Journal of General Virology (1993), 74, 2333 2338. Printed in Great Britain
2333
Tumour necrosis factor stimulates the activity of the human
cytomegalovirus major immediate early enhancer]promoter in
immature monocytic cells
Joachim Stein, ~ H a n s - D i e t e r Volk, 2 Christa Liebenthal, 2 Detlev H . Kriiger 1. and Susanna PrOsch ~
1 Institute o f Virology and 2 Institute o f Immunology, Humboldt University Medical School (Char#e),
Schumannstrasse 20/21, D-10098 Berlin, Germany
Both tumour necrosis factor ~ (TNF-~) and phorbol 12myristate 13-acetate (PMA) stimulated human cytomegalovirus (HCMV) major immediate early (IE)
enhancer/promoter activity in the HL-60 granulocyte/
monocyte progenitor cell line when added to transfected
cells. In U-937 monocytic cells, by contrast, TNF-~ had
no stimulatory effect and the addition of PMA produced
only marginal stimulation. In the mature THP-1 monocytic cell line and in differentiated HL-60 cells, addition
of TNF-~ caused inhibition of the IE enhancer/promoter
activity. The stimulating effect of PMA, as observed in
the other cell lines, however, remained. Thus the effect
of TNF-~ on the major IE enhancer/promoter activity is
determined by the degree of differentiation of the
infected cells. Unlike TNF-~ and PMA, the interleukins
IL-1, IL-3, IL-6 as well as the cytokine GM-CSF were
found to have no detectable influence on the activity of
the IE enhancer/promoter activity which, likewise, was
not affected by the presence of the modulator sequence.
Since premonocytic cells are suggested to be sites of
HCMV latency, the stimulation by TNF-~ could be of
potential pathophysiological significance.
Human cytomegalovirus (HCMV) is the single most
important infectious agent in patients under immunosuppressive therapy, particularly organ transplant recipients. Despite numerous investigations our understanding of the pathogenesis of HCMV infection is still
incomplete. However, peripheral blood mononuclear
cells (PBMCs), especially monocytes, are believed to play
a central role in the pathogenesis of HCMV infection. It
was suggested that monocytes may harbour the latent
virus in healthy seropositive blood donors (TaylorWiedeman et al., 1991). During systemic HCMV infection in normal hosts both granulocytes and mononuclear leukocytes contain the virus (Saltzman et al.,
1988; Schrier et al., 1985). Furthermore, HCMV can
infect bone marrow progenitor cells in vitro causing
abortive or productive infection and interfere with
normal haematopoiesis (Einhorn & Ost, 1984; Reiser et
al., 1986; Simmons et al., 1990; Sing & Ruscetti, 1990).
Very little is known about the regulation of HCMV gene
expression in monocytic cells and the factors precipitating reactivation of latent virus when immunosuppressive treatments are given.
Infection of promonocytic/monocytic cell lines such
as HL-60, U-937 and THP-1 results in abortive infection
(Rice et al., 1984). However, transition to productive
infection may be achieved by stimulation of these cells
with 12-O-tetradecanoylphorbol 13-acetate or phorbol
12-myristate 13-acetate (PMA) (Dudding & Garnett,
1987; Weinshenker et al., 1988). Unrestricted replication
of HCMV in hydrocortisone-treated macrophages was
recently reported (Lathey & Spector, 1991).
There is some evidence that HCMV expression in
monocytic cells strongly depends on their stage of
differentiation (Sinclair et al., 1992; Weinshenker et al.,
1988). Sinclair et at. (1992) identified a cellular factor
which inhibits HCMV expression in immature monocytic
cells by binding to the HCMV immediate early (IE)
modulator. It is quite possible that other cellular factors
also influence HCMV expression.
Differentiation of bone marrow stem cells into
monocytes/macrophages or granulocytes is triggered by
several different cytokines. Monocytes/macrophages and
granulocytes produce cytokines regulating the haematopoiesis of stem cells, but they and their haematopoietic
precursors themselves are targets of cytokines produced
by other cells in the bone marrow stroma or immune
organs.
For human immunodeficiency virus type 1 (HIV-1)
several different cytokines were found to be important
mediators of virus replication in chronically infected
promonocytic cells (Fauci, 1990). Since HCMV is a
monotropic virus we studied several cytokines pertaining
0001-1580 © 1993 SGM
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2334
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Table 1. Phenotypic characterization o f monocytic cell
lines
Marker (%) on cellline*
Marker
HL-60
U-937
THP-1
HL-60*t
HL-60**t
CD34
CDI4
CDlla
CDllb
CDllc
CD18
CD71
15
2
3
7
8
5
94
2
63
98
13
0
29
73
3
46~
47
73
0
52
89§
3
7
3
54
58
41
73§
1
95
86
95
0
88
99§
* The cell surface markers were determined by cytofluorometry
using monoclonal antibodies as described in the text. Data given are
mean averages of four independent experiments.
t For HL-60* and HL-60** see Fig. 3 legend.
:~ The density of CD14 antigen expression (mean fluorescence) was
about three times higher on THP-1 cells as compared with HL-60**
and U-937 cells.
§ The density of CD71 antigen expression was lower as compared
with HL-60 and U-937 cells.
to monocytic cells with regard to their effect on the
activity of the major IE promoter of HCMV.
The major IE promoter plays a central role in HCMV
replication initiation by controlling HCMV IE protein
expression. IE genes are the first to be transcribed
following virus entry into the host cell, and this occurs
independently of de novo synthesis of viral proteins.
Then, a switch from restricted to unrestricted, extensive
transcription of the genome takes place. The HCMV
promoter is strongly influenced by an upstream enhancer
region with binding sites for various cellular transcription
factors (Boshart et al., 1985; Niller & Henninghausen,
1991; Stinski & Roehr, 1985; Thomsen et al., 1984).
Upstream of the enhancer region there is a modulator
region which regulates the cell-type-specific expression of
HCMV by binding further cell-specific transcription
factors (Lubon et al., 1989).
For these experiments we used two different constructs, the plasmids pRR55 and pMP32. The former,
kindly provided by Drs B. Fleckenstein and T. Stamminger (Erlangen, Germany), contains nucleotides + 52
to - 6 7 2 representing the 65 bp IE promoter and the
complete enhancer region (Fickenscher et al., 1989). The
latter, which was constructed in our laboratory, includes
an additional 553 nucleotides, upstream of the enhancer,
representing the HCMV modulator. The modulator was
derived from the HCMV plasmid clone pCM5018
(Fleckenstein et al., 1982) by H p a l I - S s t I digestion and
ligation to the HindIII SstI-digested plasmid pRR55,
generating pMP32. The structure of the construct was
confirmed by dideoxynucleotide sequencing.
For transfection experiments we used the celt lines
HL-60, U-937 and THP-1 (ATCC CCL 240, CRL 1593
and TIB 202), each representing a different stage of
differentiation. The cell lines were shown to be
mycoplasma-free by the Mycoplasma Detection Kit
(Boehringer Mannheim). HL-60 cells are closely related
to early progenitor cells and have retained the ability to
differentiate
into
granulocytes
or monocytes/
macrophages. As was shown by flow cytometry, HL-60
cells expressed CD34 and low levels of typical differentiation antigens such as adhesion molecules (CD1 la, 1lb,
llc, 18) and the monocytic marker CD14 on their
surface (Table 1). The U-937 and THP-1 cells could be
classified as being of the monocyte/macrophage lineage
because they expressed typical monocytic differentiation
markers. THP-1 cells seemed to be at a later differentiation stage than U-937 cells since they expressed a higher
level of CD14 and other differentiation markers (Table
1). Spontaneous and experimentally induced cytokine
secretion in the supernatant of 106 cells after 4 h of
incubation under standard culture conditions was
assessed by using enzyme-linked immunoassays (Quantikine).
Cells were transfected by the DEAE dextran method
(Sambrook et al., 1989). Briefly, cells were transfected
with 5 lag plasmid per 107 cells in buffer containing
25 mM-Tris-HC1 pH 7.4, 137 mu-NaC1, 5 mM-KC1,
0.7 mM-CaC12, 0"5 mN-MgC12, 0.6 mN-Na2HPO 4 and
5 mg/ml DEAE dextran (Pharmacia LKB Biotechnology) for 1 h at 37 °C. Subsequently the cells were
washed with RPMI medium. Samples of 107 cells were
cultured in RPMI-1640 medium (Biochrom) supplemented with FCS (Biochrom), selected for low endotoxin
content, at a concentration of 10 %, and the appropriate
cytokine at 37 °C in a humidified atmosphere (5 % CO~)
for 2 days. Transfection efficiencies for all the three cell
lines were confirmed as all being equivalent in control
experiments using 3~P-labelled plasmids. Cells from an
identical transfection experiment without cytokine served
as an appropriate control. The cells were then washed
with PBS and disrupted by repeated freezing and
thawing. After centrifugation, samples of the supernatants were assayed for protein (Bradford, 1976). Equal
quantities of protein were used in the chloramphenicol
acetyltransferase (CAT) assay as described by Gorman
et
al.
(1982). Lysates were incubated with
[14C]chloramphenicol (Amersham); the acetylated products were quantified after separation by thin-layer
chromatography using a thin layer scintillator. Promoter
activities were estimated relative to the conversion rate of
[14C]chloramphenicol under the influence of cytokines
and compared with the control samples. Tumour necrosis
factor (TNF-~) and IL-3 receptor expression was
determined with ~25I-labelled cytokines (Amersham). ILl, IL-6 and GM-CSF receptors were detected using
Fluorokine kits (Biermann) according to the supplier's
recommendation. Phenotypic analysis of the cell lines
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2335
100
(c)
(b)
80
o 60
e~
O
40
20
pRR55
pMP32
pRR55
pMP32
L
pRR55
pMP32
Fig. 1. CAT conversions (%) in different monocytic cell lines, HL-60 (a), U-937 (b) and THP-1 (c), transfected with the plasmid pRR55
or pMP32 and incubated with TNF-~ (100 units/ml or 5 ng/ml; N) or P M A (10 ng/ml; D) or control (11). For specification of the
TNF-c~ effect, TNF-c~ was preincubated with the monoclonal antibody Di62 (10 gg/ml; IN) for 30 min. Results of one representative
experiment are shown in each case, each experiment being repeated at least twice.
was performed using various monoclonal antibodies
(Becton Dickinson).
Results are shown for HL-60, U-937 and THP-1 cells
in Fig. 1 (a, b and c, respectively). All three cell lines were
transfected with the constructs pRR55 or pMP32. In the
HL-60 cell line, the IE enhancer/promoter activity with
and without the modulator was significantly increased by
recombinant TNF-~ and P M A (Fig. 1 a). In the presence
of T N F - e we measured a five- to sevenfold increase of
gene expression after transfection of the cells with
pRR55 or pMP32. Addition of P M A resulted in a sevento ninefold increase in the level of CAT expression.
Simultaneous application o f both TNF-~ and P M A did
not result in any additional increase of CAT expression
(data not shown). In U-937 cells transfected with either
pRR55 or pMP32 no stimulatory effect of TNF-~ was
seen (Fig. 1 b). In contrast to this, P M A did stimulate the
IE enhancer/promoter in the absence of the modulator.
However, the degree of stimulation was lower than that
found in HL-60 cells (by a factor of 2.5 to three) (Fig.
l b). The stimulation by P M A was absent when the
modulator was present, indicating that the presence of
the modulator diminished the positive P M A effect on the
H C M V IE enhancer/promoter in U-937 cells.
In the mature monocytic THP-1 cell line TNF-~
induced an inhibition of the H C M V IE enhancer/
promoter activity, irrespective of the presence or absence
of the modulator (Fig. 1 c). The P M A effect, by contrast,
varied according to whether or not the modulator
sequence was present. Whereas P M A stimulated the IE
enhancer/promoter activity in pRR55-transfected THP1 cells (two- to threefold), the CAT activity measured in
pMP32-transfected THP-1 cells was mainly unaffected
by P M A (Fig. 1 c). When incubating pRR55-transfected
THP-1 cells with both TNF-~ and P M A the inhibitory
effect of TNF-~ was neutralized (data not shown).
Table 2. Endogenous and PMA-induced synthesis of
TNF-o~ in monocytic cells*
Cell
line
Stimulator
HL-60
PMA
U-937
THP-1
HL-60*
PMA
PMA
PMA
Synthesis
of TNF-c~
(pg per ml)
62
56
28
1269
< 10
569
230
1450
* Monocytic cells (10 s cells/ml) were cultured for 4 h in the presence
or absence of P M A (10 ng/ml) and the supernatants were collected.
Synthesis of cytokines was measured by ELISA. For definition of
HL-60* see Fig. 3 legend. Data of one out of three representative
experiments are shown.
The specificity of these TNF-0~ effects was confirmed
by preincubation of TNF-~ with the neutralizing
monoclonal anti-TNF-e antibody Di62 (kindly provided
by Dr T. Diamantstein, Berlin, Germany); the results
demonstrated that antibodies to TNF-~ can inhibit both
the stimulating effect of TNF-0c in HL-60 ceils and its
inhibitory effect in THP-1 cells (Fig. 1 a to c), whereas the
irrelevant IgG1 mouse monoclonal antibody, TSH, had
no influence (data not shown). The P M A effect in HL-60
cells was unlikely to be TNF-c~-mediated because P M A
did not induce TNF-~ synthesis in HL-60 cells (Table 2)
and antibody Di62 did not abolish the P M A effect (data
not shown). In pMP32- and pRR55-transfected THP-1
cells Di62 increased the small PMA-mediated stimulation
by a factor of two and five respectively, indicating that
the slight P M A effect occurring in this cell line may
result, at least partially, from the induction of large
amounts of TNF-0~ which seem to counteract the P M A
effect (data not shown).
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2336
Short communication
21
100
(a)
80
g
.~ 60
~= 40
>
10
20
5
0
0
100
10
2
0.08
0 100 10
TNF (units/ml)
1
0.1 0.01
Fig. 2. Effect of different concentrations of TNF-0¢ (1 unit/ml or
50 pg/ml) and control (m) on CAT conversion (%) in HL-60 (a) and
THP-1 (b) cells transfected with the plasmid pRR55. The diagrams
show the results of one out of three representative experiments.
Both the stimulatory effect of TNF-~ in HL-60 cells
and its inhibitory effect in THP-1 cells were clearly
dependent on concentration (Fig. 2a, b).
We also studied the influence of interleukins IL-1,
IL-3 and IL-6 as well as the cytokine G M - C S F on the
expression of CAT under control of the H C M V IE
enhancer/promoter in the presence or absence of the
modulator in the three cell lines. Despite the presence of
a sufficient number of receptors for these cytokines on all
the three cell lines (not shown), neither G M - C S F nor
any interleukin altered the H C M V enhancer/promoter
activity even when given in excess ( > 1 ng/ml) (data not
shown). Furthermore, like TNF-7, IL-lfl, IL-6 and GMCSF inhibited U-937 and THP-1 cells, suggesting
functional activity of the appropriate cytokine receptor.
HL-60 cells were affected only by TNF-~, IL-6 and GMCSF but not by IL-lfl.
In summary, we were able to demonstrate that the
cytokine TNF-c~ is involved in the regulation of H C M V
IE enhancer/promoter activity in monocytic cells. In our
experiments the effect of TNF-~ depended strongly on
the cell line studied. TNF-~-induced stimulation of the
IE
enhancer/promoter
activity
in
immature,
granulocyte/monocyte/macrophage progenitor cell-related HL-60 cells contrasted with the inhibition of this
activity observed in mature monocytic cells like THP-1.
Therefore, the stimulating effect of TNF-c~ on the H C M V
IE enhancer/promoter decreases with increasing cell
differentiation.
This suggestion was confirmed by studying the effect
of TNF-~ on HL-60 cells induced to differentiate along
the monocyte/macrophage lineage in vitro (see Table 1).
Differentiated HL-60 cells were obtained either by
cultivating cells in the presence of TNF-0~ prior to
transfection (HL-60*) or by selection of a spontaneously
differentiated subclone (HL-60**). As was expected in
differentiated HL-60 cells the response of the H C M V IE
Fig. 3. CAT conversion (%) in differentiated HL-60 cells transfected
with the plasmid pRR55 after treatment with TNF-c~ (N, 100 units/ml
or 5 ng/ml), PMA (G, 10 ng/ml) or control ( . ) . The diagrams show
the results of one out of three representative experiments. (a) HL60*/pRR55, HL-60 ceils induced for differentiation by incubation for
48 h in the presence of 100 units/ml TNF-c~ before transfection with the
plasmid pRR55. (b) HL-60**/pRR55, spontaneously differentiated
HL-60 clone (see Table 2) transfected with plasmid pRR55.
enhancer/promoter to T N F - e and P M A was similar to
that in THP-1 cells (Fig. 3a, b).
The differences between the cell lines do not seem to be
due to variations in TNF-~ responsiveness. HL-60, U937 and THP-1 cells expressed similar amounts of T N F receptors on their surface (3575, 3002, 4603 receptors
per cell, respectively) and their expression of adhesion
molecules was increased by TNF-~ (data not shown).
Furthermore, endogenous TNF-~ production was even
lower in U-937 and THP-1 cells than in HL-60 cells
(Table 2). In conclusion, it seems unlikely that the U-937
and THP-1 cells failed to respond to exogenous TNF-~
because of either high endogenous TNF-c~ levels or the
absence of receptors.
P M A affected the H C M V IE enhancer/promoter
activity in a more complex manner than T N F - ~ did.
Whereas PMA stimulated gene expression in all three cell
lines transfected with pRR55, the level of stimulation
decreased as the cells became more differentiated. When
the IE enhancer/promoter was coupled to the modulator,
significant stimulation by P M A occurred only in the
immature cell line HL-60 (Fig. 1 a to c). Therefore, the
stimulatory effect of P M A on the IE enhancer/promoter
was counteracted by the modulator in more differentiated
cells.
Both TNF-c~ and PMA are known to be inducers of
the transcription factor NF-~cB (Lenardo & Baltimore,
1989) and Sp-1 (Hamanaka et al., 1992). H C M V IE
enhancer has four NF-KB binding sites and five Sp-1
binding sites (Cherrington & Mocarski, 1989). As our
results confirm, TNF-c~ and P M A have distinct modes of
action. Unlike the activation induced by TNF-~, PMAinduced activation of the transcription factor NF4cB
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Short communication
involves the protein kinase C system (Meichle et al.,
1990; Sen & Baltimore, 1986). TNF-~ effects sphingomyelin hydrolysis generating ceramide, which in turn
may act as an inducer of NF-KB. Sphingomyelin
hydrolysis was observed both in HL-60 and U-937 cells
(Kirn et al., 1991).
Therefore, we propose that during differentiation of
early progenitor cells into monocytes a factor accumulates which, in combination with TNF-~, may induce
inhibition of HCMV enhancer/promoter activity in
mature cells such as THP-1 and differentiated HL-60
cells. In vitro experiments have shown that TNF-~
induces an antiviral state or antiviral cytotoxicity against
HCMV-infected fibroblasts (Duncombe et al., 1990; Ito
& O'Malley, 1987). The molecular mechanism of this
antiviral action of TNF-0~ is unknown but if this effect in
fibroblasts can be attributed to the inhibition of the IE
enhancer/promoter as observed in our model, this should
be of considerable interest.
Similar effects of TNF-0~ were described as occurring
in HIV-1 replication. TNF-c~ stimulates the long terminal
repeat (LTR) of HIV-1 in vitro and induces HIV-1
replication in latently infected immature promonocytic
cells (e.g. U-937 and HL-60 cells), whereas in mature
monocytic cells (e.g. THP-1 cells and freshly isolated
human monocytes) no stimulation by TNF-a of HIV-1
production was observed (Griffin et al., 1991 ; Mellors et
al., 1991; Tadmori et al., 1991). This phenomenon was
discussed as being a result of constitutive TNF-c~
expression in HIV-l-infected mature monocytic cells
(Griffin et al., 1991). As shown for the expression of
HIV-1, the activity of the HCMV IE enhancer/promoter
can be stimulated by PMA in both immature and mature
cells. There are obvious similarities between the effects of
TNF-~ and PMA on the HIV-1 LTR and HCMV IE
enhancer/promoter. In both systems the TNF-~ effect
correlated with the differentiation stage of infected or
transfected cells. In this context, the stimulation of HIV1 LTR and HCMV IE enhancer/promoter by TNF-a in
immature, early progenitor-related cells seems of particular interest.
TNF-a, a cytokine produced by monocytes/
macrophages and T lymphocytes, is thought to play an
important role in the host response to inflammation and
infection (Beutler & Cerami, 1987). Elevated levels of
TNF-c~ have also been reported in serum taken from
most patients after renal or bone marrow transplantation
and from patients with septicaemia (Segolchi et al.,
1991). For transplant recipients, particularly bone marrow recipients, a high prevalence of systemic HCMV
infection in the post-transplantation phase is well
documented (Pr6sch et al., 1992; Rubin, 1990; Winston
et al., 1990). Apart from primary infection or reinfection
of the patient through the graft or blood transfusions,
2337
reactivation of latent HCMV is thought to be the most
important mode of infection. If HCMV is able to infect
progenitor cells latently in vivo, increased TNF-~ levels
may mediate the reactivation of latent virus in these cells.
This hypothesis was confirmed by our observation that
approximately 80 % of patients with documented septicaemia also developed active H C M V infection, as
diagnosed by the presence of HCMV antigen and HCMV
DNA in PBMCs (W. D. D6cke et al., unpublished).
We would like to thank B. Fleckenstein and T. Stamminger
(Erlangen, Germany) for kindly providing the plasmids pRR55 and
pCM5018, K. Muske and K. Staak for experimental help and F. Kern
(Berlin, Germany) and C. Schroeder (London, U.K.) for their support
in preparing the manuscript. This work was supported by a grant from
the Deutsche Forschungsgemeinschaft (DFG Pr-337/1-1).
References
BEUTLER, B. & CERAMI, A. (1987). Cachectin: more than a tumor
necrosis factor. New England Journal of Medicine 316, 379 385.
BOSrIART, M., WEBER, F., JAHN, G., DORSCrI-HKSLER, K., FLECKENSTEIN, B. & SCHAVFNER,W. (1985). A very strong enhancer is located
upstream of an immediate early gene of human cytomegalovirus.
Cell 41, 521-530.
BRADFORD, M . M . (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Analytical Biochemistry 72, 248-254.
CHERRINGTON,J. M. & MOCARSKI,E. S. (1989). Human cytomegalovirus IE 1 transactivates the alpha promoter-enhancer via an 18-basepair repeat element. Journal of Virology 63, 1435-1440.
DUDDING, L.R. & GARNETT, H.M. (1987). Interaction of strain
AD169 and a clinical isolate of cytomegalovirus with peripheral
monocytes: the effect of lipopolysaccharide stimulation. Journal of
Infectious Diseases 155, 881-886.
DUNCOMBE, A.S., MEAGER, A., PRENTICE, H.G., GRUNDY, J.E.,
HESLOP, H.E., HOFFBRAND, A.V. & BRENNER, M.K. (1990).
Gamma-interferon and tumor necrosis factor production after bone
marrow transplantation is augmented by exposure to marrow
fibroblasts infected with cytomegalovirus. Blood 76, 104(~1053.
EINHORN, L. & OST, A. (1984). Cytomegalovirus infection of human
blood cells. Journal of Infectious Diseases 149, 207-214.
FAUCI, A.S. (1990). Cytokine regulation of HIV expression.
Lymphokine Research 9, 527-531.
FICKENSCHER,H., STAMMINGER,T., R~'GER, R. & FLECKENSTEIN,B.
(1989). The role of a repetitive palindromic sequence element in the
human cytomegalovirus major immediate early enhancer. Journal of
General Virology 70, 107-123.
FLECKENSTEIN, B., M/2LLER, I. & COLLINS, J. (1982). Cloning of the
complete human cytomegalovirus genome in cosmids. Gene 18,
3946.
GORMAN, C.M., MOFFAT, L.F. & HOWARD, B.H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in
mammalian cells. Molecular and Cellular Biology 2, 1044~1051.
GRIFFIN, G. E., LEUNG, K., FOLKS, T. M., KUNKEL, S. & NABEL, G. J.
(1991). Induction of NF-~cB during monocyte differentiation is
associated with activation of HIV-gene expression. Research in
Virology 142, 233-238.
/-{AMANAKA,R., KOHNO, K., SEGUCHI, T., OKAMURA, K., MORIMOTO,
A., ONO, M., OGATA, J. & KUWONA, M. (1992). Induction of low
density lipoprotein receptor and a transcription factor SP-1 by tumor
necrosis factor in human microvascular endothelial ceils. Journal of
Biological Chemistry 267, 13160-13165.
Ixo, M. & O'MALLEY, J. A. (1987). Antiviral effects of recombinant
tumor necrosis factor. Lymphokine Research 6, 309 318.
KIM, M.Y., LINARDIC, C., OBEID, L. & HANNUN, Y. (1991).
Identification of sphingomyelin turnover as an effector mechanism
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:14:24
2338
Short communication
for the action of tumor necrosis factor and gamma-interferon.
Journal of Biological Chemistry 226, 484M89.
LATrmy, J. L. & SVECTOR, S.A. (1991). Unrestricted replication of
human cytomegalovirus in hydrocortisone-treated macrophages.
Journal of Virology 65, 6371-6375.
LENARDO, M.J. & BALTIMORE, D. (1989). NF-KB: a pleiotropic
mediator of inducible and tissue-specific gene control. Cell 58,
227 229.
LUBON, H_, GHAZAL,P., HENNINGHAUSEN,L., REYNOLDS-KOHLER, C.,
LOCKSH1N, C. & NELSON, J. (1989). Cell-specific activity of the
modulator region in the human cytomegalovirus major immediateearly gene. Molecular and Cellular Biology 9, 1342-1345.
ME1CHLE, A., SCnOTZE, S., HENSEL, G., BRt3~SING,D. & KR6NKE, M.
(1990). Protein kinase C-independent activation of nuclear factor/cB
by tumor necrosis factor. Journal of Biological Chemistry 265,
8339-8343.
MELLORS, J.W., GRIFHTH, B.P., ORITZ, M.A., LANDRY, M.L. &
RYAN, J.L. (1991). Tumor necrosis factor c~/cachectin enhances
human immunodeficiency virus type 1 replication in primary
macrophages. Journal of Infectious Diseases 163, 78-82.
N~LLER, H. H. & HENNINOHAUSEN, L. (1991). Conformation of several
specific nucleoprotein complexes on the human cytomegalovirus
immediate early enhancer. Nucleic Acids' Research 19, 3715 3721.
PROSCH, S., KIMEL, V., DAWYDOWA, I. & KRi)GER, D.H. (1992).
Monitoring of patients for cytomegalovirus after organ transplantation by centrifugation culture and PCR. Journal of Medical
Virology 38, 246-251.
REISER, H., K/2HN, J., DOERR, H.W., KIRCHNER, H., MUNK, K. &
BRAUN, R. (1986). Human cytomegalovirus replicates in primary
human bone marrow cells. Journal of General Virology 6"7,
2595-2604.
RICE, G. P. A., SCHRIER, R. D. & OLDSTONE, M. B. A. (1984). Cytomegalovirus infects human lymphocytes and monocytes: virus
expression is restricted to immediate early gene products. Proceedings
of the National Academy of Sciences, U.S.A. 81, 6134-6143.
RUBIN, R. H. (1990). Impact of cytomegalovirus infection on organ
transplant recipients. Reviews of Infectious Diseases 12, 5754-5766.
SALTZMAN,R. L., QUIRK, M. R. & JORDAN,M. C. (1988). Disseminated
cytomegalovirus infection: molecular analysis of virus and leucocyte
interaction in viremia. Journal of Clinical Investigation 81, 75-81.
SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Transfection
mediated by DEAE~:textran. In Molecular Cloning: A Laboratory
Manual, 2nd edn, pp 16.41-16.46. New York: Cold Spring Harbor
Laboratory.
SCrrRmR, R. D., NELSON, J. A. & OLDSTONE,M. B. A. (1985). Detection
of human cytomegalovirus in peripheral blood lymphocytes in a
natural infection. Science 230, 1048-1051.
SEGOLCHI,G., VERCELLONE,A., MONES, M., TETTA, C. & CAMUSSI,G.
(1991). Levels of TNF in sera of renal allograft recipients. Clinical
Transplantation 5, 10~106.
SEN, R. & BALTIMORE, D. (1986). Inducibility of/c immunoglobulin
enhancer-binding protein NF-KB by a posttranslational mechanism.
Cell 47, 705-716.
SIMMONS,P., KAUSHANSKY,K. & TOROK-STORB, B. (1990). Mechanisms
of cytomegalovirus-mediated myelosuppression: pertubation of
stromal cell function versus direct infection of myeloid cells.
Proceedings of the National Academy of Sciences, U.S.A. 87,
1386~ 1390.
SINCLAIR,J. H., BAILLIE, J., BRYANT, L. A., TAYLOR-WIEDEMAN,J. A.
& SISSONS, J. G. P. (1992). Repression of human cytomegalovirus
major immediate early gene expression in a monocytic cell line.
Journal of General Virology 73, 433-435.
SING, G.K. & RUSCETTI, F.W. (1990). Preferential suppression of
myelopoiesis in normal human bone marrow cells after in vitro
challenge with human cytomegalovirus. Blood 75, 1965-1973.
STINSKI, M.F. & ROEHR, T.J. (1985). Activation of the major
immediate early gene of human cytomegalovirus by cis-acting
elements in the promoter-regulatory sequence and by virus-specific
trans-acting components. Journal of Virology 55, 431-441.
TADMORI, W., MONDAL, D., TADMORI, I. & PRAKASH, O. M. (1991).
Transactivation of human immunodeficiency virus type 1 long
terminal repeats by cell surface tumor necrosis factor cc Journal of
Virology 65, 6425-6429.
TAYLOR-WIEDEMAN, J., SISSONS, J . G . P . , BORYSIEWICZ, L.K. &
SINCLAIR, J. H. (1991). Monocytes are a major site of persistence of
human cytomegalovirus in peripheral blood mononuclear cells.
Journal of General Virology 72, 2059-2064.
THOMSEN, D.R., STENBERG,R.M., COINS, W. F. & STINSKI,M. F.
(1984). Promoter-regulatory region of the major immediate early
gene of human cytomegalovirus. Proceedings of the National
Academy of Sciences, U.S.A. 81,659-663.
WEINSHENKER, B. G., WILTON, S. & RICE, G. P. A. (1988). Phorbolester-induced differentiation permits productive human cytomegalovirns infection in a monocytic cell line. Journal of Immunology
140, 1625-1631.
WINSTON, D.J., WINSTON, G. H. & CHAMPLIN,R. E, (1990). Cytomegalovirus infections after allogenic bone marrow transplantation.
Reviews of Infectious Diseases 12, 5776-5792.
(Received 18 January 1993; Accepted 7 July 1993)
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