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JOURNAL OF VIROLOGY, May 2003, p. 5145–5151 Vol. 77, No. 9
0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.9.5145–5151.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Expression of Inducible Nitric Oxide Synthase and Elevation of
Tyrosine Nitration of a 32-Kilodalton Cellular Protein in Brain
Capillary Endothelial Cells from Rats Infected with a
Neuropathogenic Murine Leukemia Virus
Atsushi Jinno-Oue,
1
† Susan G. Wilt,
2
‡ Charlotte Hanson,
1
Natalie V. Dugger,
2
Paul M. Hoffman,
2
Michiaki Masuda,
3
and Sandra K. Ruscetti
1
*
Basic Research Laboratory, National Cancer Institute, Frederick, Maryland 21702
1
; Research Service, Department of Veterans
Affairs Medical Center and Department of Neurology, University of Maryland, Baltimore, Maryland 21201
2
; and
Department of Microbiology, School of Medicine, Dokkyo University, Tochigi 321-0293, Japan
3
Received 7 October 2002/Accepted 4 February 2003
PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) which
causes a rapidly progressive spongiform neurodegenerative disease in rodents. The primary target of PVC-211
MuLV infection in the brain is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV
infection. Previous studies have shown that changes in the envelope gene of PVC-211 MuLV confer BCEC
tropism to the virus. However, little is known about how infection of BCECs by PVC-211 MuLV induces
neurological disease. Previous results suggest that nitric oxide (NO), which has been implicated as a potential
neurotoxin, is involved in PVC-211 MuLV-induced neurodegeneration. In this study, we show that expression
of inducible nitric oxide synthase (iNOS), which produces NO from
L-arginine, is induced in BCECs from
PVC-211 MuLV-infected rats. Furthermore, elevated levels of a 32-kDa cellular protein modified by 3-nitro-
tyrosine, which is a hallmark of NO production, were observed in virus-infected BCECs. BCECs from rats
infected with BCEC-tropic but nonneuropathogenic PVF-e5 MuLV, which is a chimeric virus between PVC-211
MuLV and F-MuLV, fail to induce either iNOS expression or elevation of tyrosine nitration of a 32-kDa
protein. These results suggest that expression of iNOS and nitration of tyrosine residues of a 32-kDa protein
in PVC-211 MuLV-infected BCECs may play an important role in neurological disease induction.
A number of murine leukemia viruses (MuLVs) have been
shown to induce diseases of the central nervous system (CNS)
that are characterized by progressive loss of neuronal function
(35, 39). The major cell types within the CNS that are prom-
inently infected with the MuLVs are glial and endothelial cells,
with neurons being infrequently infected. The most commonly
observed pathological changes are gliosis, neuronal loss, and
demyelination. The mechanism(s) by which the MuLVs induce
neurological diseases remains to be elucidated.
PVC-211 MuLV is a neuropathogenic variant of the leuke-
mia-inducing Friend MuLV (F-MuLV) (21). Infection of sus-
ceptible rats with PVC-211 MuLV causes a rapidly progressive
neurodegenerative disease characterized by tremor, spasticity,
ataxia, and hind limb paralysis. Neuropathological changes in-
clude widespread perivascular gliosis, neuropil vacuolation
without inflammation, and neuronal degeneration in the brain
stem, cerebellum, and spinal cord (19, 28).
The primary target of PVC-211 MuLV infection in the CNS
is the brain capillary endothelial cell (BCEC), which is resis-
tant to F-MuLV infection (19). The determinant of the BCEC
tropism of PVC-211 MuLV was mapped to two amino acids
(G
116
and K
129
) which lie within the putative receptor binding
domain of the envelope surface glycoprotein (SU) (30). Within
the CNS, reactive astrocytes and degenerating neurons showed
no evidence of virus infection (19). BCEC tropism of the virus
has been shown to be necessary for neuropathogenesis (29),
suggesting that CNS injury is indirect and that molecular
events in virus-infected BCECs play a very important role in
neurological disease induction.
Nitric oxide (NO) is an important messenger and effector
molecule involved in a number of biological functions (31). NO
is synthesized from
L-arginine by three isoforms of NO syn-
thases (NOS). Endothelial cell NOS (eNOS) and neuronal
NOS are constitutively expressed, and their activities are reg-
ulated by Ca
2⫹
. In contrast, inducible NOS (iNOS) is inducible
and Ca
2⫹
independent (13). In the CNS, NO may play impor
-
tant roles in neurotransmitter release, neurotransmitter re-
uptake, neurodevelopment, synaptic plasticity, and regulation
of gene expression, although excessive production of NO can
lead to neurotoxicity (9, 27). iNOS is an attractive candidate
for mediating NO-associated neurotoxicities, because long
bursts of large amounts of NO are produced by iNOS (7, 9, 32).
Indeed, elevated iNOS expression has been demonstrated in
such human neurological diseases as Alzheimer’s disease (24)
and Parkinson’s disease (26).
Recently, the spongiform vacuolation observed in PVC-211
MuLV-infected brains was reported to be associated with ox-
idative damage as detected by increased immunoreactivity for
* Corresponding author. Mailing address: Building 469, Room 205,
National Cancer Institute at Frederick, Frederick, MD 21702-1201.
Phone: (301) 846-5740. Fax: (301) 846-6164. E-mail: ruscetti@ncifcrf
.gov.
† Present address: Department of Virology and Preventive Medi-
cine, Gunma University School of Medicine, Gunma 371-8511, Japan.
‡ Present address:R&DProgram 151, Department of Veterans
Affairs Medical Center, Bronx, NY 10468.
5145
3-nitrotyrosine (NTyr) in infected brains (43). NTyr is widely
used as an indicator of NO formation, because nitration of
tyrosine is mediated by reactive nitrogen species derived from
NO (2, 11, 15). Elevated expression of NTyr has also been
reported in human neurodegenerative diseases such as familial
amyotrophic lateral sclerosis (41), Alzheimer’s disease (17),
Parkinson’s disease (12), and human immunodeficiency virus
type 1 dementia complex (5). In this study, we examined ex-
pression of iNOS and elevated expression of NTyr in PVC-211
MuLV-infected BCECs to evaluate the contribution of NO
produced by infected BCECs to the neuropathogenicity in-
duced by PVC-211 MuLV infection.
MATERIALS AND METHODS
Viruses. Neuropathogenic PVC-211 and PVF-e5 MuLV, a nonneuropatho-
genic variant of PVC-211, were grown in NIH 3T3 cells as described previously
(30). The viral supernatants had titers of 10
5
to 10
6
PFU/ml as determined by an
XC assay (40). Virus samples were stored at ⫺80°C until use.
Animals. Pregnant Fisher 344 (F344) rats were obtained from Charles River
(Raleigh, N.C.) and housed in the Small Animal Facility at the Department of
Veterans Affairs Medical Center (Baltimore, Md.). All experiments were per-
formed in accordance with Public Health Service guidelines, using an IACUC-
approved protocol (P. M. Hoffman). Two-day-old F344 rats were inoculated
intracerebrally with 0.03 ml of supernatant from virus-producing NIH 3T3 cells.
Cells. Primary rat BCECs were isolated from the brains of virus- or medium-
inoculated 3-week-old F344 rats as described previously (6, 19) and grown for 2
weeks in minimum essential medium (MEM) with
D-valine (Life Technologies,
Inc., Gaithersburg, Md.) supplemented with 20% fetal calf serum, 2 mM
L-
glutamine, 50 U of penicillin/ml, 50 g of streptomycin/ml, 1 mM MEM nones-
sential amino acids solution (Life Technologies), 1 mM vitamin solution (Life
Technologies), 50 g of endothelial mitogen (Biomedical Technology Inc.,
Stoughton, Mass.)/ml, and 16 U of heparin (Life Technologies)/ml at 37°Cina
5% CO
2
humidified incubator for 2 weeks. Isolated BCEC populations were
⬎95% pure as determined by immunohistochemistry with the endothelial cell
marker factor VIII.
Immunohistochemistry on brain sections. Brain tissue was collected from
medium- or virus-inoculated rats following intracardiac perfusion. The tissues
were then fixed, and serial sections were stained for expression of iNOS, eNOS,
and MuLV SU (gp70). Briefly, sections were first incubated for 48 h with mouse
anti-iNOS (610329 from BD Transduction Laboratories, Lexington, Ky.); mouse
anti-eNOS (610296 from BD Transduction Laboratories); or goat anti-Rauscher
MuLV (R-MuLV) gp70 (National Cancer Institute, Bethesda, Md.). After wash-
ing in Tris-buffered saline, sections were incubated for 1 h with the appropriate
biotinylated secondary antibody (Southern Biotechnology, Birmingham, Ala.).
Antibody complexes were visualized using the Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, Calif.). Sections were then dehydrated and cover-
slipped with Permount (Sigma, St. Louis, Mo.). Three to six sections per rat brain
were analyzed. Images were collected using a Nikon Eclipse 600 equipped with
a Nikon DMX 1200 digital camera.
RT- PCR analysis. To examine expression of NOS isoforms, we obtained total
cellular RNA from BCECs isolated from the brains of rats inoculated with
medium, PVC-211 MuLV, or PVF-e5 MuLV by using the RNA-STAT60 reagent
(TEL-TEST, Inc., Friendswood, Tex.). RNA from BCECs stimulated with lipo-
polysaccharide (LPS) was used as a positive control for iNOS expression. RNA
(2 g) was reverse transcribed in the presence of 50 mM random hexamers using
200 U of Superscript II reverse transcriptase (RT; Invitrogen, Carlsbad, Calif.)
according to the protocol supplied by the manufacturer. PCR was performed to
amplify iNOS, eNOS, or -actin sequences. Specific primer pairs used for each
amplification were as follows: iNOS sense, 5⬘-ATGGAACAGTATAAGGCAA
ACACC-3⬘; iNOS antisense, 5⬘-GTTTCTGGTCGATGTCATGAGCAAAGG-
3⬘; eNOS sense, 5⬘-TACGGAGCAGCAAATCCAC-3⬘; eNOS antisense, 5⬘-GA
TCAAAGGACTGCAGCCTG-3⬘; -actin sense, 5⬘-CGTAAAGACCTCTATG
CCAA-3⬘; and -actin antisense, 5⬘-AGCCATGCCAAATGTCTCAT-3⬘.
Each amplification reaction mixture contained 100 ng of cDNA, 400 ng of each
specific primer, a 0.2 mM concentration of each deoxyribonucleoside triphos-
phate, 2.5 U of Taq polymerase (Invitrogen), and 10⫻ reaction buffer. Amplifi-
cation conditions were as follows: 95°C for 5 min for 1 cycle; 30 cycles of 95°C for
1 min, 55°C for 1 min, and 72°C for 1 min; and 72°C for 10 min for 1 cycle.
Western blot analysis. To prepare cell lysates, cells were washed with cold
phosphate-buffered saline and lysed with immunoprecipitation buffer (50 mM
Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% sodium dodecyl sulfate [SDS], 10 mM EDTA, 10 g of aprotinin/ml, and
1 mM phenylmethylsulfonyl fluoride). The protein samples were prepared in
buffer containing 625 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM
dithiothreitol (SDS-sample buffer) and boiled for 5 min. Then, SDS-polyacryl-
amide gel electrophoresis (SDS-PAGE) was performed, and protein samples
were electrophoretically transferred to nitrocellulose membranes (Invitrogen).
The membranes were then incubated with goat antibody to R-MuLV gp70 or p30
(National Cancer Institute); rabbit antibody to iNOS (Santa Cruz Biotechnology,
Santa Cruz, Calif.); rabbit antibody to NTyr (Upstate Biotechnology, Lake
Placid, N.Y.); or mouse antibody to -tubulin (Sigma). Bound antibodies were
detected with peroxidase-labeled secondary antibodies (DAKO, Carpinteria,
Calif.) by using the enhanced chemiluminescence system (Amersham Pharmacia
Biotech, Piscataway, N.J.). Blocking experiments for NTyr immunoreactivity
were performed by preincubating (25°C, 30 min) the anti-NTyr antibody with 10
mM NTyr (Sigma) prior to incubation with the membrane. For reprobing the
membrane, antibodies bound to the membrane were removed by incubating with
buffer containing 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercap-
toethanol at 55°C for 30 min. After washing the membrane, subsequent proce-
dures for the binding of antibodies were carried out as described above. A cell
lysate from BCECs stimulated with LPS was used as a positive control for iNOS
expression. Stimulation was performed by incubation with 100 ng of LPS/ml for
24 h.
Detection of iNOS enzymatic activity. iNOS enzymatic activity was measured
by production of
L-[
3
H]citrulline from L-[
3
H]arginine (14) with the use of the
NOSdetect kit (Stratagene, La Jolla, Calif.) according to the manufacturer’s
specifications. The reaction was performed in the presence of EGTA (1 mM) to
determine the calcium-independent activity of the inducible enzyme. Boiled
samples were used to determine the background level.
Inoculation of PVC-211 MuLV to primary BCECs in vitro. One day before
inoculation (day 0), primary rat BCECs (passage 2) were seeded at 10
4
per well
into 24-well tissue culture plates (Costar, Cambridge, Mass.). The following day
(day 1), PVC-211 MuLV (multiplicity of infection ⫽ 10) was incubated with cells
in the presence of Polybrene (5 g/ml) for1hat37°C. Then the cells were
washed with medium, and fresh culture medium was added. This inoculation step
was performed once a day and completed by day 4. At day 14, cell extracts were
prepared and analyzed for expression of viral protein and iNOS by Western
blotting as described above.
RESULTS
Specific expression of iNOS in brains and BCECs from
PVC-211 MuLV-infected rat brain. To evaluate the molecular
events in PVC-211 MuLV-infected brains that may be contrib-
uting to neuropathogenicity, we examined brains from virus-
infected rats for expression of iNOS. As a control, we used
brains from rats infected with PVF-e5 MuLV, a chimeric virus
between F-MuLV and PVC-211 MuLV that efficiently infects
BCECs but fails to induce neurological disease (29, 43). We
first used immunohistochemistry to examine brain sections for
expression of iNOS. As shown in Fig. 1, iNOS could be de-
tected only in brains from PVC-211 MuLV-infected rats, and
the protein was expressed predominantly in BCECs (Fig. 1A).
Although only one time point is shown (14 days postinfection),
iNOS immunoreactivity was also observed at 21 and 28 days
after PVC-211 MuLV inoculation (data not shown) and was
not restricted to specific brain regions. Roughly 20 to 30% of
all BCECs in microvessels exhibited iNOS immunoreactivity,
compared with 90 to 100% of all BCECs in microvessels that
express the viral SU gp70 glycoprotein (Fig. 1G). Other cell
populations known to be activated following PVC-211 MuLV
infection, either microglia (43) or astrocytes (19), failed to
express iNOS immunoreactivity. In contrast to brains from
PVC-211 MuLV-infected rats, iNOS immunoreactivity could
not be detected in brains from rats infected with the nonneu-
5146 JINNO-OUE ET AL. J. VIROL.
ropathogenic PVF-e5 MuLV at the day 14 time point shown
(Fig. 1B) or at other time points examined (21 days and 16
weeks postinfection [data not shown]), despite comparable
levels of expression of viral SU gp70 (compare Fig. 1G with H).
As expected, brain sections from medium-inoculated rats ex-
hibited essentially no iNOS immunoreactivity at any time point
examined (Fig. 1C, 14 days postinfection). Neither PVC-211
MuLV (Fig. 1D) nor PVF-e5 MuLV (Fig. 1E) infection af-
fected the expression of eNOS compared to that observed in
brains from medium-inoculated rats (Fig. 1F) at the day 14
postinfection time point shown or at other time points exam-
ined (21 days and 16 weeks postinfection [data not shown]).
To further examine the expression of iNOS in virus-infected
BCECs, we isolated primary BCECs from virus-infected rat
brains and propagated them in vitro for 2 weeks, which is the
minimum time required to obtain sufficient cell numbers for
subsequent analysis. As a positive control, we used BCECs
stimulated with LPS, a known inducer of iNOS. When RT-
PCR analysis using specific primers was carried out on BCEC
RNA from rats inoculated with medium, PVC-211 MuLV, or
PVF-e5 MuLV, iNOS transcripts could be detected in BCECs
stimulated with LPS as well as in BCECs from PVC-211
MuLV-infected rats, but not in BCECs from rats injected with
PVF-e5 MuLV or medium (Fig. 2A). eNOS was expressed at
equivalent levels in all samples (Fig. 2A). Consistent with this
result, a 130-kDa iNOS protein was specifically detected in
only LPS-stimulated BCECs or BCECs from PVC-211 MuLV-
infected rats (Fig. 2B). The successful infection of BCECs with
both PVC-211 MuLV and PVF-e5 MuLV was demonstrated
by the expression of viral envelope SU proteins (gPr85 and
gp70) (Fig. 2B). All samples expressed low levels of eNOS
protein (data not shown).
Detection of iNOS enzymatic activity in PVC-211 MuLV-
infected BCECs. iNOS catalytic activity was also directly mea-
sured in extracts of BCECs from either virus-infected or me-
dium-inoculated rats. iNOS enzymatic activity was measured
by the conversion of
L-arginine to NO and L-citrulline. Since it
has been reported that the expression of ecotropic MuLV
envelope proteins can affect the intracellular arginine concen-
tration due to down-modulation of the cationic amino acid
transporter CAT-1 (42), iNOS enzymatic activity was deter-
mined by measuring the conversion of radioactive
L-arginine
into
L-citrulline in the presence of EGTA to inhibit Ca
2⫹
-
dependent eNOS activity. As a positive control, we used a
murine macrophage cell line, RAW264.7, which showed a sev-
enfold increase in iNOS activity after LPS stimulation (data
FIG. 1. Expression of iNOS in PVC-211 MuLV-infected brains. Brain sections (cerebellum) from rats inoculated 14 days previously with
PVC-211 MuLV (A, D, and G), PVF-e5 MuLV (B, E, and H) or medium (C, F, and I) were fixed and stained for iNOS (A to C), eNOS (D to
F), or viral envelope proteins (G to I) using the ABC peroxidase technique, with diaminobenzidine as substrate. Panels A, B, C, G, H, and I show
40-m-thick frozen fixed sections. Panels D, E, and F show 8-m-thick paraffin sections. Final magnification for all panels is ⫻25.
V
OL. 77, 2003 iNOS LEVELS AND Tyr NITRATION IN MuLV-INFECTED BCECs 5147
not shown). As shown in Fig. 3, the iNOS activities in extracts
of BCECs from both medium- and PVF-e5 MuLV-inoculated
rats were almost undetectable. In contrast, a marked increase
in iNOS activity (⬎20-fold) was detected in extracts of BCECs
from PVC-211-infected rats, in agreement with the results ob-
tained for the specific expression of iNOS mRNA and protein
(Fig. 1 and 2). This difference is significant as determined by
Student’s t test (P ⬍ 0.00001).
Detection of tyrosine-nitrated protein in PVC-211 MuLV-
infected BCECs. NTyr is a biomarker for the presence of NO.
Since elevated immunoreactivity to NTyr was recently reported
in brain sections from PVC-211 MuLV-infected rats (43), we
tested more precisely for the presence of tyrosine-nitrated pro-
teins in extracts of BCECs from PVC-211 MuLV-infected rats.
The specificity of the anti-NTyr antibody was confirmed by the
recognition of tyrosine-nitrated bovine serum albumin (NTyr-
BSA) by Western blot analysis (Fig. 4A). This antiserum de-
tected multiple bands with a wide range of molecular masses,
mostly between 20 and 94 kDa, in all BCEC samples (Fig. 4B,
lanes 1 to 3). Interestingly, a more intense immunoreactive
band with a molecular mass of 32 kDa was found in PVC-211
MuLV-infected BCECs (lane 2). This 32-kDa protein appears
to be a cellular protein, not a viral protein, because it migrates
slower than MuLV p30
gag
(lane 4) and, unlike MuLV p30, it is
not present in a 65-kDa precursor (p65
gag
). Specific binding of
anti-NTyr was confirmed by blocking experiments using excess
NTyr (lanes 5 to 7).
Infection of BCECs by PVC-211 MuLV in vitro fails to
induce iNOS expression. We next examined whether infection
of primary BCECs by PVC-211 MuLV in vitro induces iNOS.
In vivo, BCECs are repeatedly infected by circulating virus in
the blood. To create a similar situation in vitro, BCECs were
seeded on day 0 and inoculated with PVC-211 MuLV one time
(on day 1), two times (on days 1 and 2), or four times (on days
1, 2, 3, and 4) (Fig. 5). Compared to a single inoculation,
inoculation of BCECs on two or four separate occasions sig-
FIG. 2. Specific expression of iNOS in PVC-211 MuLV-infected BCECs. (A) RNA was isolated from BCECs cultured from the brains of rats
inoculated with medium, PVC-211 MuLV, or PVF-e5 MuLV. RT-PCR was then carried out using specific primers to detect iNOS, eNOS, and
-actin. RNA from LPS-stimulated BCECs was used as a positive control for iNOS. (B) Protein extracts (20 g) of BCECs cultured from the brains
of rats inoculated with medium, PVC-211 MuLV, or PVF-e5 MuLV were separated by SDS–8% PAGE, and Western blot analysis was performed
using anti-iNOS antibody. The anti-iNOS antibody was stripped from the membrane, and it was reprobed with goat anti-MuLV envelope SU
(gp70) antibody. Viral envelope proteins, gp70 and its noncleaved precursor gPr85, were detected. The same membrane was reprobed with
anti--tubulin antibody as an internal control. LPS-stimulated BCECs were analyzed separately as a positive control for iNOS.
FIG. 3. Measurement of enzymatic iNOS activity. Cell extracts of
BCECs from rats inoculated with medium or virus were tested for
iNOS enzymatic activity as described in Materials and Methods. All
measurements were determined in triplicate, and the data are shown
as means ⫾ standard deviations. iNOS activity detected in BCECs
from rats inoculated with PVC-211 MuLV is significantly higher (P ⬍
0.00001 by Student’s t test) than that in BCECs from rats inoculated
with medium or PVF-e5 MuLV.
5148 JINNO-OUE ET AL. J. VIROL.
nificantly improved the efficiency of virus infection (compare
lane 4 to lanes 5 and 6). Almost equivalent levels of viral
envelope proteins were detected between BCECs infected with
PVC-211 MuLV in vivo and in vitro (compare lane 2 to lanes
5 and 6). The expression of iNOS protein, however, was not
detected in BCECs infected with virus in vitro (lanes 4 to 6).
This result suggests that an additional factor(s) induced in vivo
after PVC-211 MuLV infection might be necessary to induce
iNOS in BCECs.
DISCUSSION
Our previous studies have shown that neurodegeneration
induced by PVC-211 MuLV is indirect and occurs after the
infection of BCECs by the virus. Since excessive production of
NO can lead to neurotoxicity, we carried out studies to deter-
mine if infection of BCECs results in activation of iNOS or
eNOS, which produce NO from
L-arginine. Not only could we
detect iNOS immunoreactivity localized to capillaries in brain
sections from PVC-211 MuLV-infected rats, but also BCECs
isolated from these brains expressed elevated levels of iNOS
RNA, protein, and enzymatic activity compared to levels in
BCECs from medium-inoculated rats or from rats infected
with the nonneuropathogenic PVF-e5 MuLV. This is in con-
trast to eNOS, which continues to be expressed at a low,
constitutive level after PVC-211 MuLV infection. Since NTyr
is a biomarker for the presence of NO, we also examined
infected BCECs for proteins modified by tyrosine nitration and
detected elevated amounts of a 32-kDa tyrosine-nitrated cel-
lular protein in BCECs from PVC-211 MuLV-infected rats.
Thus, our data suggest that PVC-211 MuLV may be causing
neuronal death indirectly by infecting BCECs and triggering,
through iNOS activation, the production of large amounts of
NO.
The precise mechanism by which PVC-211 MuLV induces
the expression of iNOS in BCECs remains to be elucidated.
One possibility is that a viral protein expressed within BCECs
is initiating a cascade of events culminating in activation of
iNOS. However, we failed to detect elevated iNOS protein in
BCECs after in vitro infection with PVC-211 MuLV, suggest-
ing that expression of a viral protein(s) in these cells is not
sufficient to activate iNOS. Recently, Wilt et al. (43) reported
the appearance of activated microglia adjacent to PVC-211
MuLV-infected BCECs before neuronal damage. Activation
of microglia appears to be important for disease induction,
since activated microglia could not be detected in brains from
rats infected with the nonneuropathogenic PVF-e5 MuLV
(43). Activated microglia release a variety of inflammatory
molecules which are known mediators of iNOS induction (16).
FIG. 4. Expression of tyrosine-nitrated proteins in BCECs from PVC-211 MuLV-infected rats. (A) BSA (0.1 g) (lane 1) or tyrosine-nitrated
BSA (0.1 g) (lane 2) was separated by SDS–4-to-20% PAGE, and Western blot analysis using anti-NTyr antibody was performed. The arrow
represents the migration of BSA. (B) Protein extracts (20 g) of cultured BCECs from medium- (lanes 1 and 5), PVC-211 MuLV- (lanes 2 and
6), or PVF-e5 MuLV- (lanes 3 and 7) inoculated rats were separated by SDS–4-to-20% PAGE, and Western blot analysis was performed using
anti-NTyr antibody. The asterisk represents a 32-kDa protein. The anti-NTyr antibody was stripped from the membrane, and it was reprobed with
goat anti-MuLV gag capsid (p30
gag
) antibody. Gag proteins, p30
gag
and its noncleaved precursor p65
gag
, were detected (lane 4). The right panel
is the result of Western blot analysis performed using anti-NTyr antibody pretreated with 10 mM NTyr. The same membrane was reprobed with
anti--tubulin antibody as an internal control.
V
OL. 77, 2003 iNOS LEVELS AND Tyr NITRATION IN MuLV-INFECTED BCECs 5149
If such cytokines or chemokines are released from the acti-
vated microglia in the brains of PVC-211 MuLV-infected rats,
they may bind to receptors on BCECs and induce the expres-
sion of iNOS. It is unclear how microglia become activated in
the brains of PVC-211 MuLV-infected rats. Virus cannot be
detected in the microglia (19), but the virus or a viral protein
could be interacting with a transmembrane receptor on these
cells that results in their activation. An attractive candidate is
TLR4, the transmembrane component of the receptor complex
mediating the cellular response to LPS (3). It was recently
reported that the envelope protein of Moloney MuLV, a ret-
rovirus highly related to PVC-211 MuLV, can bind to TLR4
(36) and that TLR4 is expressed on rat microglia (25). Thus,
activation of microglia in the brains of PVC-211 MuLV-in-
fected rats could be due to interaction of the viral envelope
protein with TLR4 on these cells. BCECs may also express
TLR4 that can be activated by the viral envelope protein. This
could lead to direct activation of iNOS in the BCEC, since one
of the signals activated by TLR4 binding is NF-B, a transcrip-
tional activator of iNOS (45). The failure of PVF-e5 MuLV to
cause neurological disease could be due to the inability of its
envelope glycoprotein, which differs from that of PVC-211
MuLV, to interact with TLR4 on microglia or BCECs. Exper-
iments to test these hypotheses are in progress. Interestingly,
C3H/HeJ mice, which do not express TLR4 due to a missense
mutation in the gene (34), are resistant to PVC-211 MuLV-
induced neurological disease (19), suggesting that TLR4 may
indeed play a role in PVC-211 MuLV disease induction.
The exact pathways by which excessive NO production by
PVC-211 MuLV-infected BCECs causes neuronal death are
not known. Reactive nitrogen species potentially generated
from NO by multiple pathways (8, 11, 15, 27) can modify
proteins by nitration of tyrosine residues (1, 2). Tyrosine ni-
tration may alter a protein’s conformation, structure, catalytic
activity, and/or susceptibility to protease digestion (23, 38, 46)
and has been shown to disrupt protein tyrosine kinase-related
signal transduction (10, 18, 22). In this study, we showed in-
creased anti-NTyr immunoreactivity of a 32-kDa cellular pro-
tein in PVC-211 MuLV-infected BCECs. It is possible that the
modification of this protein by nitration might alter its physi-
ological properties and affect the function of the BCECs. Since
BCECs are a component of the blood-brain barrier and play an
important role in maintaining the ion homeostasis of the CNS
(33), disruption of their function by NO could indirectly lead to
the death of neurons. Alternatively, NO released from PVC-
211 MuLV-infected BCECs may move to the site of neurons
where it generates reactive nitrogen species that directly dam-
age the neurons. Although neurons are spatially separated
from BCECs, models of potential diffusion of NO indicate that
it can diffuse as far as 300 m from its site of origin, which
could include as many as 2 million synapses (44).
In addition to affecting BCECs and neurons, NO produced
by PVC-211 MuLV-infected BCECs may also have other ef-
fects in the brain. NO generated by iNOS in human microvas-
cular endothelial cells has been shown to inhibit the rolling and
adhesion of leukocytes (4), and NO has been shown to inhibit
proliferation of T cells that invade the CNS (20). Since PVC-
211 MuLV-induced neurodegeneration is not associated with
either inflammation or infiltration of leukocytes (19, 21), NO
produced in PVC-211 MuLV-infected BCECs may be inhibit-
ing adhesion and infiltration of leukocytes into the brain pa-
renchyma or proliferation of T cells that do infiltrate. In addi-
tion, NO produced in PVC-211 MuLV-infected BCECs may
also inhibit virus replication. Data from a number of labora-
tories using both RNA and DNA viruses suggest that NO may
inhibit an early stage in viral replication (37). The virus titer in
brains recovered from rats infected with PVC-211 MuLV is
always significantly lower than that in the spleen (19, 21, 43). In
addition, microglia or neurons adjacent to the infected BCECs
show no evidence of PVC-211 MuLV infection despite the
release of virions into the basement membrane of infected
BCECs. Thus, it is possible that NO released from PVC-211
MuLV-infected BCECs inhibits replication and spread of the
virus in the brain.
In summary, our results show that infection of BCECs with
PVC-211 MuLV in vivo induces functionally active iNOS pro-
tein and elevates tyrosine nitration of a 32-kDa cellular pro-
tein. Our failure to detect iNOS expression in BCECs after in
vivo infection with the nonneuropathogenic but BCEC-tropic
PVF-e5 MuLV suggests that iNOS activation plays a crucial
role in the development of neurological disease induced by
PVC-211 MuLV. The use of iNOS inhibitors and the genera-
FIG. 5. Infection of BCECs by PVC-211 MuLV in vitro fails to
induce iNOS. Primary BCECs (twice passaged) were inoculated in
vitro with PVC-211 MuLV either one time, two times, or four times,
24 h apart. Fourteen days after the initial inoculation, cell extracts were
prepared and compared with extracts of BCECs obtained from rats
inoculated with PVC-211 MuLV in vivo. Lane 1, BCECs from rats
inoculated in vivo with medium; lane 2, BCECs from rats inoculated in
vivo with PVC-211 MuLV; lane 3, mock-infected BCECs; lane 4,
BCECs infected in vitro with PVC-211 MuLV one time; lane 5, BCECs
infected in vitro with PVC-211 MuLV two times; lane 6, BCECs
infected in vitro with PVC-211 MuLV four times. Extracts (10 g)
were separated by SDS–4-to-20% PAGE, and Western blot analysis
was performed using goat anti-MuLV envelope SU (gp70) antibody
(upper panel). The anti-MuLV gp70 antibody was stripped from the
membrane and reprobed with anti-iNOS antibody (middle panel). The
same membrane was reprobed with anti--tubulin antibody as an in-
ternal control (bottom panel).
5150 JINNO-OUE ET AL. J. V
IROL.
tion of iNOS-deficient rats should help to clarify how iNOS is
involved in the pathological process.
ACKNOWLEDGMENTS
We thank Takashi Yugawa, Karen Rulli, and Joan Cmarik for help-
ful advice.
REFERENCES
1. Beckman, J. S., T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman.
1990. Apparent hydroxyl radical production by peroxynitrite: implications for
endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci.
USA 87:1620–1624.
2. Beckman, J. S. 1996. Oxidative damage and tyrosine nitration from peroxyni-
trite. Chem. Res. Toxicol. 9:836–844.
3. Beutler, B. 2000. TLR4: central component of the sole mammalian LPS
sensor. Curr. Opin. Immunol. 12:20–26.
4. Binion, D. G., S. Fu, K. S. Ramanujam, Y. C. Chai, R. A. Dweik, J. A. Drazba,
J. G. Wade, N. P. Ziats, S. C. Erzurum, and K. T. Wilson. 1998. iNOS
expression in human intestinal microvascular endothelial cells inhibits leu-
kocyte adhesion. Am. J. Physiol. 275:G592–G603.
5. Boven, L. A., L. Gomes, C. Herry, F. Gray, J. Verhof, P. Portegies, M.
Tardieu, and H. S. Nottet. 1999. Increased peroxynitrite activity in AIDS
dementia complex: implications for the neuropathogenesis of HIV-1 infec-
tion. J. Immunol. 162:4319–4327.
6. Bowman, P. D., A. L. Betz, J. S. Wolinsky, J. B. Penny, R. R. Shivers, and
G. W. Goldstein. 1981. Primary culture of capillary endothelium from rat
brain. In Vitro 17:353–362.
7. Bredit, D. S., and S. H. Synder. 1994. Nitric oxide: a physiologic messenger
molecule. Annu. Rev. Biochem. 63:175–195.
8. Brennan, M.-L., W. Wu, X. Fu, Z. Shen, W. Song, H. Frost, C. Vadseth, L.
Narine, E. Lenkiewicz, M. T. Borchers, A. J. Lusis, and S. L. Hazen. 2002. A
tale of two controversies: defining both the role of peroxidases in nitroty-
rosine formation in vivo using eosinophil peroxidase and myeloperoxidase-
deficient mice, and the nature of peroxidase-generating reactive nitrogen
species. J. Biol. Chem. 277:17415–17427.
9. Dawson, T. M., and V. L. Dawson. 1998. Nitric oxide in neurodegeneration.
Prog. Brain Res. 118:215–229.
10. Di Stasi, A. M., C. Mallozzi, G. Macchia, T. C. Petrucci, and M. Minetti.
1999. Peroxynitrite induces tyrosine nitration and modulates tyrosine phos-
phorylation of synaptic proteins. J. Neurochem. 73:727–735.
11. Eiserich, J. P., C. E. Cross, A. D. Jones, B. Halliwell, and A. van der Vliet.
1996. Formation of nitrating and chlorinating species by reaction of nitrite
with hypochlorous acid. J. Biol. Chem. 271:19199–19208.
12. Ferrante, R. J., P. Hantray, E. Brouillet, and M. F. Beal. 1999. Increased
nitrotyrosine immunoreactivity in substantia nigra neurons in MPTP treated
baboons is blocked by inhibition of neuronal nitric oxide synthase. Brain Res.
823:177–182.
13. Forstemann, U., E. I. Closs, J. S. Pollock, M. Nakane, P. Schwarz, I. Gath,
and H. Kleinert. 1994. Nitric oxide synthase isozymes. Characterization,
purification, molecular cloning, and functions. Hypertension 23:1121–1131.
14. Galea, E., D. L. Feinstein, and D. L. Reis. 1992. Induction of calcium-
independent nitric oxide synthase activity in primary rat glial cultures. Proc.
Natl. Acad. Sci. USA 89:10945–10949.
15. Gaut, J. P., J. Byun, H. D. Tran, W. M. Lauber, J. A. Carroll, R. S. Hotchkiss,
A. Belaaouaj, and J. W. Heinecke. 2002. Myeloperoxidase produces nitrating
oxidants in vivo. J. Clin. Investig. 109:1311–1319.
16. Gehrmann, J., Y. Matsumoto, and G. W. Kreutzberg. 1995. Microglia: in-
trinsic immuno-effector cell of brain. Brain Res. Rev. 20:269–287.
17. Good, P. F., P. Werner, A. Hsu, C. W. Olanow, and D. P. Perl. 1996. Evidence
for neuronal oxidative damage in Alzheimer’s disease. Am. J. Pathol. 49:21–
27.
18. Hevel, J. M., K. A. White, and M. A. Marletta. 1991. Purification of the
inducible murine macrophage nitric oxide synthase. Identification as a fla-
voprotein. J. Biol. Chem. 266:22789–22791.
19. Hoffman, P. M., E. H. Cimino, D. S. Robbins, R. D. Broadwell, J. M. Powers,
and S. K. Ruscetti. 1992. Cellular tropism and localization in the rodent
nervous system of a neuropathogenic variant of Friend murine leukemia
virus. Lab. Investig. 67:314–321.
20. Juedes, A. E., and N. H. Ruddle. 2001. Resident and infiltrating central
nervous system APCs regulate the emergence and resolution of experimental
autoimmune encephalomyelitis. J. Immunol. 166:5168–5175.
21. Kai, K., and T. Furuta. 1984. Isolation of paralysis-inducing murine leuke-
mia viruses from Friend virus passaged in rats. J. Virol. 50:970–973.
22. Kong, S. K., M. B. Yim, E. R. Stadtman, and P. B. Chock. 1996. Peroxynitrite
disables the tyrosine phosphorylation regulatory mechanism: lymphocyte-
specific tyrosine kinase fails to phosphorylate nitrated cdc2(6–20)NH2 pep-
tide. Proc. Natl. Acad. Sci. USA 93:3377–3382.
23. Kuo, W. N., J. M. Kreahling, V. P. Shanbhag, P. P. Shanbhag, and M.
Mewar. 2000. Protein nitration. Mol. Cell. Biochem. 214:121–129.
24. Lee, S. C., M. L. Zhao, A. Hirano, and D. W. Dickson. 1999. Inducible nitric
oxide synthase immunoreactivity in the Alzheimer disease hippocampus:
association with Hirano bodies, neurofibrillary tangles, and senile plaques.
J. Neuropathol. Exp. Neurol. 58:1163–1169.
25. Lehnardt, S., C. Lachance, S. Patrizi, S. Lefebvre, P. L. O. Follett, F. E.
Jensen, P. A. Rosenberg, J. J. Volpe, and T. Vartanian. 2002. The toll-like
receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte
injury in the CNS. J. Neurosci. 22:2478–2486.
26. Liberatore, G. T., V. Jackson-Lewies, S. Vukosavic, M. Vila, W. G. McAuliffe,
T. M. Dawson, and S. Przedorski. 1999. Inducible nitric oxide synthase
stimulates dopaminergic neurodegeneration in the MPTP model of Parkin-
son disease. Nat. Med. 5:1403–1409.
27. Lipton, S. A. 1999. Neuronal protection and destruction by NO. Cell Death
Differ. 6:943–951.
28. Masuda, M., M. P. Remington, P. M. Hoffman, and S. K. Ruscetti. 1992.
Molecular characterization of a neuropathogenic and nonerythroleukemic
variant of Friend murine leukemia virus PVC-211. J. Virol. 66:2798–2806.
29. Masuda, M., P. M. Hoffman, and S. K. Ruscetti. 1993. Viral determinants
that control neuropathogenicity of PVC-211 murine leukemia virus in vivo
determine brain capillary endothelial cell tropism of the virus in vitro. J. Vi-
rol. 67:4580–4587.
30. Masuda, M., C. A. Hanson, W. G. Alvord, P. M. Hoffman, S. K. Ruscetti, and
M. Masuda. 1996. Effect of subtle changes in the SU protein of ecotropic
murine leukemia virus on its brain capillary endothelial cell tropism and
interference properties. Virology 215:142–151.
31. Moncada, S., R. M. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol. Rev. 43:109–142.
32. Nathan, C., and Q.-W. Xie. 1994. Nitric oxide synthases: roles, tolls, and
controls. Cell 78:915–918.
33. Pardridge, W. M. 1999. Blood-brain barrier biology and methodology.
J. Neurovirol. 5:556–569.
34. Poltorak, A., X. He, I. Smirnova, M.-Y. Liu, C. Van Huffel, X. Du, D.
Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freudenberg, P. Ricciardi-
Castagnoli, B. Layton, and B. Beutler. 1998. Defective LPS signaling in
C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:
2085–2088.
35. Power, C. 2001. Retroviral diseases of the nervous system: pathogenic host
response or viral gene-mediated neurovirulence? Trends Neurosci. 24:162–
169.
36. Rassa, J. C., J. L. Meyers, Y. Zhang, R. Kudaravalli, and S. R. Ross. 2002.
Murine retroviruses activate B cells via interaction with toll-like receptor 4.
Proc. Natl. Acad. Sci. USA 99:2281–2286.
37. Reiss, C. S., and T. Komatsu. 1998. Does nitric oxide play a critical role in
viral infections? J. Virol. 72:4547–4551.
38. Roberts, E. S., H. L. Lin, J. R. Crowley, J. L. Vuletich, Y. Osawa, and P. F.
Hollenberg. 1998. Peroxynitrite-mediated nitration of tyrosine and inactiva-
tion of the catalytic activity of cytochrome P450 2B1. Chem. Res. Toxicol.
11:1067–1074.
39. Rosenberg, N., and P. Jolicoeur. 1997. Retroviral pathogenesis, p. 475–586.
In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
40. Rowe, W. P., W. E. Pugh, and J. W. Hartley. 1970. Plaque assay techniques
for murine leukemia viruses. Virology 42:136–138.
41. Sasaki, S., N. Shibata, T. Komori, and M. Iwata. 2000. iNOS and nitroty-
rosine immunoreactivity in amyotrophic lateral sclerosis. Neurosci. Lett.
291:44–48.
42. Wang, H., E. Dechant, M. Kavanaugh, R. A. North, and D. Kabat. 1982.
Effects of ecotropic murine retroviruses on the dual-function cell surface
receptor/basic amino acid transporter. J. Biol. Chem. 267:23617–23624.
43. Wilt, S. G., N. V. Dugger, N. D. Hitt, and P. M. Hoffman. 2000. Evidence for
oxidative damage in a murine leukemia virus-induced neurodegeneration.
J. Neurosci. Res. 62:440–450.
44. Wood, J., and J. Garthwaite. 1994. Models of the diffusional spread of nitric
oxide: implications for neural nitric oxide signalling and its pharmacological
properties. Neuropharmacology 33:1235–1244.
45. Xie, Q. W., Y. Kashiwabara, and C. Nathan. 1994. Role of transcription
factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem.
269:4705–4708.
46. Yamakura, F., H. Taka, T. Fujimura, and K. Murayama. 1998. Inactivation
of human manganese-superoxide dismutase by peroxynitrite is caused by
exclusive nitration of tyrosine 34 to 3-nitrotyrosine. J. Biol. Chem. 273:
14085–14089.
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