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Introduction:
Human
cytomegalovirus (HCMV), a betaherpesvirus, is capable of
establishing lifelong latent infection after a primary
infection in vivo (1).
The latent
virus is reactivated under condition of
immunosuppression and sometimes causes devastating
diseases such as congenital malformation in the newborn
and interstitial pneumonia in the immunocompromised
host.
Numerous
pathogenic changes in infected organs stem from virus
replication in permissive cells resulting in their
lyses.
In
contrast, HCMV may contribute to pathogenesis due to
altered cellular gene expression that occur independent
of virus replication. Moreover, immediately after
infection HCMV activates NFkB
and other transcription factors (2) that are required
for viral DNA synthesis, thus allowing the productive
infection of quiescent, differentiated cells that do not
express these factors in sufficient amounts.
The effects of HCMV on NFkB
are reminiscent to those of growth factors and hormones
that result in transcription of numerous viral and
cellular genes.
More specifically, HCMV-mediated
NFkB
activation may occur on the level of A) binding of viral
glycoproteins to cellular receptors (3), B) introduction
of constituents of the virion (i.e., tegument protein
pp71) with transactivation activity (4), or C)
transactivation of the NFkB
gene via HCMV immediate-early proteins 1 and 2 (IE1 and
IE2) which are produced in infected cells before
initiation of virus replication (5).
In vitro, the only cells fully permissive for
replication of laboratory strains are human skin or lung
fibroblasts, whereas clinical isolates replicate
preferentially on endothelial cell cultures.
Some
transformed cell lines derived from glioblastomas, as
well as primary arterial smooth muscle cells, support
productive infection, although at lower levels than in
fibroblasts.
The cause of the reduction in the level of virus
production in the transformed cells is not well known.
In
this study the productive infection of HCMV was
monitored in normal human fibroblasts obtained from a
whole embryo (KMS-6) and in its malignantly transformed
(KMST-6) cells which transformed with 60Co gamma rays
(6).
Materials and Methods
Cells and virus
KMS-6 and KMST-6 cells
were cultured with Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% newborn calf serum and 2 mM
L-glutamine in 5% CO2 incubators at 37°C.
All experiments were done
using confluent KMS-6 cells that were between passages
18 and 23 and KMST-6 cells that were between passages
117 and 127.
To reduce the basal levels
of cellular gene expression, KMS-6 and KMST-6 cells were
serum-starved before HCMV infection.
The Towne strain of
HCMV was propagated in human embryonic lung (HEL)
cells by infection at a multiplicity of below 0.001
plaque-forming unit (PFU) per cell.
Two HCMV stocks containing
1.3 x 107 and 2.5 x 107 PFU/ml
were used in the present experiments.
Compounds and antisera
LY294002 was obtained from Sigma. The stock solution of
LY294002 was made by dissolving in dimethyl sulfoxide at
the concentrations of 20 mM. This compound was further
diluted in DMEM prior to use.
The mouse monoclonal antibody (mAb) MAB810 which
recognizes the HCMV MIE (IE1 and IE2) gene products (Mazeron
et al., 1992), mouse anti-actin mAb C4 (MAB1501)
and rabbit polyclonal antibodies which can recognize
c-Jun and c-Fos were obtained from Chemicon. The mouse
mAbs which react with HCMV early protein (pUL44) or late
protein pp65 (pUL83) were purchased from Fitzgerald and
ViroGen, respectively.
The rabbit polyclonal
antibodies which can recognize Akt or only Akt that is
phosphorylated on Ser473 were purchased from Cell
Signaling Technology.
Virus infection and
treatment of the infected cells with inhibitors
KMS-6 and KMST-6 cells
were grown to confluent state in a growth medium and
then serum-starved for 48 h in DMEM. These cells were
infected with HCMV at a multiplicity of 1 PFU/cell
unless otherwise indicated. After a 1-hr adsorption
period, the infected cells were washed with Hanks’
balanced salt solution (HBSS) and incubated in the
growth medium for the indicated times after infection.
When cells were treated with LY294002, these drugs were
present during the 2-h adsorption period and from 1 h
until 5 days.
Virus growth study
Confluent,
serum-starved KMS-6 and KMST-6 cells, grown in 25-cm2
culture flasks, were infected with HCMV at various
multiplicities of infection as indicated in each
experiment. After a 1-h viral adsorption period, cells
were washed with HBSS and maintained in the growth
medium. At 1 to 5 days after infection, the total amount
of infectious HCMV (the virus released into the medium
plus the cell-associated virus) was determined after
disrupting the infected cells by freezing and thawing
once, and by sonically treatment by plaque assay on HEL
cells.
Western blotting analysis
For preparation of
total cell lysates, mock- and HCMV-infected cells were
washed twice with ice-cold phosphate-buffered saline
(PBS) and lysed in sodium dodecyl sulfate (SDS) lysis
buffer (50 mM Tris [pH6.8], 2% SDS, 3%
2-mercaptoethanol, 10% glycerol). The lysates were
sonically treated briefly, boiled for 3 min, and
clarified by centrifugation at 15,000 rpm for 15 min.
The supernatants were stored at –85ºC until use.
Proteins (50-80 μg) were separated on SDS-7.5%
polyacrylamide gels and transferred to Hybond-ECL
nitrocellulose membrane. The membrane was blocked with
5% skim milk in Tris-buffer saline (TBS). HCMV-specific
MIE, early and late proteins and actin protein (used as
an internal marker) were detected by the reaction with
MAB810 (diluted 1:400 in TBS plus 5% skim milk),
anti-pUL44 (diluted 1:800 in the same), anti-pp65
(diluted 1:500 in the same), or anti-actin antibody
(diluted 1:1,000 in the same), respectively, followed by
reaction with horseradish peroxidase (HRP)-labeled goat
secondary antibody to mouse IgG (Santa Cruz; diluted 1:
5,000 in the same). Cellular c-Jun and c-Fos proteins
were detected by the incubation with rabbit polyclonal
antibodies specific to c-Jun (diluted 1:200 in the same)
or c-Fos (diluted 1:200 in the same), followed by the
reaction with HRP-labeled goat antibody to rabbit IgG
(diluted 1: 2,000 in the same). The visualization of
signals was ECL (Amersham).
Preparation
of nuclear extracts
Mock- or HCMV-infected
KMS-6
and KMST-6
cells
were washed 3 times with ice-cold PBS, scraped off by
using a rubber cell scraper, and collected by
centrifugation. Two packed volumes of Buffer A (10 mM
HEPES [pH 7.9], 1.5 mM MgCl2, 10
mM KCl, 1 mM dithiothreitol [DTT], and proteinase
inhibitor mixture [Sigma]) were added to the cells
and kept on ice for 5 min to swell the cells. Vortex was
then used to rupture the cell membranes, and nuclei
were collected by centrifugation at 5,000 rpm for
1 min at 4 °C. The pellet was resuspended in a
high salt buffer C (20 mM HEPES [pH 7.9], 25%
glycerol, 0.42 M NaCl) and kept at 4 °C for
30 min before centrifugation at 5,000 rpm for 15 min.
The supernatants were stored at –85°C as
nuclear extracts in small aliquots until use.
Electrophoretic mobility shift assay (EMSA)
Using Dig Gel Shift Kit
(Roche) EMSA was performed according to the
manufacture’s protocol. In brief, the nuclear extracts
(10 μg) were incubated with 20 μl of reaction
mixture (20 mM HEPES [pH 7.6], 30 mM KCl, 1 mM
EDTA, 10 mM (NH4)2SO4,
1 mM DTT, 0.2% Tween 20, 0.1 μg of poly-L-lysine, 1 μg
of poly [d (I-C)] DNA and digoxigenin-labeled
oligonucleotide probe specific to NFkB
[Santa Cruz]) for 15 min at room temperature.
DNA-protein
complexes were electrophoretically resolved
on 8% polyacrylamide gels. After blotting on to a
positive nylon membrane, signals were detected by
reaction with sheep anti-digoxigenin polyclonal antibody
conjugated with alkaline phosphatase.
Reverse transcription (RT)-PCR
Total RNA was prepared
from mock- and HCMV-infected cells using ISOGEN (Nippon
Gene). RT-PCR was performed using the Superscript
One-Step RT-PCR system (Invitrogen). Total RNA (0.1 μg)
was used for a single reaction. Nucleotide sequences of
oligonucleotide primers for the IE1 and IE2 DNA used for
RT-PCR were described elsewhere (Shirakata et al.,
2002).
The reverse
transcriptase reaction was performed at 55°C
for 30 min. To amplify the IE1 and IE2 cDNAs,
each sample was denatured at 94°C
for 30 s, annealed at 50°C
for 30 s and extended at 72°C
for 2 min.
The RT-PCR products were
subjected to agarose gel electrophoresis and stained by
ethidium bromide.
Results
HCMV
replication in KMS-6 and KMST-6 cells
In the
first experiment the question was whether there is any
difference in HCMV replication between the KMS-6 and
KMST-6 cells. For this, confluent, serum-starved KMS-6
and KMST-6 cells were infected with HCMV and infectious
progeny virus and syntheses of viral proteins were
analyzed every day for 5 days. Typical growth kinetics
of HCMV cells was shown in KMS-6 and KMST-6 cells (Fig.
1A). Although, in KMST-6 cells HCMV replication at 3 to
5 days was lower by approximately 3- to 5 logs.
Expression level of the IE1 protein in both cells at 1
to 5 days pi was basically the same (Fig. 1B upper vs
Fig. 1C upper). In KMS-6 cells synthesis of the IE2
protein appeared at 1 day pi and high levels of this
protein were continuously detectable during the 5-day
period of this experiment (Fig. 1B upper). The early and
late proteins, first synthesized at 2 days pi,
increased in amount with time after infection (Fig. 1B
middle and lower), whereas in KMST-6 cells IE2 was
detected at day 2 pi (Fig. 1C upper) and early and late
protein synthesis was at 4 days pi (Fig. 1C middle and
lower).
C-June and
c-Fos synthesis and NFκB activity in infected KMS-6 and
KMST-6 cells.
To
determine wither the blockage of the viral replication
and gene expression in the transformed cells were due to
inhibition of transcriptional factors, c-Jun and c-Fos
synthesis were determined at the time indicated times pi
in infected normal and transformed cells (Fig. 2A), and
the nuclear extracts were prepared, and activation of
NFκB was assessed by DNA binding activity to the NFκB
probe using EMSA at the times indicated times pi.
Infection of KMS-6 cells with HCMV enhanced c-Jun and c-Fos
synthesis and NFkB
activity at day one pi. In KMST-6 cells c-Jun synthesis
was enhanced at day one pi but at lower pattern than
normal cell (Fig. 2A). On the other hand, c-Fos
synthesis and NFkB activity were detected at 2 days pi
with lower pattern than KMST-6 (Fig. 2A&B).
Effect of
PI3-K inhibitor on synthesis of the MIE proteins and
infectious HCMV
PI3-K is
strongly activated immediately after HCMV infection of
quiescent human fibroblasts and the PI3-K activity is
required for activation of the transcriptional factor
NFκB (7). Therefore, I examined the effect of LY294002,
a specific inhibitor of PI3-K kinase activity (King
et al., 1997; Wennstrom & Downward, 1999), on
expression of the MIE proteins and infectious HCMV (Fig.
3). The results indicated that synthesis of the IE1 and
IE2 proteins and the infectious HCMV at 5 days pi was
markedly blocked in KMS-6 cells treated with LY294002
(Fig. 3 left).
Surprisingly, in the contrary, LY294002 treatment
enhanced the synthesis of the MIE proteins and HCMV
replication at 5 days pi (Fig. 3 right).
Effect of
LY294002 on the NFkB activity and synthesis of IE1 and
IE2 mRNAs in KMS-6 and KMST-6 cells
To
investigate the mechanisms by which LY249002 down
regulates MIE protein synthesis in KMS-6 cells and
up-regulates it in KMST-6 cells, I
first examined whether the inhibitory effect of LY249002
on expression of the MIE genes occurs at the
transcriptional or translational level. HCMV-infected
KMS-6 and KMST-6
fibroblasts were maintained in the presence or absence
of LY294002 and total RNA was isolated at 1 day pi.
Synthesis of the MIE mRNAs was evaluated by RT-PCR. The
results indicated that there is little, if any,
difference in the level of the IE1 mRNA synthesized in
untreated and LY294002-treated KMS-6 and KMST-6 cells
(Fig. 4). On the contrary, expression level of the IE2
mRNA in LY294002-treated cells was markedly reduced in
KMS-6 cells and enhanced in KMST-6 cells compared to
that in untreated cells. This indicated that expression
of the MIE genes in LY294002-treated cells were blocked
or enhanced at the transcriptional level.
Activation of NFκB is thought to be important for
expression of the MIE promoters which encodes the IE1
and IE2 proteins. I tested if the effects of LY294002
were due to NFkB
activity. As shown in Fig. 4B the activity of NKkB
was markedly inhibited in the normal cells treated with
LY294002 and markedly enhanced in the transformed cells.
This indicated that expression of the MIE genes was
under the effect of NKkB
activity.
Discussion
The present study has
shown that replication of HCMV is markedly blocked in
the transformed human embryonic (KMST-6) cells compared
to the normal (KMS-6) cells (Fig. 1).
Consistent with the previous
reports (8), infection of fibroblast cells with HCMV
induced activation of NFκB, and this induction was
markedly inhibited in transformed KMST-6 cells (Fig.2).
The hallmark of HCMV infection in different human
cell types is a rapid induction of NFkB
DNA binding activity (5). This initial and very rapid
increase in binding activity appears to be due to the
release of preformed cytosolic NFkB
heterodimers resulting from the binding of the major
HCMV envelope glycoproteins, gB and gH, to their cognate
cellular receptors (3). However, HCMV infection was also
shown to transactivate the promoters for the two NFkB
subunits, p105/p50 and p65, which may be important for
the sustained increase in NFkB
activity during the course of the infection. The
transactivation of NFkB
genes occurs through modulation of cellular factors
(independent of viral gene expression) and/or through
transactivation activity of the major HCMV IE gene
products (3). It has been widely accepted that HCMV
involves the NFkB
pathway to support HCMV DNA replication.
In contrast, HCMV-induced
NFkB
activity is relevant for virus associated
immunopathological mechanisms that involve NFkB-dependent
gene expression (9). In HCMV-infected fibroblasts, NFkB
is up-regulate by PI3-K pathway (7). When KMS-6 cells
was treated with PI3-K specific inhibitor, the NFkB
activity was markedly inhibited and consequently the
virus protein synthesis and virus replication.
Unexpectedly, in
the transformed cells the inhibition of the PI3-K
pathway enhanced both the NFkB
activity and the virus replication (Fig. 3). The results
indicate that HCMV used different pathways to activate
the NFkB
which is crucial for viral gene expression (Fig. 4), and
transforming the fibroblast disrupt the kinases activity
of the cells which is used by the HCMV to complete its
replicative cycle. This research and future research may
open the possibility for future therapy for HCMV.
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