From the Unité des Virus Lents, CNRS URA 1930,
Institut Pasteur, 75724 Paris Cedex 15 and the ¶ Institut de
Génétique et de Biologie Moléculaire et Cellulaire,
CNRS-INSERM, 67404 Illkirch, Strasbourg, France
Received for publication, June 13, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Persistence of Borna disease virus (BDV) in the
central nervous system causes damage to specific neuronal populations.
BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the
response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV
to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal
differentiation in response to NGF. We report that persistence of BDV
led to a progressive change of phenotype of PC12 cells and blocked
neurite outgrowth in response to NGF. Infection down-regulated the
expression of synaptophysin and growth-associated protein-43, two
molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the
block in response to NGF was due in part to the down-regulation of NGF
receptors. Moreover, although BDV caused constitutive activation of the
ERK1/2 pathway, activated ERKs were not translocated to the nucleus
efficiently. These observations may account for the absence of neuronal
differentiation of persistently infected PC12 cells treated with
NGF.
Borna disease virus
(BDV)1 is a nonsegmented,
negative stranded RNA virus (1, 2), belonging to the
Bornaviridae family in the Mononegavirales order.
BDV causes central nervous system diseases characterized by behavioral
abnormalities in a wide variety of animals (3). There is evidence that
BDV infects humans, although its role in neuropsychiatric disorders
remains a matter of debate (4-9). When inoculated in adult
immunocompetent Lewis rats, BDV causes behavioral disturbances
associated with massive brain inflammation and extensive neuronal
damage (10, 11). Neonatal infection also results in a lifelong
persistent infection associated with a variety of behavioral
abnormalities but without generalized meningitis or encephalitis (10,
12-15). Therefore, this second model allows studying the consequences
of persistent BDV infection on neuronal functions in the absence of inflammation.
Many lines of evidence suggest that BDV persistence per se
can cause damage to specific neuronal populations. For example, infection of adults with BDV disrupts cortical cholinergic innervation prior to encephalitis (16), whereas neonatal infection causes alterations in postnatal development of the cerebellum and hippocampus as well as synaptic pathology (13, 14, 17, 18). In particular, neonatal
infection causes Purkinje cell death, dentate gyrus granule neuron
degeneration, and a progressive loss of cortical neurons (14,
17-19).
BDV is a noncytopathic virus, and the mechanism whereby it causes
neuronal pathology is still not understood. Neonatal infection leads to
variations in the level of proinflammatory cytokines and to changes in
monoamine tissue content as well as in the expression of molecules
regulating central nervous system plasticity (18-22). Although these
changes might be responsible for part of the pathology, they, however,
appear quite late after infection (i.e. at least 3 weeks
post-inoculation). A recent study showing that cerebellar damage is
observed only if infection occurs before postnatal day 15 suggests that
BDV also interferes with early events of postnatal brain development
(23). Therefore, we hypothesized that infection may affect the response
to neurotrophic factors that play key roles in early differentiation
and survival of neurons. In particular, we decided to investigate
whether BDV infection interferes with the response to nerve growth
factor (NGF), the prototypic member of the neurotrophin family (24). As
a first step to test this hypothesis, we studied the response to NGF of
PC12 cells infected by BDV. The PC12 cell line is a neural
crest-derived adrenal chromaffin cell line obtained from a rat
pheochromocytoma. PC12 cells exhibit several features of neuronal
differentiation following treatment with NGF. They extend neurites,
become post-mitotic, and resemble sympathetic neurons (25). Binding of
NGF to its receptor, a tyrosine kinase, triggers a cascade of protein
phosphorylation events, leading to the activation of several genes.
This signal transduction cascade and the genes involved have been
studied extensively (26, 27).
Here, we show that persistent infection of PC12 cells with BDV leads to
dramatic changes in cell morphology and expression of genes implicated
in synaptic plasticity. Moreover, infection causes a complete block in
NGF-induced neurite outgrowth and several changes in the NGF signal
transduction cascade.
Maintenance of Cells and Infection with BDV Treatment with NGF--
Infected and noninfected PC12 cells were
plated on collagen-coated supports, grown in complete medium for
16 h, and starved in medium containing 1% serum for 16 h.
They were then treated with 100 ng/ml NGF (Life Technologies, Inc.),
for the indicated times before analysis.
Northern Blot Analysis--
Total RNA was isolated using
TRI-Reagent (Sigma), size-fractionated by 2.2 M
formaldehyde-agarose gel electrophoresis, transferred by capillarity
with 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) to MagnaGraph nylon membranes (MSI,
Westboro, MA), and UV cross-linked. The blots were hybridized for
2.5 h at 65 °C in Quikhyb buffer (Stratagene, La Jolla, CA)
containing 100 µg/ml and 5 ng/ml of heat-denatured salmon sperm DNA
and DNA probe, respectively. DNA probes were labeled with
[ Protein Extracts and Western Blot Analysis--
Cells were
rapidly lysed in a buffer (29) containing 10 mM Tris-HCl,
50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM dithiothreitol, 1% Triton X-100,
5 µM ZnCl2, 100 µM
Na3VO4, and 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, a protease inhibitor
(Interchim, Montluçon, France). Insoluble material was removed by
centrifugation (13,000 rpm for 20 min at 4 °C), and protein
concentration was determined by a Bradford assay (Bio-Rad protein
assay). Cells lysates (30, 20, or 10 µg/lane depending on the
antibody used) were separated by 12% SDS-polyacrylamide gel
electrophoresis (Novex, La Jolla, CA) before transfer onto Hybond
C-extra membranes (Amersham Pharmacia Biotech). The membrane was
blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST), supplemented with 5% of either bovine serum albumin or nonfat dry milk. Subsequently, the membrane was
incubated overnight at 4 °C with the primary antibodies diluted in
blocking buffer. After washing with TBST, the blots were incubated for
1 h with either biotinylated anti-rabbit or anti-mouse antibodies (diluted 1:1000; Amersham Pharmacia Biotech), followed by a
Streptavidin-peroxydase complex (diluted 1:1000; Amersham Pharmacia
Biotech). After extensive washes in TBST and TBS, peroxydase activity
was revealed by incubating with Pierce Super signal substrate
(Interchim) and exposing to Biomax MS films (Kodak). When detecting the
phosphorylated and the total form of a protein, the blots were first
probed with the antibody specific for the phosphorylated form. After
revelation, the blot was stripped by incubating in 0.1 M
glycine, pH 2.5 (20 min, twice), blocked as described above, and
reprobed with the antibody recognizing all forms of the protein.
Photographic films were scanned on a flatbed Agfa scanner and processed
using Adobe Photoshop and Canvas softwares.
Antibodies Used for Western Blot Analysis--
We used the
following antibodies: a monoclonal antibody recognizing specifically
the phosphorylated (activated) form of mitogen-activated protein kinase
(MAPK) ERK1 and ERK2
(anti-phospho-Thr183-Tyr185 ERK, Sigma; diluted
1:10000); a rabbit polyclonal antibody recognizing total ERK1 and ERK2
(New England Biolabs, Berverly, MA; diluted 1:500); rabbit polyclonal
antibodies recognizing phosphorylated CREB
(anti-phospho-Ser133 CREB) and total CREB protein (New
England Biolabs; both diluted 1:750); rabbit polyclonal antibodies
recognizing the phosphorylated forms of MEK
(anti-phospo-Ser217/221 MEK1/2) and p90RSK
(anti-phospho-Ser381 p90RSK) proteins (New
England Biolabs; both diluted 1:500); a monoclonal antibody recognizing
the phosphorylated form of Elk-1 (anti-phospho-Ser383
Elk-1, Santa Cruz Biotechnology; diluted 1:1000); and a rabbit polyclonal antibody raised against the BDV NP (diluted 1:25000) described previously (6).
Immunofluorescence and Confocal Microscopy Analysis--
Cells
grown on glass coverslips were fixed with 4% paraformaldehyde in
phosphate-buffered saline followed by methanol-acetone (50/50) and
processed for immunofluorescence as described previously (30). Briefly,
after blocking with 2% normal goat serum and 2% horse serum for
1 h at room temperature, cells were stained with primary
antibodies. After washing in phosphate-buffered saline containing 0.1%
Triton X-100, cells were stained with secondary antibodies,
i.e. fluorescein isothiocyanate-labeled anti-rabbit IgG
(Interchim; diluted 1:500) and Cy3-labeled anti-mouse IgG (Interchim;
diluted 1:500). Cells were extensively washed as described for primary
antibodies, mounted in Vectashield (Vector Laboratories, Burlingame,
CA), and sealed with nail polish. The primary antibodies were
monoclonal antibodies to synaptophysin (Roche Molecular Biochemicals; diluted 1:5), microtubule-associated protein (MAP)-2 (Sigma; diluted 1:200), Persistence of BDV in PC12 Cells Causes Progressive Changes in Cell
Morphology--
PC12 cells were infected with BDV as described above
and analyzed at each passage after infection (Fig.
1). Early after infection, infected cells
were morphologically undistinguishable from control noninfected PC12
cells. After three passages, over 96% of the cells were infected, as
determined by detection of the viral nucleoprotein using
immunofluorescence and fluorescence-activated cell sorter analysis (not
shown). After four to five passages, cells in infected cultures changed
morphology. PC12-BV cells became flat and nonrefractile, and their
adhesion to the support increased (Figs. 1 and
2). After eight to 12 passages (depending
on the experiment), the changes concerned the whole cell population.
We also examined the response of infected PC12 cells to NGF. NGF blocks
the proliferation of noninfected PC12 cells and induces neurite
outgrowth. As shown in Fig. 2, PC12-BV cells treated with NGF failed to
develop neurites and continued to proliferate. By contrast, control
PC12 cells passaged in parallel remained unchanged and responded
normally to NGF (Fig. 2). Five independent experiments gave similar
results. The infection did not impair cell viability, as determined by
trypan blue staining (not shown). Infected cells did not lose BDV upon
passaging, and viral infection did not impair cell growth. The doubling
time of PC12-BV cells under normal serum conditions was slightly
shorter than that of control cells (44 ± 2 h.
versus 65 ± 3 h., n = 9, p < 0.0005 by Mann-Whitney test).
We next asked whether NGF-treated PC12-BV cells still expressed
neuronal markers. Cells were grown on coverslips, treated with NGF,
fixed, and processed for immunofluorescence. Compared with control PC12
cells, PC12-BV cells expressed comparable levels of MAP-2, Tau protein,
and neuron-specific Impaired Gene Expression in PC12 Cells Infected with BDV--
We
then examined the level of mRNA coding for synaptophysin in PC12
and PC12-BV cells, together with the level of mRNA encoding another
plasticity-related protein, the GAP-43 (31). We also examined the
expression of the tyrosine hydroxylase (TH) gene, a delayed-early gene
induced by NGF in PC12 cells (32). TH is an enzyme that catalyzes the
rate-limiting step in the catecholamine biosynthetic pathway leading to
dopamine synthesis. It is expressed in catecholaminergic neurons as
well as in neural crest-derived chromaffin cells. Finally, Northern
blots were also probed for the housekeeping gene GAPDH and for viral RNA.
As shown in Fig. 5, PC12 cells expressed
relatively high constitutive levels of synaptophysin, and exposure to
NGF was accompanied by the characteristic increase (33) in GAP-43 RNA
levels. Likewise, TH mRNA was readily detected in untreated PC12
cells, and expression increased following NGF treatment. In contrast,
mRNAs for synaptophysin, TH and GAP-43 were not, or barely,
observed by Northern blotting in PC12-BV cells and were not
up-regulated in response to NGF (Fig. 5), whereas mRNA levels for
GAPDH remained unaffected. As expected, we only detected BDV-specific
RNAs in PC12-BV cells. Hence, BDV persistence in PC12 cells
specifically alters the expression of genes coding for molecules
involved in synaptic plasticity as well as in catecholamine
biosynthesis.
Expression of NGF Receptors Is Down-regulated in BDV-infected PC12
Cells--
We next investigated whether the absence of response of
PC12-BV cells to NGF could be due to a down-regulation of the
expression of the NGF receptors. Two structurally unrelated NGF
receptors are known (34). The low affinity neurotrophin receptor p75 is a transmembrane glycoprotein that also binds the other neurotrophins BDNF, NT3 and NT4/5. NGF also binds and activates the tyrosine kinase
receptor TrkA, and it has been suggested that both p75 and TrkA may be
required for the formation of the high affinity NGF-binding site and
for the full NGF-mediated effect (34, 35). We analyzed the expression
of mRNAs encoding p75 and TrkA using Northern blot (Fig.
6A). In PC12 cells, we
observed a significant increase in the level of mRNAs encoding p75
and TrkA molecules following NGF treatment. In contrast, in PC12-BV
cells, mRNAs levels were greatly decreased for p75 and were below
the threshold of detection for TrkA. We next studied the expression of
p75 by indirect immunofluorescence (Fig. 6B). The p75
molecule was strongly expressed after NGF treatment in PC12 cells,
whereas expression was uneven in BDV-infected PC12 cells. In the
latter, some cells had levels of p75 comparable with those of the
noninfected cells, whereas others had levels that were barely
detectable. Levels of p75 did not appear to correlate with viral
nucleoprotein load. TrkA expression was below the threshold of
detection by immunofluorescence, even in the control PC12 cells.
Alterations in NGF Signal Transduction Cascade in PC12 Cells
Infected with BDV--
The block in neuronal differentiation observed
in PC12-BV can be explained in part by the down-regulation NGF receptor
expression. However, results from Northern blot and immunofluorescence
showed that some PC12-BV cells still expressed low levels of receptor (at least for p75) and nevertheless did not differentiate in response to NGF. Moreover, analysis of mRNA expression with a sensitive reverse transcriptase-polymerase chain reaction assay showed that infected cells still expressed low levels of TrkA mRNA (data not shown). Therefore, we decided to examine the effects of BDV infection on the NGF signal transduction cascade (27) (Fig.
7A). Binding of NGF to the
TrkA receptor triggers sequential phosphorylations that propagate the
signal to the nucleus. Phosphorylation of the MAP/ERK kinases (MEK)
isoforms (the 45-kDa MEK1 and the 46-kDa MEK2) stimulates the mitogen
activated protein kinases ERK1 and ERK2 (p42 and p44 MAPK). Following
their activation, the ERKs phosphorylate a large number of regulatory
proteins including the transcription factor Elk-1 that will
subsequently bind to serum response elements located in several
promoters (36). In addition, the ERKs will phosphorylate the
p90RSK kinase (37), that will in turn phosphorylate targets
such as CREB and activate the transcription of immediate early genes
such as c-fos by binding to cAMP response element-binding
sites (38, 39).
The existence of commercially available antibodies specific for
activated MAPK proteins (antibodies that will only recognize double-phosphorylated substrates) allowed us to study in detail the
effects of BDV infection on the NGF signal transduction cascade. Because most events occur within minutes after addition of NGF, PC12
and PC12-BV cells were rapidly lysed at different times after adding
NGF, and equal amounts of proteins (as determined by Bradford assays)
were analyzed by Western blot for the expression of the above-mentioned
activated kinases. In some cases, the blots were stripped after
detection of the activated form of the kinase and reprobed with an
antibody that detects all forms of the kinase (phosphorylated and
nonphosphorylated). This experiment was repeated at least five times,
and the results were consistently similar to those shown in Fig.
7B. We observed no basal expression of activated MEK or ERK
in control PC12 cells prior to adding NGF and only minimal CREB
phosphorylation at this point. After adding NGF, both MEK and ERK were
rapidly and strongly induced. This was followed by activation of Elk1
and of the p90RSK/CREB cascade and ultimately by
transcription of c-fos, the latter being analyzed by
Northern blotting (Fig. 7B). Several marked differences were
observed in PC12 cells persistently infected with BDV (Fig.
7B). First, we observed that MEK1/2 and ERK1/2 were
constitutively activated, even in the absence of NGF. This was
consistently observed, even if cells were serum-starved for long
periods of time (not shown). It should be emphasized that activated
forms of MEK and ERK were never observed in noninfected PC12 cells
before NGF treatment, even after increasing the quantity of protein on
the blot. Second, PC12-BV cells responded to NGF treatment with an
increase in the amount of activated MEK and ERK proteins to levels
comparable with those of the control PC12 cells. This strongly suggests
that the TrkA receptor molecule is still present in PC12-BV cells and
is capable of initiating the MAPK signaling cascade. Nevertheless, this
increased activation was more transient than in control PC12 cells and
levels returned rapidly to baseline. Third, activation of the
p90RSK/CREB pathway was significantly decreased, leading to
only low level of c-fos transcription. Fourth, there was
constitutive phosphorylation of Elk1 in PC12-BV cells, and treatment
with NGF caused accelerated additional phosphorylation of this
transcription factor.
The Nuclear Translocation of Phosphorylated ERKs Is Impaired in
BDV-infected PC12 Cells--
The finding that the MAPK pathway is
constitutively activated in PC12-BV cells is in apparent contradiction
with the fact that these cells do not differentiate (40). However, we
also observed that activation of the p90RSK/CREB cascade
was decreased in PC12-BV cells. Because this activation requires the
nuclear translocation of activated ERKs, we hypothesized that BDV
infection may interfere with the nuclear translocation of activated
ERK1/2. To examine this possibility, PC12 and PC12-BV cells were plated
on collagen-coated coverslips, treated with NGF, and analyzed for the
localization of the activated ERK1/2 proteins by indirect
immunofluorescence and confocal microscopy (Fig.
8A). The results shown are
representative of a large number of cells analyzed for each time point,
and the experiment was repeated several times. Consistent with the
results of Western blots, there was no detectable levels of activated
ERK1/2 in PC12 cells prior to NGF treatment. As early as 5 min after
NGF addition, activated ERK1/2 was strongly expressed and was localized
both in the nucleus and in the cytoplasm of PC12 cells. The vast
majority of cells were strongly positive for activated ERKs by 10 min
after treatment. In most of them, the signal was mainly nuclear.
Rapidly thereafter, the signal decreased, and by 4 h the signal
was barely detectable. In PC12-BV cells, activated ERKs expression was
again detected even prior to NGF treatment, confirming our Western blot results. Expression levels were variable from cell to cell, and the
proportion of cells strongly expressing ERKs increased upon NGF
treatment. By 30 min, levels were back to those observed before NGF.
Interestingly, the distribution of activated ERKs staining was very
different from that in control cells. Confocal analysis (by image
overlay and three-dimensional scan, also performed after staining with
a nuclear-specific dye; data not shown) revealed that staining was
almost exclusively cytoplasmic and that there was very little activated
ERK translocated to the nucleus of PC12-BV cells.
We examined whether these changes were already present in short term
infected PC12 cells. As shown in Fig. 8B, the nuclear relocalization of activated ERK was already impaired in the rare infected cells present in the population early after infection. Moreover, some infected cells were already resistant to NGF-induced neurite outgrowth. Therefore, the changes occur early after infection and spread in time, together with the virus, to the entire cell population.
It has been proposed that the neurological symptoms observed in
rats infected at birth with BDV are linked to alterations of the
morphogenesis of the hippocampus and cerebellum, two brain structures
that continue to develop after birth (19, 41). It is well established
that neurotrophins such as NGF have a profound influence on the
development of the central nervous system (24). The critical role of
neurotrophins in supporting neuronal differentiation and protecting
from neuronal programmed cell death, together with recent results
suggesting that BDV neonatal persistent infection causes alterations of
synaptic plasticity and neuronal cell death by apoptosis (18, 19, 22),
led us to examine the effects of BDV on NGF signaling. Because BDV is
noncytopathic, it persists and replicates at high levels in neurons
in vivo, as well as in PC12 cells in vitro.
Therefore, this cell line is an interesting model to study the effects
of viral infection on neuronal gene expression and on the responses to
neurotrophic factors. Here, we describe an experimental system suitable
to explore the mechanisms whereby persistent BDV infection
leads to decreased neuronal gene expression and differentiation.
We observed that the persistence of BDV in PC12 cells was accompanied
by a progressive change of phenotype. Although PC12 mutant cells
with altered responses to NGF can arise spontaneously during continuous
culturing (42, 43), we can rule out that the phenotype described here
was due to culturing conditions and clonal selection of PC12 cells
because (i) we never observed a similar change of phenotype in control
PC12 cells maintained under the same conditions and for the same number
of passages and (ii) a similar change of phenotype was obtained in
several independent experiments. Moreover, none of the PC12 mutants
reported so far exhibit the pattern of gene expression and receptor
tyrosine kinase activity observed in PC12-BV cells. Finally, at early
stages infected cells were morphologically undistinguishable from
control cells and expressed normal levels of synaptophysin. This argues
strongly against initial infection of a cell subpopulation with
characteristics different from those of normal PC12 cells. Instead, it
is most likely that viral persistence in PC12 caused a progressive
change of phenotype.
BDV infection did not change the level of expression of neuronal
markers normally expressed in PC12 cells, such as Tau or BDV-infected PC12 cells failed to extend neurites in response to NGF.
We showed that the block of NGF signaling was due in part to a strong
down-regulation of NGF receptor expression. We then examined the
effects of BDV infection on receptor tyrosine kinase signaling and
immediate early gene induction, expecting that the MAPK signaling
pathway would also be decreased. In contrast, we identified several
important changes, which can be summarized as follows: MEK1/2 and
ERK1/2 proteins were constitutively activated and responded transiently
to NGF induction; Elk-1 transcription factor activation followed the
same pattern; and phosphorylation of the components of the
p90RSK/CREB cascade was decreased and led to little or no
c-fos mRNA induction.
The MAPK signaling cascade is implicated in the response of cells to
several growth factors and mitogens. In the case of PC12 cells, the
best-studied models have been the response to NGF and to epidermal
growth factor (EGF). Although NGF causes neurite extension and
acquisition of a post-mitotic phenotype, EGF treatment leads instead to
proliferation mediated by the EGF receptor, which is also a tyrosine
kinase. Both TrkA and EGF receptor induce similar early transduction
pathways, with a strong activation of ERK proteins. The key bifurcation
between EGF and NGF-mediated signaling pathways lies in the duration of
ERK1/2 activation that they induce. Transient activation of the ERK
pathway will induce proliferation, whereas sustained ERK activation is
critical for differentiation of PC12 cells (40). Hence, we were
surprised to find that BDV infection caused a sustained activation of
ERKs, which was not followed by activation of immediate early genes and
neuronal differentiation. A detailed analysis of signaling events that
follow the phosphorylation of ERKs, including the phosphorylation of
Elk-1, p90RSK, and CREB, indicated that the ERKs failed to
transduce a strong nuclear signal in BDV-infected PC12 cells. In
particular, the expression of phosphorylated CREB, which localizes
exclusively to the nucleus, was decreased in PC12-BV cells, suggesting
that ERK1/2 might not efficiently translocate to the nucleus. The low level of CREB activation remaining in infected cells could be due to
phosphorylation by MSK-1 (45). The impaired translocation of ERKs to
the nucleus of infected cells was confirmed by immunodetection of
activated ERKs and analysis by confocal microscopy. For Elk-1, however,
we observed a constitutive activation (similar to the MEKs and ERKs
proteins) as well as an accelerated response after treatment with NGF.
This may be due to the fact that this transcription factor, which is
expressed mainly in neuronal cells, localizes both to the cytoplasm and
to the nucleus before activation (46). It may also be due to increased
MAPK-independent phosphorylation of Elk1 (47).
The precise mechanism leading to chronic activation of ERK1/2 in
PC12-BV cells is not known. We were unable to detect ERK1/2 activation
following treatment of PC12 cells with supernatants from PC12-BV cells
(data not shown). This suggests that cytokine and/or soluble factors
released by the infected cells are not responsible for an autocrine
loop leading to chronic ERK1/2 activation. A possible explanation is
that BDV replication affects the regulation of the phosphorylation or
dephosphorylation processes in the cell. For example, BDV infection
could enhance the activity of any of the components of the
Ras-dependent pathway. Similar to all the nonsegmented,
negative-stranded RNA viruses, BDV encodes a phosphoprotein that is
phosphorylated by the host cellular machinery. Although phosphorylation
of the BDV phosphoprotein does not appear to be related to the ERK1/2
pathway (48), it could nevertheless interfere with upstream events
involved in phosphorylating ERK1/2. Alternatively, dephosphorylation of
ERK1/2 is normally mediated by a substrate-specific MAPK phosphatase
(MKP-1), which is present in the nucleus (49). This phosphatase rapidly
dephosphorylates the activated ERKs after their nuclear translocation,
allowing their recycling to the cytoplasm. It is possible that impaired
nuclear translocation leads to a progressive cytoplasmic accumulation
of activated ERK in PC12-BV cells. How BDV alters nuclear translocation
of ERK remains to be established. Because it actively replicates in the
nucleus, transport of viral RNA and proteins is an important feature of the BDV life cycle. It implicates viral proteins such as the
nucleoprotein, the phosphoprotein and the protein X/p10, whose
interactions have been well studied (50-52). However, cellular factors
associated with these viral proteins in the nuclear import of viral RNP
have not been identified yet. One can hypothesize that BDV could hijack some cellular factors linked to nuclear import and divert them from
their normal functions, hence affecting the nuclear translocation of
proteins such as the activated ERKs. To our knowledge, this is the
first description of a persistent viral infection interfering with
neuronal differentiation and linked to impaired ERK nuclear translocation. It has been demonstrated that adenovirus type 5 E1a
protein causes a block of NGF differentiation, in this case linked to
association of E1a with proteins implicated in cell cycle regulation
(53, 54). Interestingly, it has recently been shown that the
ret oncogene also blocks the nuclear translocation of ERK
and renders PC12 cells resistant to NGF (55).
In the central nervous system, the Ras/ERK pathway and
MAPK-dependent transcription are important regulators of
neuronal survival and synaptic plasticity (56). If such pathways are
indeed affected in BDV-infected animals, this may lead to severe
dysfunction, particularly in the critical phases of early postnatal development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
PC12 cells
(CRL-1721; American Type Culture Collection) were grown at 37 °C in
Dulbecco's modified Eagle's medium with Glutamax II (Life
Technologies, Inc.) supplemented with 10% horse serum, 5% fetal calf
serum and 1× Bufferall (Sigma). Cells were passaged once or twice a
week using nonenzymatic cell dissociation solution (Sigma). Cells were
replated at a 1:4 to 1:6 ratio in 75-cm2 tissue culture
flasks or transferred to culture dishes or glass coverslips coated with
collagen (Sigma). PC12 cells were infected with BDV CRP4 as described
(13) and subcultured 3 days after the initial infection to establish a
persistently infected line (PC12-BV). PC12-BV cells were subcultured
(1:5) every 4-6 days under the same conditions as the noninfected
control cells.
32P]dCTP using random hexamers (Prime-it II;
Stratagene, La Jolla, CA). After high stringency washes (0.2× SSC,
0.2% SDS at 65 °C, twice), the blots were exposed to Biomax
MS films (Eastman Kodak Co.). After each hybridization, the blots were
stripped by boiling twice in stripping buffer (2 mM EDTA, 5 mM Tris, pH 7.5, 0.1% SDS) before being rehybridized. The
following probes were used: cDNA fragments from the BDV
nucleoprotein (NP), rat synaptophysin, GAP-43, and tyrosine hydroxylase
(28) genes, as well as cDNA fragments from rat p75 and TrkA
receptors (gifts from C. Tuffereau and M. Barbacid, respectively). The
signals were compared with those obtained with a cDNA probe
corresponding to the rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
III-tubulin (Sigma; diluted 1:200) and NGF receptor molecule p75 (Roche Molecular Biochemicals; diluted 1:10). We also used a rabbit
polyclonal antibody to the Tau protein (Sigma; diluted 1:200), as well
as the antibodies to the phosphorylated forms of ERK-1/ERK2 (diluted
1:200) and to BDV NP (diluted 1:300) described above. When performing
double staining, primary and secondary antibodies were used
simultaneously. After staining, cells were examined with a TCS4D Leica
laser scanning confocal microscope. Digitalized images were processed
using Adobe Photoshop and Canvas softwares.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Kinetics of BDV infection and morphological
change of PC12 cells. PC12 cells were infected with BDV, passaged,
and analyzed for BDV infection by immunofluorescence with an antibody
specific for the BDV NP. Pictures shown for passages 0, 1, and 3 (P0, P1, and P3, respectively) were
superimposed to a bright field snapshot to show the whole cell
population. After passage 3, the vast majority of cells (>96%) are
infected. The progressive morphological change of infected cells is
illustrated at passages 5 and 6 (P5 and P6).
This figure is a representative example of five independent
infection experiments. A similar change of phenotype was never observed
in noninfected cells processed in parallel.
View larger version (76K):
[in a new window]
Fig. 2.
NGF does not induce neurite outgrowth in PC12
cells persistently infected with BDV (PC12-BV). PC12 or PC12-BV
cells (passage 10 after infection) were grown on collagen-coated plates
for 3 days in the presence or absence of 100 ng/ml NGF. The cells were
fixed and briefly stained with Giemsa.
III-tubulin. The expression of MAP-2 appeared
even higher in PC12-BV cells than in the control cells (Fig.
3). In contrast, the expression of
synaptophysin, a presynaptic vesicle marker, was lost in PC12-BV cells
(Fig. 4). Interestingly, synaptophysin
expression was lost progressively as PC12-BV cells were passaged. Loss
of synaptophysin expression correlated with the morphological
alterations linked to BDV persistence (Fig. 4). At later passages, very
few cells still expressed synaptophysin and responded to NGF. These
cells were, in fact, not infected (Fig. 4, bottom panels).
All experiments described in the rest of this study were performed on
persistently infected cells that had undergone all of the
above-described phenotypic changes.
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[in a new window]
Fig. 3.
Expression of neuronal-specific markers in
PC12 and PC12-BV cells. Cells were grown of collagen-coated
coverslips for 2 days in the presence of NGF, fixed, and processed for
immunofluorescence for the detection of MAP-2, Tau, and
III-tubulin.
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[in a new window]
Fig. 4.
BDV infection causes a progressive
down-regulation of synaptophysin expression. Control PC12 cells
and cells infected with BDV and passaged 1, 6, and 9 times
(P1, P6, and P9) are shown. PC12 and
PC12-BV P9 cells were treated with NGF for 4 days. PC12-BV P1 and P6
were left untreated. All cells were doubly stained for synaptophysin
(red) and BDV NP (green). The higher density of
PC12-BV (P9) cells is due to the fact that these cells do not become
post-mitotic in response to NGF. Infected cells stained with the BDV NP
antibody exhibit a characteristic punctuate staining of the nucleus
(better seen on Z series scans; not shown), together with a diffuse
cytoplasmic staining. Note the progressive decrease of synaptophysin in
infected cells. At passage 9, synaptophysin is not detected in PC12-BV
cells, except for a single cell that is not infected (see merged
images).
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Fig. 5.
Impaired mRNA expression in PC12 cells
persistently infected with BDV (PC12-BV). RNA was extracted from
PC12 and PC12-BV cells treated or not with NGF for the indicated times
and analyzed by Northern blot hybridization with probes for
synaptophysin (Syn), GAP-43, TH, BDV NP, and GAPDH. Exposure
times for autoradiography vary depending on the probe and range from 4 to 30 h. Staining with ethidium bromide (EtBr) shows
the integrity and the amount of ribosomal 28 and 18 S loaded in each
lane. Hybridization with a probe for the housekeeping GAPDH gene
provided another control for the integrity of mRNA. Similar results
were obtained in three independent experiments.
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Fig. 6.
Expression of NGF receptor molecules in PC12
and PC12-BV cells. A, Northern blot analysis. RNA was
analyzed as described for Fig. 3 using probes specific for p75 and TrkA
genes. Up-regulation of mRNA levels for both p75 and TrkA in PC12
cells and decreased p75 and TrkA mRNA expression in PC12-BV cells
were observed in three independent experiments. B, double
immunofluorescence staining for p75 and BDV NP analyzed by confocal
microscopy, after treatment with NGF for 2 days. p75 is detected at the
membrane of PC12 cells. Variable levels of p75 in PC12-BV cells are
apparently not related to the viral NP load (see merged images).
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Fig. 7.
Analysis of the MAPK signaling pathway in
PC12 and PC12-BV cells. A, schematic representation of
intracellular events triggered by NGF binding (see text for details).
B, Western blot analysis of phosphorylated (P) or
nonphosphorylated forms of the different kinases and Northern blot
analysis of c-fos transcription. Total protein extracts were
rapidly processed at different times after NGF treatment (indicated
above the blots), and equal amounts of protein (10 µg for the
detection of ERK and NP, 20 µg for detection of MEK, and 30 µg for
the detection of CREB, ribosomal S6 kinase, and Elk-1) were analyzed in
parallel for PC12 and PC12-BV cells as described under "Experimental
Procedures." For the detection of ERK and CREB, expression of
activated (phosphorylated) proteins was analyzed first, and then total
ERK or CREB protein were immunodetected on the same blot after
stripping. BDV infection was assessed by immunodetection of the viral
NP. Arrows on the right of each blot indicate the
expected position of the different proteins. Each blot is
representative of at least five independent experiments. Moreover,
mRNA was analyzed by Northern blotting for c-fos
expression in three independent experiments, with one representative
result being shown in the figure (bottom panel).
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Fig. 8.
Analysis by confocal microscopy of the
cellular distribution of activated ERK1/2 in PC12 and PC12-BV
cells. A, cells grown on glass coverslips were left
untreated or treated with NGF for the indicated times, processed for
immunofluorescence using the antibody specific for activated
(phosphorylated) ERK1/2 and analyzed by confocal microscopy. Similar
laser voltage was used for each scan to allow for comparison in
expression levels. Z series scan confirmed that activated ERK1/2 was
almost exclusively cytoplasmic in infected cells (bottom
panels). B, PC12 cells were infected with BDV and
analyzed at one passage after infection (P1). Cells were
treated with NGF for 5 min and analyzed for the expression of both
activated ERK and BDV NP as described above. Note the impaired nuclear
translocation of ERK in the BDV-NP positive cell. PC12-BV (P1) cells
were also treated with NGF for 3 days and analyzed for synaptophysin
and BDV NP expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
III-tubulin. The increased expression of MAP-2 in PC12-BV cells may
be due to cytoskeleton rearrangements accompanying the change of
morphology and adhesion properties caused by BDV. Alternatively, it may
be due to the increased activation of ERKs, because activated ERKs can
interact with MAP-2 (44). In contrast, the expression of neuronal
plasticity-related proteins such as synaptophysin and GAP-43 was
severely impaired in PC12-BV. This is consistent with our previously
reported results showing impaired synaptophysin and GAP-43 protein
expression in the central nervous system of neonatally infected rats
(18). The expression of TH was also inhibited in PC12-BV cells. Taken
together, these results suggest that BDV infection down-regulates a set
of neuronal-specific genes linked to NGF signaling, without affecting
NGF independent neuronal gene expression.
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ACKNOWLEDGEMENTS |
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We thank Emmanuelle Perret for outstanding assistance for confocal microscopy analysis, Christine Tuffereau and Mariano Barbacid for gift of plasmids, and Jocelyne Caboche for many helpful discussions and for the gift of some of the reagents described in this study. We are also grateful to Monique Dubois-Dalcq and Jeffrey Bajramovic for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique and by a grant from the Ministère de l'Education Nationale, de la Recherche et de la Technologie (Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a doctoral fellowship from the Ministère de l'Education Nationale et de l'Enseignement Supérieur.
To whom correspondence should be addressed: Unité des
Virus Lents, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail: ddune@ pasteur.fr.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M005107200
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ABBREVIATIONS |
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The abbreviations used are: BDV, Borna disease virus; NGF, nerve growth factor; NP, nucleoprotein; GAP-43, growth-associated protein-43; TBS, Tris-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; CREB, cAMP response element-binding protein; MAP-2, microtubule-associated protein-2; TH, tyrosine hydroxylase; EGF, epidermal growth factor.
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