inhibited growth of Gram-positive as well as Gram-negative bacteria (Buchan et al.,
1989).
Lithium specifically inhibits replication of the DNA
viruses, herpes simplex virus (HSV), pseudorabies and
vaccinia viruses but not replication of the RNA, influenza
and encephalomyocarditis viruses (Skinner et al., 1980).
The effects of lithium on macromolecular synthesis in
virus-infected cells has not been extensively studied.
Ziaie & Kefalides (1989) indicated that HSV-induced
suppression of host cell polypeptide synthesis was less
marked when cells were cultured in the presence of 20 to
30 mM-lithium and demonstrated the synthesis of virusspecific polypeptides under those conditions. Unfortunately, the effect on infectious virus yield was not
0001-1240 © 1993 SGM
Methods
Tissue culture. BHK 21 cells (clone 13) (Macpherson & Stoker, 1962)
were propagated in supplemented Eagle's medium (Vantsis & Wildy,
1962) containing 10% (v/v) tryptose phosphate broth (Difco) and
l0 % (v/v) new born calf serum (Gibco) (ETC).
Viruses and virus assays. HSV type 1 strain HFEM, a derivative of
the Rockefeller strain HF (Wildy, 1955) was used. Virus was assayed by
the suspension method of Russell (1962).
Virus growth experiments. Confluent cell monolayers were infected at
an m.o.i, of 20 p.f.u./cell. Virus inocula were removed, monolayers
washed and incubated at 37 °C for up to 18 h in appropriate media.
Cells were harvested together with medium and stored at - 7 0 °C.
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1520
C. E. H a r t l e y and others
Virus titrations were at dilutions which excluded inhibition of plaque
formation by residual lithium during assay.
Metabolic labelling
(i) Polypeptides. Cultures were incubated at 37 °C in methionine-free
Eagle's medium supplemented with 2 % calf serum and polypeptides
were metabolically labelled by addition of L-[ZSS]methionine
(Amersham) to a concentration of 5 ~tCi/ml. Cultures were harvested
into 2 % SDS, 5 % 2-mercaptoethanol, 0'05 M-Tris-HC1 buffer pH 7-0,
3 % sucrose and 0.005 % bromophenol blue.
(ii) Carbohydrate. Cultures were incubated in 2 ml ETC (with only
2% calf serum) and glycoproteins radiolabeUed by addition of D[14C]glucosamine, L-[14C]fucose or D-[14C]mannose (Amersham) to a
concentration of 1 ~tCi/ml, 1 h after infection.
Polyacrylamide gel electrophoresis. Analyses of proteins by PAGE
were carried out in the discontinuous system used by Harper et al.
(1979). A standard HSV-infected cell extract was included in all gels for
consistent identification of individual polypeptides and estimation of
their Mrs.
Radiography and densitometry. Flattened, fixed and dried gels were
exposed to Kodak X-omat S film, processed using Agfa-Geveart
developer and fixative. For densitometric analysis, using the LKB
Ultroscan XL laser densitometer, Kodak X-omat S film was hypersensitized by pre-exposure to light through a red filter from an
electronic flash gun (Metz 402). Flash intensity produced an absorbance
change of 0.15 units, at a wavelength of 540 rim, on the film (Laskey &
Mills, 1975). Laser densitometry was calibrated by multiple exposures
of gels and quantification made only within the linear range of response
of the film. For DNA analysis, using z~p, exposures were carried out at
70 °C with a single calcium tungstate intensifying screen.
-
Virus particle purification. Confluent HEp2 cell cultures were infected
at a multiplicity of 20 p.f.u./cell and incubated for 72 h at 32 °C. The
culture medium was precipitated with 8 % w/v polyethylene glycol and
layered onto 5 to 45 % (w/v) sucrose gradients. The virus band was
collected and virus was pelleted by centrifugation and then resuspended
in 0-02 M-Tris-HC1 pH 7.4 and stored at - 70 °C (Powell & Watson,
1975).
DNA extraction. Cells (5 x 10r) or purified virus particles (10 TM)were
dissolved in 1 ml of extraction buffer (150 mM-NaCI, 1.5 mM-MgCI2,
1% deoxycholate, 1% Tween 20, 10 mM-Tris-HC1 pH 8.5). The
solution was extracted sequentially in phenol, phenol~hloroformisoamyl alcohol (12: 12: 1) and chloroform. DNA was precipitated with
2.5 volumes of ethanol at - 2 0 °C. The dried pellet was dissolved in
10 mM-Tri~HCI-1 mM-EDTA pH 8.0 and stored at - 7 0 °C.
Ribonuclease treatment. Ribonuclease A (Sigma) was dissolved in
10 mM-Tris-HC1 and DNase activity inactivated by incubation at
100°C for 15min. Nucleic acid preparations were treated with
ribonuclease at a concentration of 100 Ixg/ml, at 37 °C for 30 min and
DNA was re-extracted.
Nick translation. The DNA probe (32p-labelled) was prepared (BRL
Nick Translation reagent kit). DNA was separated from free
nucleotides by elution with 10mM-Tris-HCl-I mM-EDTA pH 8.0
through a 5 ml Sephadex G75 column.
Hybridization. DNA was denatured by incubation at 100 °C and
0 °C for 10 min each and the solution adjusted to 2 M-NaC1 and 0.2 Msodium citrate. Nitrocellulose membranes (Schleicher & Schuell,
0-45 Ixm) were wetted with water and washed with 2 u-NaClq).2 Msodium citrate. Nucleic acid samples were applied (Bio-Dot, Bio-Rad)
and the membrane was air-dried and baked at 80 °C under vaccum.
The membrane was incubated for 3 h at 42 °C with hybridization
buffer: 500 ~tg/ml thermally denatured DNA from salmon testes, 50 %
deionized formamide, 1 M-NaC1, 0.1 M-sodium citrate, 0"1% Ficoll,
0"1% BSA and 0.2 % polyvinylpyrrolidonein 50 mi-sodium phosphate
buffer pH 5.0. The membrane was exposed to heat-denatured 32p.
labelled HSV DNA probe (2 x 106 c.p.m. ; about 100 ng DNA) at 42 °C
with agitation for 24 h. The membrane was washed three times in
NaCl-sodium citrate solution: 0.3u~-03M for 5min at room
temperature; 0.7 M~).07 M at 65 °C for 30 min; and 0'3 M~).03 M for
1 rain at room temperature. The membrane was then air-dried and
exposed to hypersensitized film at - 7 0 °C with a single calcium
tungstate intensifying screen.
Cell extracts. For enzyme assays, infected and uninfected cells were
harvested in 5 mM-Tris-HCl pH 7.4 (5 x 107 cells/ml), disrupted by
ultrasonication and ultracentrifuged at 100000 g for 30 min.
HSV DNA polymerase. DNA polymerase (Pol) assays were performed essentially as described by Ostrander & Cheng (1980). The
reaction mixture (100~tl) contained 200mM-KC1, 4mu-MgCl~,
200 ~tg/ml BSA, 0"5 mM-DTT, 100 lag/ml thermally denatured DNA
from salmon testes, 100 laM-2'-deoxyadenosine 5'-triphosphate, 100 ~tM2'-deoxycytidine 5'-triphosphate, 100 ~tM-2'-deoxyguanosine 5'triphosphate, 1 taCi [3H]thymidine Y-triphosphate (*H TTP) and cell
extract in 50 mM-Tris-HC1 pH 8.0. Reaction mixtures were incubated
for 30 min at 37 °C and cooled on ice. BSA and ATP were added to
each assay tube to a concentration of 2 mg/ml and 400 ~tg/ml
respectively. Samples were spotted onto 2 cm filter paper discs
(Whatman no. 1), TCA-precipitated and processed for liquid scintillation. DNA Pol activity was expressed in pmol ZH TTP/mg protein.
HSV thymidine kinase (TK). TK was assayed essentially as described
by Klemperer et al. (1967) with the modifications of Larder et al.
(1983). The reaction mixture (250 ~tl) contained 5 mM-MgC12, 5 mMATP, 2 mg/ml BSA, 2 mM-DTT, 30 mM-[14C]thymidineand cell extract
in 20 mu-sodium phosphate buffer pH 6'0. Reaction mixtures were
incubated for 30 min at 37 °C, heated to 100 °C for 2 min and then
cooled rapidly on ice. Fifty I11 samples were spotted onto filter discs
(DE81, Whatman), air-dried, washed three times for 20 min in 1 mMammonium formate, air-dried and processed for liquid scintillation.
TK activity was expressed in nmol thymidine monophosphate
generated per mg protein.
X-ray microanalysis. BHK 21 cells were grown to confluence on
Thermanox coverslips (Bio-Rad). After 24 h incubation in appropriate
media, coverslips were rinsed briefly in isotonic ammonium acetate and
carefully blotted dry. The cells were immediately frozen by 10 to 15 s
immersion in liquid propane in a Reichert-Jung cryostat. Coverslips
were transferred to 2 cm ~ porous plastic containers, stoppered and
rapidly transferred to liquid nitrogen. The cells were freeze-dried at
5.0 x 10-~ Torr overnight and the coverslips mounted on graphite bulk
holders. Bulk holders were coated with carbon to a thickness of
150pm in a Polaron sputter coater and graphite DAG (Emscope
Laboratories) was used to seal the bulk holders around the coverslip.
The carbon-coated specimens were stored under vacuum with silica gel
until required.
Energy dispersive X-ray microanalysis was performed with a
JEOL 120 CX TEMSCAN and a LINK 860 series 2 X-ray microanalysis system (Warley et al., 1983). An accelerating voltage of 20 kV
was used with the specimen tilted at 30 ° to incident electrons. Analysis
proceeded for a live time of 100 s with a window file set for the
characteristic K X-ray peaks of Na, Mg, P, S, C1, K and Ca. Spectra
were processed using the Quantem FLS program for background
subtraction of the data.
The data obtained from the spectra enabled peak:background ratios
to be calculated for each element under consideration using the formula
Peak-background ratio = net integral/(gross integral-net integral).
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Effect of Li and K & HSV-infected cells
1521
There was normal representation of host polypeptides in
uninfected cells cultured with lithium at a concentration
of 60 mM as adjudged by the number and intensity of
polypeptide bands seen in radiography. In HSV-infected
cells, virus-induced suppression of host cell polypeptides
was less marked in the presence of 60 mM-lithium, a
concentration that completely inhibited virus replication.
Virus-infected cell-specific polypeptide (ICSP) synthesis was quantified by laser densitometry and the
amounts synthesized in the absence and presence of
60 mM-lithium were compared (Fig. 1, Table 1). Every
ICSP was synthesized in the presence of lithium but
many were synthesized in decreased amounts (e.g. those
of Mrs81K, 28K and 105K) whereas others were
synthesized in increased quantities (e.g. peptides of
Mrs 35K and 22K).
The effect of lithium on the synthesis of individual
HSV glycoprotein species was examined using
[laC]glucosamine. The major HSV glycoprotein species
were tentatively identified from their apparent M r and
the amount synthesized in the absence and presence of
60 mM-lithium was compared (Fig. 2, Table 2). Synthesis
of glycoproteins gB, gD and gE was reduced 50% or
more and glycoprotein C 90 %.
(a)
The effect of lithium on H S V DNA synthesis
Results
Effect of lithium on polypeptide synthesis
1
2
3
4
5
6
7
8
i!;i:i:ii~::
:,:Li
(b)
Nucleic acids were extracted from uninfected and
infected cells cultured in the absence or presence of
60 mM-lithium. Equivalent amounts of HSV-infected cell
DNA were probed with purified HSV DNA. HSV DNA
was detected by solid phase DNA:DNA hybridization
using ~P-labelled purified HSV DNA probe and was
quantified by two-dimensional laser densitometry (Fig.
3, Table 3). An extract prepared from cells immediately
after infection facilitated quantification of input viral
DNA. In the absence of lithium there was a thousandfold
increase over the input viral DNA. There was less than
the input level of viral DNA in extracts of cells cultured
with lithium.
In an attempt to identify the molecular mechanism of
inhibition of viral DNA synthesis, the effects of lithium
on the synthesis and activities of enzymes involved in
viral DNA replication were investigated.
H S V TK
The effect of lithium on TK synthesis was examined by
comparing the in vitro TK activities of extracts of
infected cells cultured in the absence and presence of
lithium (Table 4). There was no significant difference in
TK activity in the extract of cells cultured in lithium.
The effect of lithium on TK activity in vitro was
examined by adding lithium to an assay of an extract of
infected cells cultured in the absence of lithium. Lithium
had no effect on TK activity at a lithium concentration
as high as 80 mM (data not shown).
Fig. 1. The effect of lithium on HSV-1 ICSP synthesis. Cells were pulselabelled for 40 min at alternate 40 rain intervals in the absence (a) and
presence (b) of 60 mra-lithium. ICSPs were separated by 9 % PAGE and
each was grouped in a kinetic category of synthesis approximating to
the ct, fl and ), designations (Honess & Roizman, 1975). Lane 1, 0 to
40 vain; lane 2, 80 to 120 min; lane 3, 160 to 200 rain; lane 4, 240 to
280 rnin; lane 5, 320 to 360 min; lane 6, 400 to 440 rain; lane 7, 480 to
520 rain; lane 8, 560 to 600 min.
H S V Pol synthesis.
The effect of lithium on HSV DNA polymerase synthesis
was examined (Table 4). There was considerable inherent
variation in the Pol activities of these crude cell extracts,
but the highest (and lowest) activities were observed
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C. E. Hartley and others
1522
Table 1. The effect of lithium on H S V ICSP* synthesis
Mr
( x 10-3)
Synthesis
(% control)
Mr
( x 10-z)
Synthesis
(% control)
Mr
( x 10-~)
Synthesis
(% control)
101/96
91
81
73
60
58
52
42
35
33
30
24
78
64
46
87
97
88
71
105
132
85
119
98
146
130
62
56
48
45
44
40
38
34
28
27
26
22
77
78
82
85
77
88
87
109
92
89
47
78
78
124
155
120
105
89
63
31
25
68
60
42
82
59
79
58
* Polypeptides are grouped according
(ct, fl or 7; Honess & Roizman, 1975).
1
2
3
4
to
their
kinetic
5
category
of
synthesis
Table 2. The effect of 60 raM-lithium on H S V
glycoprotein synthesis
Yield
Glycoprotein
I gC
IgB
IpgB
gC
gB
pgB
pgC
gE
gD
M r ( x 10-z)
143 to
125 to
115 to
106 to
85 to
53 to
131
122
112
100
71
39
(% inhibition)
90
45
50
30
40
35
I pgC
I gE
I
i gD
in assays which contained correspondingly similar
protein concentrations. Pol activity in the extract of
cells cultured with lithium ranged from 25 to 30%
of the control activity (highest observed activities,
91 and 352pmol/mg; lowest observed activities, 25
and 70 pmol/mg).
Effect of potassium on H S V replication; interaction with
lithium
Fig. 2. The effect of lithium on HSV glycoprotein synthesis. HSV-1infected cells were incubated in media containing various concentrations of lithium with glycoproteins metabolically labelled with
[14C]glucosamine. Polypeptides were separated by 9 % PAGE. Lane 1,
uninfected, 0 mM; lane 2, infected, 0 mM; lane 3, infected, 30 mM; lane
4, infected, 60 mM; lane 5, infected, 90 raM.
HSV-infected cells were incubated in media containing
various concentrations of KC1, NaC1, CaC12 or MgC12,
in the absence or presence of 20 mM-LiC1 which alone
decreased virus yields by 10-fold to approximately
10 p.f.u./cell. This permitted the detection of increases in
virus yields (greater than 10 p.f.u, per cell) and decreases
in virus yields (less than 10 p.f.u, per cell) brought about
by additional substances. In the presence of additional
potassium, virus yields were increased and reached
control levels at 40 mM-KCI. Additional potassium alone
did not inhibit virus replication except at higher,
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Effect of Li and K in HSV-infected cells
Time p.i.
N
Table 4. The effect of lithium on H S V TK and Pol
synthesis
1/5
lh
Lithium
Volume
of cell
extract
(lal)
18 h
Potassium-starved 18 h
1/100
18 h
1/5000
~:~[[l~
Potassium-starved 18 h
and then replaced
Fig. 3. The effect of lithium and potassium on HSV DNA synthesis in
infected cells. DNA extracts were hybridized to z2P-labelled purified
HSV DNA probe, in duplicate. The probe did not react with uninfected
cell DNA. N, Not diluted.
Table 3. Quantification of H S V DNA synthesis
Cell
extract
Time p.i.
(h)
Infected
1
Infected
18
Infected, plus
60 mM-LiC1
18
1523
Extract
dilution
Total HSV
DNA yield
(logx0 pg)
N*
1/5
1/ 500
1/2500
N*
1/5
4.1
4.2
6"9
7.1
3"6
3.7
* N, Not diluted.
cytotoxic levels, as adjudged by direct light microscopy.
In both the absence and presence of lithium, the cations
sodium, calcium and magnesium did not affect virus
Amount
of
protein
(lag)
TK activity
(nmol
TMP/mg
protein)
Pol activity
(pmol 3H
TTP/mg
protein)
Infected cells ir~
absence of
lithium
27
9
3
1
156
52
17
5.7
5.9
12-9
11.8
8.8
68
205
352
70
Infected cells in
presence of
60 mM-lithium
27
9
3
1
219
73
24
8
4.03
10.14
6"25
7"05
25
55
91
25
replication except at higher cytotoxic concentrations i.e.
100 mM, 50 m~a and 50 mM respectively.
Potassium moderated the inhibition of virus replication by lithium. It was unclear, however, whether
inhibition of virus replication was attributable to
increased intracellular lithium or to decreased intracellular potassium brought about by lithium influx.
Intracellular elemental profiles of cells incubated for
24 h in media containing 5 mM-KC1 and concentrations
of 30, 60 or 120 mM-LiC1 or in medium which was
free of both potassium and lithium were investigated
by X-ray microanalysis (Fig. 4, Table 5). Intracellular
potassium levels were decreased in an approximately
linear relationship relative to lithium concentration.
Potassium deprivation decreased intracellular potassium
concentrations to a similar level to that achieved by
60 mM-lithium treatment. On the other hand, concentrations of phosphorus and chlorine were only slightly
decreased whereas levels of sodium, calcium and sulphur
were unchanged.
It was important to examine the effect of potassium
deprivation on HSV D N A synthesis. Uninfected cells
were starved of potassium by incubation in potassiumfree medium (with three changes) for 18 h and were then
infected and incubated in potassium-free or potassiumcontaining media (Fig. 3). In potassium-starved cells
there was no HSV D N A synthesis, but following
replacement of potassium HSV D N A synthesis was
restored to control levels where there was about a
thousandfold increase over input viral DNA. It is
possible that decreased HSV DNA yields from cells
starved of potassium for 18 h before and after infection
were due to irreversible cytotoxicity. However, cells
starved of potassium for a total of 36 h before infection
supported virus replication to usual levels after potassium was replaced.
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1524
C. E. Hartley and others
0-5
oo
OO
0.4
V
V
0
0
o%
0
oo
g
0.3
o
g
o
o
o
O0
O0
O0
o
oo
oo
go
o
0.2
go
o
o
o
o
o
o
oo
o
o
o
o
o
o
o
0.1
COCO0
0
OCO
OOCO
I
I
0
30
P
OCO0
I
O0
OCO
0
O0
I
60 120
Na
I
I
0
30
I
I
60 120
P
I
0
I
I
I
I
I
I
I
30 60 120 0 30 60 120
Lithium concentration (raM)
S
C1
0
CO:COO0 0 ~ 0
O0
I
I
I
I
I
I
I
I
0
30
60
120
0
30
60
120
K
Ca
Element
Fig. 4. The effect of lithium on intracellular elemental profiles of uninfected BHK 21 cells. Each circle denotes data derived from a field
containing approximately 500 cells. Cells were incubated in lithium-free medium (0) or in medium containing lithium (30, 60 or 120 mM,
as indicated on the x-axis).
Table 5. The effect of lithium and potassium on
intracellular potassium levels in uninfected B H K 21 cells
Lithium
Potassium
Peak-background
ratio
(potassium
X-ray peak)
mean ± S.D.
0
30
60
0
5"3
5"3
5-3
0
0"41 +0"05
0"29 + 0"04
0"25 -t-0"03
0"26___0'04
Concentration (mM)
Discussion
There was synthesis of all ICSPs in the presence of
lithium. Synthesis of y-polypeptides in the presence of
inhibitors of DNA synthesis is decreased (Powell et al.,
1975; Honess & Roizman, 1975), and the stringency of
the requirement for DNA synthesis has led to the subclassifications yl and y2 (Silver & Roizman, 1985;
Holland et al., 1980). For example, under conditions of
inhibited viral DNA synthesis (a 20-fold reduction) the
synthesis of gD (yl) was inhibited less severely (fivefold)
than the synthesis of a y2 polypeptide of M r 21K (50fold; Johnson et al., 1986). In this study, the hybridization system was sufficiently sensitive to detect viral
DNA at a concentration below that attributable to input
viral DNA and equivalent to a thousandfold reduction in
viral D N A synthesis. Under these conditions, in the
presence of lithium, there was reduced synthesis of gC
(Table 3) and so it is unlikely that there is an absolute
requirement of DNA synthesis for y2 gene expression. It
has been suggested that the promoters of transcription of
the late genes are relatively weak and that high copy
numbers are required for their efficient expression
(Johnson et al., 1986). In addition to this, regulation of
synthesis of gC involves the ssDNA-binding protein,
ICP8 (UL29). Using a mutant of HSV deficient in ICP8
under conditions of inhibited DNA synthesis, Godowski
& Knipe (1985) found over-expression ofgC mRNA and
concluded that ICP8 acts to repress late gene expression.
Although the effect of lithium on gC synthesis is therefore
a consequence of inhibited viral DNA synthesis the
precise regulatory mechanism(s) involved is unclear.
A possible mechanism by which lithium inhibits viral
D N A synthesis might relate to reduced synthesis or
activity of DNA replicative enzymes. Synthesis of
HSV Pol in the presence of lithium remained at 25 to
35% of control levels and was thus available for
HSV DNA synthesis; moreover, D N A Pol activity in
vitro was stimulated by lithium (Randall et al., 1991).
Similarly, HSV TK synthesis was only slightly decreased
with no effect on TK activity in vitro. Thus, the effects of
lithium on Pol and TK cannot account for the absence of
HSV DNA synthesis.
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Effect of Li and K in HSV-infected cells
Secondly, lithium may mediate stimulation of phosphatidyl inositol degradation in a similar way to
interferon-induced stimulation (Cernescu et al., 1988).
This seems unlikely since lithium had no effect on the
replication of influenza and encephalomyocarditis viruses (Skinner et al., 1980) which are notably susceptible
to interferon. A third possibility is that replacement of
magnesium ions by lithium might affect ATP formation
and so reduce D N A replication (Bach, 1987), but
inhibition of virus replication was unaltered by
additional magnesium.
It seems likely that intracellular potassium depletion
may play a significant role. Potassium depletion in the
presence of lithium was confirmed by X-ray microanalysis of uninfected cells (Table 5) and would almost
certainly occur in HSV-infected cells. Indeed, the
intraceUular sodium and potassium ion concentrations in
mock-infected and HSV-infected Vero cells are similar
(Hackstadt & Mallavia, 1982). Moreover, when potassium was restored to potassium-depleted infected cells
HSV D N A synthesis was restored to normal levels.
Therefore, it appears that lithium inhibits HSV D N A
synthesis by displacement of potassium from a potassium-dependent biochemical event or through other
physiological change following the loss of cellular
potassium. Our data cannot exclude the possibility that
lithium may also directly inhibit a virus replicative
event(s).
References
BACH, R.O. (1987). Lithium and viruses. Medical Hypotheses 23,
15%170.
BUCHAN, A., RANDALL, S., HARTLEY, C.E., SKrNNER, G. R. B. &
FULLER,A. (1989). Effect of lithium salts on the replication of viruses
and non-viral micro-organisms. In Lithium: Inorganic Pharmacology
and Psychiatric Use, pp. 83-90. Oxford & Washington D.C. : IRL
Press.
CERNESCU, C., POPESCU, L., CONSTANTINESCU,S. t~ CERNESCU, S.
(1988). Antiviral effect of lithium chloride. Review of Rumanian
Medical Virology 39, 93-101.
GODOWSKI, P.J. & KI'~n'E, D . M . (1985). Identification of a herpes
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(Received 16 July 1992; Accepted 5 April 1993)
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