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B in Neuronal Cells: Implications on the Immune Response to Viral Infections in the Central Nervous System1
Department of Neurology, University of Maryland at Baltimore, Baltimore, MD 21201; and Department of Veterans Affairs, Baltimore, MD 21201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding to the
positive regulatory domain II element of the IFN-ß promoter,
which is essential for virus-induced IFN-ß gene activity. In this
study, we demonstrate that the failure to activate NF-B in neuronal
cells is due to the inability of MV to induce phosphorylation and
degradation of IB, the inhibitor of NF-B. In contrast, TNF-
induced degradation of IB in the neuronal cells, suggesting that
failure to induce IB degradation is likely due to a defect in
virus-mediated signaling rather than to a defect involving neuronal
IB. Like MV, mumps virus and dsRNA failed to induce IB
degradation in the neuronal cells, suggesting that this defect may be
specific to viruses. Autophosphorylation of the dsRNA-dependent protein
kinase, a kinase possibly involved in virus-mediated IB
phosphorylation, was intact in both cell types. The failure of virus to
induce IB phosphorylation and consequently to activate NF-B in
neuronal cells could explain the repression of IFN-ß and class I gene
expression in virus-infected cells. These findings provide a potential
mechanism for the ability of virus to persist in neurons and to escape
immune surveillance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Demonstration of failure to induce IFN-ß in neuronal but not glial
cells was associated with lack of NF-B binding to the positive
regulatory domain II (PRDII) element of the IFN-ß promoter (4)
necessary for virus inducibility of the IFN-ß gene promoter activity
(6). This defect is virus specific as TNF-, but MV, mumps virus
(MPS), and dsRNA were all unable to induce NF-B DNA-binding activity
(4). NF-B, a member of the Rel family of proteins, is
involved in the regulation of both HLA class I and IFN-ß gene
expression (6, 7, 8). The Rel family of proteins includes p50 (NF-B1),
p52 (NF-B2), and p65 (RelA) and form homodimeric or heterodimeric
complexes of which the p50/p65 dimer is most abundant and is strongly
transactivating. In unstimulated cells, NF-B is sequestered in the
cytoplasm by complexing with IB, a family of proteins that includes
IB, IBß, Bcl-3, and p105. IB predominantly inhibits
NF-B. IB is a constitutively phosphorylated protein and
hyperphosphorylation induced by a variety of agents including
cytokines, virus, or dsRNA signals ubiquitination and subsequent
degradation by the 26S proteasome complex (9, 10, 11, 12, 13, 14, 15, 16). This allows NF-B
to translocate into the nucleus and to bind the target B site.
Phosphorylation of IB by the dsRNA-dependent protein kinase (PKR)
is believed to be involved in the virus-induced activation of NF-B
(13). PKR is a serine-threonine kinase involved in growth inhibition
and NF-B activation through phosphorylating eIF2a and IB,
respectively (17). PKR functions as a self-associating dimer with an
N-terminal dsRNA-binding domain that requires autophosphorylation for
its activity. Autophosphorylation but not transphosphorylation of PKR
requires dsRNA. PKR is induced by IFNs and is believed to mediate some
of the antiviral and antiproliferative effects of these cytokines (17).
PKR activity can also be inhibited by some cellular and viral gene
products (18, 19). Studies in PKR knockout mice showed that the
induction of type I IFN by dsRNA was unimpaired in these mice and that
the antiviral response induced by dsRNA and IFN-
was reduced.
However, in embryo fibroblasts from these mice, the induction of type I
IFN and NF-B activation by dsRNA were strongly impaired but
partially restored with IFN (20). Thus, although PKR is involved in
dsRNA-induced activation of NF-B and type I IFN expression, PKR
activity may not be required for virus to induce IFN-ß and
NF-B.
Because NF-B is involved in the regulation of both IFN-ß and class
I genes, and because both genes are suppressed in virus-infected
neuronal cells, we investigated the mechanism responsible for the
failure of MV to activate NF-B in these cells. In this study, we
demonstrate that the lack of NF-B activation in neuronal cells by MV
is due to a defect in the phosphorylation and degradation of the
NF-B inhibitor, IB.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The neuroblastoma cell line IMR-32 clone was a gift from Dr. Richard J. Ziegler (University of Minnesota, Duluth, MN). This cell line retains a number of neuronal characteristics such as expression of nicotinic and muscarinic cholinergic receptors and neurotransmitter expression (21). The neuroblastoma cell line CHP-126 (22) was used in limited experiments and was a gift from Dr. Lois A. Lampson (Harvard Medical School, Boston, MA). The neural cell lines were grown in MEM medium supplemented with 5% FCS, glutamine, HEPES buffer, penicillin, streptomycin, and gentamicin. The human astrocytoma cell line U-251 MG was a gift from Dr. Darryl Bigner (Duke University, Durham, NC). The characteristics of this cell line and the culture conditions have been previously described (23).
Reagents
The Edmonston strain of MV and MPS were obtained from the
American Type Culture Collection (ATCC, Manassas, VA), grown in
confluent vero cells (monkey kidney fibroblasts from the ATCC), and
titrated by plaque assay according to standard methods. The stock titer
was 5 x 107 plaque forming units/ml for MV and
108 plaque forming units/ml for MPS. When monolayers of
neuronal and glial cells were 
80% confluent, cells were stimulated
with varying doses of MV or MPS in serum-free medium for the indicated
time periods. In some experiments, MV was inactivated by UV irradiation
(400 µW/cm2 x 8 min). dsRNA polyinosinic polycytidylic
acid (PIPC) was from Pharmacia (Piscataway, NJ). Human recombinant
TNF- was from Genzyme (Cambridge, MA). Rabbit polyclonal IgG to p65
NF-B subunit, IB, and PKR were from Santa Cruz Biotechnology
(Santa Cruz, CA). Calf intestinal phosphatase (CIP) was from Sigma (St.
Louis, MO).
Immunoprecipitation and Western blot analyses
Monolayer cells were grown in 75-cm2 flasks to
80% confluency (2 x 107 cells/flask). Cells
were washed twice with PBS, and complete medium was added before
infection with MV at a moi of 2.0 or treatment with 500 U/ml of TNF-
for the indicated time periods. After washing four times in ice-cold
PBS, cells were lysed in 1 ml of TNT-E (Tris-NaCl-Triton
X-100-EDTA) lysis buffer (12) containing the following protease
inhibitors: 50 mM NaF, 0.1 mM Na orthovanaolate, 20 mM
ß-glycosophosphate, 10 mM molybdic acid, 21 µg/ml aprotinin, and
0.2 mM 4-(2-aminoethyl)-benzene sulfonyl fluoride (AEBSF; Sigma). Cell
lysates were centrifuged at 10,000 x g for 10 min at
4°C. The supernatants were mixed with 50 µl of protein A-Sepharose
(Pharmacia) for 1 h at 4°C. After centrifugation at 10,000
x g for 10 min at 4°C, the clarified lysates were
incubated with the primary or control Abs for 16 h at 4°C. Fifty
microliters of protein A-Sepharose was then added to each tube and
incubated with constant shaking for 2 h at 4°C. The tubes
were spun again, and the supernatants were discarded. The precipitates
were then washed three times with TNT-E buffer containing protease
inhibitors. Fifty microliters of 2x sample buffer (Promega, Madison,
WI) was then added to the precipitate and boiled for 5 min. The
immunoprecipitates were then electrophoresed on a 15%
SDS/polacrylamide gel at 75 V for 16 h. The proteins were then
transferred to a Hybond ECL membrane at 30 V overnight. Filter strips
were blocked with 5% milk-TBS at 4°C overnight and then incubated
with .5 µg/ml primary Ab of milk-TBS for 1 h at room
temperature. The blots were washed three times with TBS-milk, then
incubated with anti-IgG HRP conjugate (1:10,000 dilution) for
1 h. After three washes with TBS, the blots were developed using
the Amersham enhanced chemiluminescence system (Arlington Heights, IL).
PKR autophosphorylation assay
Autophosphorylation of PKR was determined as previously described (13) with the following modifications. Neuronal and glial cells (107 cells/75-cm2 flask) were infected with 2 moi of MV for 20 h. Cells were washed with ice-cold PBS and lysed with TNT-E buffer containing AEBSF. PKR was then immunoprecipitated with anti-PKR mAb on protein A-Sepharose beads and washed with kinase reaction buffer (20 mm HEPES (pH 7.5), 50 mM Kcl, 5 mM 2 mercaptoethanol, 1.5 mM Mg (OAC)2, 1.5 mM MnCl2, and 10 µCi [32P]ATP). The beads were then suspended in 25 µl kinase reaction buffer containing 10 µCi of [32P]ATP and incubated at 30°C for 30 min. After washing with ice-cold kinase buffer, the beads were suspended in 50 µl SDS-PAGE sample loading buffer and boiled at 100°C for 5 min. The samples were then electrophoresed on a 10% SDS-PAGE gel and visualized by radiography.
Statistical analysis
The densities of the bands observed on Western blot analyses
were quantitated by densitometry using NIH Image 1.61 software
(National Institutes of Health, Bethesda, MD). For statistical
analysis, the densities of the bands observed with treatment were
normalized against the density of the IB band immunoprecipitated
from untreated cells. The phosphorylated IB band (IBp)
density was expressed relative to the density of the hypophosphorylated
band (IBp/IB) in each lane. The Student’s
t test was used to examine the statistical significance of
treatment effects using Statworks Mcintosh software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B degradation and dissociation
from NF-B in glial cells
We have examined IB phosphorylation and degradation in glial
cells after MV infection to explore the hypothesis that failure to
activate NF-B by MV in neuronal cells but not in glial cells may be
due to a failure of IB phosphorylation and degradation in
neuronal cells. IB was immunoprecipitated from unstimulated or
MV-stimulated glial cells using rabbit anti-sera to IB or
anti-p65 subunit of NF-B. The blots were probed with
anti-IB Ab and visualized by enhanced chemiluminescence (Fig. 1
A). As expected, an 37-kDa
band corresponding to IB was immunoprecipitated by
anti-IB and by anti-p65 but not by normal rabbit serum in
unstimulated glial cells. After MV infection, a double band
corresponding to IB could be immunoprecipitated at 5 min.
IB bands associated with NF-B were no longer coprecipitated
with anti-p65 Ab at 20 min (Fig. 1A). The upper band
represents hyperphosphorylated IB, because it could be almost
abolished by treatment with CIP (24) (Fig. 1B). To examine
the kinetics of IB degradation, IB phosphorylation and
degradation were examined at serial time points in the presence of MV.
As shown in Fig. 1C, IB was hyperphosphorylated within
5 min and degradation occurred between 10 and 30 min after MV
infection. After 2 h of viral infection, IB appeared to be
regenerated, thus entering a new phosphorylation and degradation cycle.
The mean effect of MV on IB phosphorylation and degradation in
the glial cells obtained from three experiments are presented in Fig. 2. IB phosphorylation occurred as
early as 5 min and significant degradation at 15 and 30 min after MV
infection.
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B phosphorylation and degradation in neuronal cells
in response to MV
To determine whether the inability of MV to activate NF-B in
neuronal cells is due to a failure of IB phosphorylation and
degradation, IB immunoprecipitated from unstimulated or
MV-stimulated neuronal cells using rabbit anti-sera to IB or
to p65 was examined. In contrast to glial cells, IB is not
hyperphosphorylated nor degraded in neuronal cells infected with MV for
up to 120 min, as it could be immunoprecipitated with anti-p65 as
shown in Fig. 3A. Identical
results were obtained when anti-IB Ab was used to
immunoprecipitate IB (data not shown). Subsequent experiments
showed that IB was not phosphorylated nor degraded at time points
as late as 24 h after MV stimulation (data not shown). However,
neuronal cells stimulated with TNF- showed a hyperphosphorylated
IB after 5 min and degradation within 30 min (Fig. 3B). The average effects of MV infection or TNF-
stimulation of the neuronal cell line IMR-32 on IB expression
obtained from three experiments are presented in Fig. 4. Although TNF- produced significant
degradation of IB beginning 10 min after stimulation, MV failed
to do so at time points varying from 5 to 60 min. To ascertain that
these findings are not peculiar to the neuronal cell lines IMR-32, we
examined the effects of MV and TNF- stimulation on IB
degradation in another neuronal cell line CHP-126. As shown in Fig. 5, TNF- but not MV stimulation
resulted in phosphorylation and degradation of IB at time points
ranging from 10 to 60 min, which is consistent with the results
obtained with the IMR-32 cells.
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B degradation in neuronal cells infected
with MPS
To determine whether differential IB degradation in neuronal
and glial cells is MV specific, another neurotropic virus, MPS, was
tested in both cell lines. Additional conditions included stimulation
with MV, TNF-, or a combination of MV and TNF- for comparison. A
representative of two experiments is shown in Fig. 6. In all conditions, cells were
stimulated for 15 min. All stimuli resulted in IB degradation in
the glial cell line U-251 MG (Fig. 6A). In contrast, neither
MPS nor MV virus resulted in appreciable IB degradation in the
neuronal cells IMR-32 (Fig. 6B). As observed previously,
TNF- resulted in phosphorylation and degradation of IB in the
neuronal cell line, which was not inhibited when cells were stimulated
with MV and TNF- simultaneously (Fig. 6B).
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B degradation in the neuronal
cells
The inability of MV to induce IB degradation in neuronal
cells may involve a defective kinase activity critical for IB
phosphorylation such as PKR. PKR is a kinase activated by dsRNA and
also by viral RNA and is implicated in the signal cascade leading to
IB phosphorylation and NF-B activation (13, 17, 25). Thus,
neuronal and glial cells were treated with the dsRNA PIPC for 20
h. As PIPC may not readily penetrate the cell membrane, cells were
pretreated with dextran before PIPC stimulation. IB
phosphorylation and partial degradation were observed in the glial but
not in the neuronal cells after exposure to PIPC (Fig. 7). Similar results were obtained with
PIPC stimulation for shorter periods of time (1, 2, 4, or 8 h)
(data not shown).
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PKR activation requires autophosphorylation before phosphorylating
its target protein (17). Therefore, we examined the effect of MV
stimulation on PKR autophosphorylation in neuronal and glial cells.
Cells were exposed to MV for 20 h before the assay to allow
sufficient time for viral dsRNA formation. Cells were then harvested,
and PKR was immunoprecipitated with rabbit anti-PKR Ab followed by
the in vitro autophosphorylation assay. A representative of two
experiments is shown in Fig. 8. MV
stimulation in both cell types resulted in phosphorylation of a 68-kDa
protein consistent with the molecular mass of PKR. This band was
specifically immunoprecipitated with the anti-PKR, but not the
control Ab.
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B degradation by UV-inactivated MV
The observation that MV stimulation of glial cells resulted in
IB phosphorylation within 5 min suggested that early signaling
events generated by MV binding to its cellular receptor may be involved
in NF-B activation. Thus, UV-inactivated MV was examined for its
ability to induce IB degradation. Cross-linking viral RNA by UV
inhibits viral transcription and replication without affecting receptor
binding (our unpublished data). U-251 MG glial cells stimulated
with 5 moi of MV or the equivalent UV-MV for 1 h were examined for
IB degradation. A representative of duplicate experiments is
shown in Fig. 9. UV-MV was able to induce
IB degradation (Fig. 9A) and NF-B DNA-binding
activity (Fig. 9B) with similar efficiency as that of live
virus. UV inactivation of MV was ascertained by the absence of live
virus in a standard plaque assay. These findings suggested that NF-B
activation by MV may occur as a result of an early signal generated
from the binding of the MV to its receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NF-B is a transcription factor involved in the induction of a number
of cytokine genes including IFN-ß and possibly the up-regulation of
the HLA class I gene (7, 8). We have previously shown that in contrast
to glial cells lack of IFN-ß induction in neuronal cells by MV
correlates with lack of NF-B binding to PRDII (5). This does not
appear to be due to lack of NF-B expression in neuronal cells as
TNF- induces NF-B binding to PRDII in these cells and is not due
to lack of MV receptor expression or infectivity of these cells (5). In
this study, we demonstrated that the lack of activation of NF-B in
neuronal cells after viral infection correlates with the failure of
phosphorylation and degradation of the inhibitor IB. This finding
is not specific to the IMR-32 neuronal cells, because similar findings
were obtained with another neuronal cell line, CHP-126.
Information on the cellular defects responsible for the inability of
virus to induce IB phosphorylation and degradation is critical to
understand neuronal responses to viral infection and persistency. Our
findings may exclude a number of possibilities. It is unlikely that MV
activates a phosphatase that can dephosphorylate IB, because MV
does not block TNF--induced IB phosphorylation (Fig. 6
). It is
also unlikely that neuronal cells contain mutant IB isoforms,
because TNF- is able to phosphorylate and degrade IB in these
cells. Currently, we favor the hypothesis that virus infection of
neuronal cells fails to generate a signal pathway required for IB
phosphorylation and degradation.
In signaling, we may also exclude a failure of MV to activate the IB
kinase complex (IKK) or NK-B inducing kinase (NIK) MAP
kinase. Activation of IKK, a recently described kinase complex that
phosphorylates IB at the critical serine residues S32 and S36,
has been implicated for activation of NF-B (27, 28, 29, 30). IKK, which
exists in two identical forms, IKK1 or (85 kDa) and IKK2 or ß (87
kDa), are part of a larger protein complex (700 kDa) that associates
with IB. In TNF--stimulated cells, this complex is believed to
be recruited to the intracellular domain of the TNF- receptor and
phosphorylated by a MAP kinase known as NIK, which is required for
TNF--dependent NF-B activation (31, 32, 33). Thus, the ability of
TNF- to induce IB phosphorylation in the neuronal cells may
exclude defects in NIK or IKK in these cells.
PKR is a kinase activated by viral RNA and has been implicated in the
signaling cascade leading to IB phosphorylation (13, 17, 26). As
IB is not phosphorylated in neuronal cells even after 24 h
of exposure to MV, we assayed PKR autophosphorylation at the 20-h time
point that corresponds to the duration of the MV replicative cycle.
Therefore, the 20-h time point was chosen to ensure the availability of
sufficient levels of dsRNA in the infected cells. Our findings that MV
was unable to phosphorylate IB in neuronal cells despite PKR
activation suggests either a failure to activate a signal distal to PKR
in the signaling cascade that leads to IB phosphorylation or the
existence of a PKR-independent pathway used by virus to phosphorylate
IB. In fact, recent studies in PKR knockout mice support the
latter possibility (20). In addition, IB phosphorylation in
response to MV as early as 5 min poststimulation and the ability of
UV-inactivated MV to induce IB phosphorylation and NF-B
activation in glial cells indicate that viral transcription,
replication and dsRNA formation may not be necessary to induce IB
phosphorylation by MV. In addition, it is unlikely that PKR is
activated in MV-infected cells within 5 min of exposure to virus,
because MV nucleocapsid is detectable 4 h postinfection at the
earliest (M. Shin, unpublished observation). This finding would
suggest the existence of a signaling pathway for MV-induced IB
phosphorylation that is independent of dsRNA and PKR activation and
that this pathway may not be activated by MV in neuronal cells. Our
long-term goal is to identify this pathway and to determine whether it
is impaired in virus-infected neuronal cells.
Our findings have implications regarding the understanding of
mechanisms that favor virus persistence in neurons. IFN-ß induction
in an infected cell is an important host defense mechanism and is
dependent on NF-B activation. Therefore, our finding, implicating an
inability of MV to activate the signaling pathway that would normally
lead to IB phosphorylation and subsequently NF-B activation,
represents a step toward understanding cell-specific events that favor
viral persistence in neurons.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Suhayl Dhib-Jalbut, Department of Neurology, University of Maryland Hospital, Rm. N4W46, 22 S. Greene Street, Baltimore, MD 21201. E-mail address:
3 Abbreviations used in this paper: MV, measles virus; AEBSF, 4-(2-aminoethyl)-benzene sulfonyl fluoride; CIP, calf intestinal phosphatase; IB, inhibitor B; MPS, mumps virus; PIPC, polyinosinic polycytidylic acid; PKR, PRDII, positive regulatory domain II; dsRNA-dependent protein kinase; SSPE, subacute sclerosing panencephalitis.
Received for publication October 5, 1998. Accepted for publication December 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B regulation: the emerging role of phosphorylation and proteolysis. Immunity 3:263.[Medline]
B- proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485.B activation by IB. EMBO J. 12:4685.[Medline]
B by phosphorylating IB. Proc. Natl. Acad. Sci. USA 91:6288.ßa in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol. Cell. Biol. 14:1401.
B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol. Cell. Biol. 9:2424.B transcription factor by dissociating the cytoplasmic NF-B-IB complex. Proc. Natl. Acad. Sci. USA 90:9247.-induced phosphorylation of IB is a signal for its degradation but not dissociation from NF-B. Proc. Natl. Acad. Sci. USA 91:12740.B kinase. Cell 90:373.[Medline]
B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B kinases essential for NFB activation. Science 278:860.B kinase-ß: NF-B activation and complex formation with IB kinase- and NIK. Science 278:866.B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B kinase: beginning, not the end. Proc. Natl. Acad. Sci. USA 94:11758.B kinase complex. Science 278:818.This article has been cited by other articles:
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