From the Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Département de Biologie médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec G1V 4G2, Canada
Received for publication, March 16, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Previous studies have shown that
human immunodeficiency virus type-1 (HIV-1) can incorporate several
surface proteins of host origin. Recent findings indicate that
host-encoded cell surface constituents retain their functionality when
found embedded into the viral envelope. The primary objective of the
current study was to define whether interaction between some specific
virion-bound host proteins with their natural cognate ligands present
on target cells could mediate intracellular signaling cascade(s). For
this purpose, we have generated a whole series of isogenic virus stocks (NL4-3 backbone) bearing or not bearing on their surface foreign CD28,
CD54 (ICAM-1), CD80 (B7-1) or CD86 (B7-2) proteins. Our results
indicate that incubation of human T lymphoid cells with virions bearing
host-derived B7-2 proteins and anti-CD3 antibody can potently activate
HIV-1 long terminal repeat-driven gene expression. This up-regulating
effect necessitates the involvement of nuclear factor- The attachment of HIV-11
to target cells is occurring via high affinity binding between the
external viral envelope gp120 and the cell surface CD4 glycoprotein.
Recently, several studies have identified chemokine receptors as major
fusion cofactors for T cell- and macrophage-tropic HIV-1 isolates
(reviewed in Ref. 1). It is known that a threshold number of
interactions between viral and cellular surface molecules is necessary
to achieve an efficient viral infection because the surfaces of both
CD4+ T cells and HIV-1 are highly negatively charged (2). McKeating and
co-workers (3) have reported that the observed spontaneous shedding of gp120 in vitro was linked to a loss of HIV-1 infectivity.
The shedding of gp120 in vitro is also taking place in
vivo (4). Thus, one may assume that the infection process will be
greatly jeopardized if the number of gp120 molecules is too low to
provide the threshold binding energy required to overcome the repulsive electrostatic forces between cellular and viral membranes (2). Changes
in infectivity have dramatic consequences in viral output. For
instance, a reduction in infectivity of 50% will diminish the viral
production to 0.0032% of the expected output after only five
replicative cycles (5). The binding of virus-associated gp120 to
cellular CD4 is often weak, and most cell types that are permissive for
HIV-1 infection express low levels of CD4. Thus, other interactions
between the viral entity and the host cell surface could play a
dominant role in the attachment process. Others and we have postulated
that host-derived proteins present on the viral surface could influence
the initial interaction between the virion and its target (6-8).
The incorporation of cellular constituents in newly formed viruses has
been demonstrated to occur in retroviruses (9-13). Similar studies
were extended to HIV-1, and a vast array of cell membrane proteins was
found to be acquired by this retrovirus such as the HLA-DR, -DP, and
-DQ determinants of major histocompatibility complex class-II (MHC-II),
ICAM-1, LFA-1, Although it is clear now that virion-anchored foreign proteins play an
essential role in the attachment process of HIV-1 to its target, there
is no information available yet with respect to putative signaling
events that could be mediated by interaction between HIV-1-bound host
proteins and their physiologic counter-receptors located on the cell
surface. This is somewhat surprising, based on previous observations
suggesting that intracellular signal transduction events can be
mediated upon the virus attachment process. Indeed, interaction between
the HIV-1 envelope and CD4/chemokine coreceptors has been shown to
mediate several intracellular signaling events, including
phosphorylation of phosphatidylinositol 3-kinase (PI 3-kinase),
tyrosine phosphorylation of Pyk2, focal adhesion kinase and CCR5,
activation of PI 4-kinase, Raf-1, and several mitogen-activated protein
kinase pathways (e.g. mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase, c-Jun N-terminal kinase, p38) (17-28). Thus, it can be proposed that binding
of some virally incorporated host proteins with their normal
counter-receptors can also lead to signal transduction into target cells.
Thus, the primary goal of this study was to define whether different
intracellular biochemical events can be initiated depending of the
foreign host cell surface constituent that is found embedded within the
HIV-1 envelope. Our experiments were performed using isogenic virions
bearing or not bearing on their surface host-derived B7-1 (CD80), B7-2
(CD86), CD28, and ICAM-1 (CD54) proteins. We demonstrate that the
nature of virion-anchored host protein is indeed of utmost importance
with respect to HIV-1-mediated signal transduction pathway that is seen
upon virus-cell attachment. Furthermore, we provide evidence that such
signaling events can positively modulate HIV-1 LTR-driven gene
expression via activation of nuclear factor- Cell Lines and Culture Conditions--
The 1G5 T cell line, a
Jurkat E6.1 derivative that harbors two stably integrated constructs
constituted of the luciferase gene under the control of the
HIV-1SF2 LTR, was obtained from Dr. Estuardo
Aguilar-Cordova and Dr. John Belmont through the AIDS Research and
Reference Reagent Program (Division of AIDS, NIAID, National Institutes
of Health, Bethesda, MD) (29). The human T lymphoid cell line Jurkat
clone E6.1 was used in this study because it is considered as a model
cell line for the study of T cell signaling machinery (30). We have
also used DT30, which are murine Fc receptor-bearing mastocytoma P815
cells stably expressing cell surface human B7-1 (CD80) proteins (31).
Both cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in RPMI 1640 medium supplemented with
10% heat-inactivated fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/ml penicillin G, 100 µg/ml
streptomycin, 0.22% NaHCO3, and were maintained at
37 °C under a 5% CO2 humidified atmosphere. DT30 cells
were maintained under the pressure of 1 mg/ml selective agent G418
(Life Technologies, Inc.). DT30 cells were fixed briefly in 1%
paraformaldehyde, washed extensively with phosphate-buffered saline
(PBS), and then stored frozen in aliquots at a density of 2 × 106/ml of PBS. Since such cells are fixed, they do not grow
or secrete factors that could mediate signal transduction in studied
target cells. For our experiments aimed at stimulating Jurkat cells, 2 × 104 DT30 was added to 105 Jurkat cells.
Plasmids--
We have used pLTR-Luc (HIV-1 LTR from strain HXB2)
and pm Antibodies and Purified Proteins--
Anti-CD3 hybridoma OKT3
(specific for the Production of Virus Stocks--
Isogenic virus preparations
bearing or not bearing some specific host-derived proteins were
produced by calcium phosphate (CaPO4) transfection of 293T
cells as we described previously (6, 42-44). In brief, 293T cells were
transfected with pNL4-3 alone (virus stock called NL4-3/Null) or were
cotransfected with either pCDL-SR DEAE-dextran Transfections--
Transient transfections were
done using the DEAE-dextran method. In brief, cells (5 × 106) were first washed once in TS buffer (25 mM
Tris-HCl (pH 7.4), 5 mM KCl, 0.6 mM
Na2HPO4, 0.5 mM MgCl2,
and 0.7 mM CaCl2) and resuspended in 0.5 ml of
TS containing 15-30 µg of used plasmid(s) and 500 µg/ml of
DEAE-dextran (final concentration). The cells/TS/plasmid/DEAE-dextran mix was incubated for 25 min at room temperature. Thereafter, cells
were diluted at a concentration of 1 × 106/ml using
complete culture medium supplemented with 100 µM of chloroquine (Sigma) and transferred into six-well plates. After 45 min
of incubation at 37 °C, cells were centrifuged, resuspended in
complete culture medium, and incubated at 37 °C for 24 h. To minimize variations in plasmid transfection efficiencies, cells were
transfected in bulk and were next separated into various treatment groups.
Detection of Virion-bound Host B7-2 Proteins by a Virus Capture
assay--
The presence of virion-bound host B7-2 proteins was
semiquantitatively estimated using a modified version of a previously described virus capture assay (45). Briefly, 12.5 × 106 magnetic beads (BioMag, Fc-specific; PerSeptive
Diagnostics, Inc., Cambridge, MA), previously coated with the anti-B7-2
antibody (BU-63), were incubated with similar amounts of studied virus preparations standardized in terms of the viral core p24 protein (2500 pg of p24) in a final volume of 1 ml of binding medium (PBS + 0.1%
bovine serum albumin). This mixture was incubated for 1 h at
4 °C on a rotating plate. The beads were washed three times in
binding medium with a magnetic separation unit and were resuspended in
100 µl of binding medium. The amount of immunocaptured HIV-1 particles was assessed by measuring viral p24 protein content found
associated with the immunomagnetic beads by a commercial p24 enzymatic
assay. Magnetic beads coated with an isotype-matched antibody
(i.e. IgG2a) specific human CD45RO (clone
UCHL-1) were used as a negative control because CD45 has been shown to
be excluded from HIV-1 envelope (47).
Stimulations and Reporter Gene Assays--
Transiently
transfected Jurkat E6.1 cells were seeded at a density of 2 × 105 cells/well (100 µl) in 96-well flat-bottom plates.
Cells were next incubated for 1 h on ice with virions bearing or
not bearing host B7-1 (NL4-3/B7-1), B7-2 (NL4-3/B7-2), CD28
(NL4-3/CD28), or ICAM-1 (NL4-3/ICAM-1) (10-200 ng of p24). Cells
inoculated with HIV-1 particles were next either left untreated or were
treated with the anti-CD3 antibody (clone OKT3) at 3 µg/ml. As
controls, uninfected cells were either left unstimulated or were
treated in a final volume of 200 µl with the following stimuli:
anti-CD3 antibody (clone OKT3 at 3 µg/ml), anti-CD28 antibody (clone
9.3 at 1 µg/ml), or the combination of anti-CD3 antibody and DT30 cells (2 × 104 DT30/105 transfected
Jurkat cells). Finally, cells were incubated at 37 °C for 7 h
unless otherwise specified. For some experiments, before the addition
of activators, virions were either left untreated or were pretreated
with CTLA-4 Ig or by freeze-thaw cycles. Following the incubation
period, 100 µl of cell-free supernatant were drawn from each well and
25 µl of cell culture lysis buffer (25 mM Tris phosphate
(pH 7.8), 2 mM dithiothreitol, 1% Triton X-100, and 10%
glycerol, final concentrations) were added before incubation at room
temperature for 30 min. The extracts (20 µl) were analyzed for
luciferase activity in 96-well plates using a Dynex MLX luminometer. Each well was injected with 100 µl of luciferase assay buffer (20 mM Tricine, 1.07 mM
(MgCO3)4·Mg(OH)2·5H2O,
2.67 mM MgSO4, 0.1 mM EDTA, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP, and 33.3 mM dithiothreitol). Light
output was measured for 20 s with a 2-s delay. Values are
expressed in terms of relative light units as measured by the
apparatus. Results shown are expressed as -fold inductions relative to
basal luciferase activity in untreated/uninfected control cells.
Virus Infection--
Similar amounts of each recombinant
luciferase-encoding virus stocks (130 ng of p24 for NL4-3/Null and
NL4-3/B7-2 virions) were used to infect to 2 × 105
1G5 cells in a 96-well flat bottom tissue culture plate in a final
volume of 200 µl. After 48 h of infection, cells were lysed and
luciferase activity was monitored using a microplate luminometer (MLX;
Dynex Technologies, Chantilly, VA).
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared according to the microscale preparation protocol. Briefly,
Jurkat E6.1 cells were left untreated, treated with stimuli, or
incubated with virus stocks (150 ng of p24) in the presence of anti-CD3
for 60 min at 37 °C. The incubation of cells with the stimulating
agents and/or viruses was terminated by the addition of ice-cold PBS
and nuclear extracts were then prepared as described previously (48,
49). Six micrograms of nuclear extracts were used to perform
electrophoretic mobility shift assay. Nuclear extracts were incubated
for 30 min at room temperature in 15 µl of binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol, 5 mM
EDTA, 250 mM NaCl, 2 µg of poly(dI-dC), 10 µg of
nuclease-free bovine serum albumin fraction V) containing 1 ng of
Incubation of Cells with B7-2-bearing Virions and Anti-CD3 Antibody
Leads to Induction of HIV-1 LTR-mediated Activity--
In an attempt
to define whether attachment of HIV-1 particles bearing some specific
host-encoded cell surface proteins can lead to signaling events, the
TCR/CD3-, CD4-, CD28-, LFA-1-, and CXCR4-expressing human T lymphoid
cell line Jurkat was incubated with isogenic virions bearing or not
bearing foreign B7-1, B7-2, CD28, and ICAM-1 proteins. Transfection of
such cells with a vector made of the luciferase reporter gene driven by
the regulatory element of HIV-1 (pLTR-Luc) prior to incubation with
virus preparations allowed us to assess the putative up-regulating
effect on virus transcription mediated by the binding step. Our initial
set of experiments was performed by incubating transiently transfected Jurkat cells with similar amounts of isogenic virions standardized in
terms of p24. As expected, HIV-1 LTR transcription was not activated
neither by antibody-mediated cross-linking of cell surface TCR/CD3
complex nor by multivalent occupancy of CD28 by using B7-1-expressing
DT30 cells (Fig. 1A). However,
the occupancy of both TCR/CD3 and CD28 led to a marked activation of
HIV-1 LTR-dependent luciferase expression. These results
are in agreement with previous observations indicating that the
CD28-mediated signal transduction pathway is considered as one of the
dominant costimulatory pathways to achieve the complete activation of
the T cell and induction of HIV-1 transcription/expression (50-52).
The HIV-1 long terminal repeat was not modulated by attachment of
infectious HIV-1 particles not bearing (NL4-3/Null) or bearing host
B7-1 (NL4-3/B7-1), B7-2 (NL4-3/B7-2), CD28 (NL4-3/CD28), and ICAM-1
proteins (NL4-3/ICAM-1).
Given that effective activation of T lymphocytes necessitates the
coupling of antigen-nonspecific signal with antigen-specific interactions of the TCR/CD3 complex, we next performed similar experiments in the presence of soluble anti-CD3 antibodies. Again, the
biochemical signals provided by the ligation of TCR/CD3 complex ( The HIV-1 Enhancer Region Is the Target for LTR Activation by
B7-2-bearing Virions and Anti-CD3 Antibody--
It has been shown that
the HIV-1 enhancer is the main responding region of the LTR and is
primarily responsible for the transcriptional increase observed
following T cell activation (53). The HIV-1 enhancer is composed of two
NF-
To demonstrate that the major positive effect on HIV-1 LTR element by
B7-2-bearing viruses is due to signaling from the cell surface as
opposed to an increased efficiency of virus entry and infection
(e.g. Tat-mediated effect), Jurkat cells transfected with
p The Combined Action of Soluble Anti-CD3 Antibody and Binding of
NL4-3/B7-2 Virions to Host T Cells Results in Activation of NF-
To confirm NF-
Recent findings indicate that NFAT can synergize with NF- Trans-dominant Negative Mutants of I
We next compared the degree of virion-anchored B7-2 proteins in viruses
originating from our transient-and-expression system (i.e.
293T cells) versus virions expanded in a more physiological cellular milieu. The incorporation of foreign B7-2 into the HIV-1 envelope was assessed using a recently developed virus precipitation assay. The validity of this test was first tested by cotransfecting 293T cells with pNL4-3 and increasing concentrations of the vector coding for human B7-2. Flow cytometry analysis revealed that increasing concentrations of B7-2 expression vector resulted in a concomitant enhancement of the expression of B7-2 on the surface of 293T cells (data not shown). Viruses produced by such transiently transfected 293T
cells were next subjected to our virus capture assay. The amount of
virus captured was found to be in linear correlation with the level of
expression of B7-2 on the surface of 293T cells (Fig.
7A). These results suggest
that our virus precipitation assay is not limiting and that the level
of B7-2 expression on the producer cells influences the incorporation
rate of B7-2 in the HIV-1 envelope. The degree of incorporation of
host-encoded B7-2 within virions expanded in a more natural cellular
reservoir was next monitored. For this purpose, HIV-1NL4-3
was grown in human tonsil histocultures, a model system that preserves
and maintains the mixed cell populations found in secondary lymphoid organs, including T cells, B cells, macrophages, and dendritic cells.
This tissue culture system was selected because lymphoid tissue is
known as the major site of HIV-1 replication in vivo. Results from a virus capture assay revealed that anti-B7-2 antibodies (clone BU-63) as efficiently captured HIV-1NL4-3 particles produced by 293T cells as viruses produced by histocultures of human
tonsils from two different healthy donors (Fig. 7B). These data suggest that comparable levels of host-encoded B7-2 proteins are
found embedded on viruses produced either by transiently transfected 293T cells or by human lymphoid tissue following a normal infection with HIV-1.
HIV-1 attachment to host cells has been found to profoundly affect
the immune system. For example, HIV-1 envelope glycoproteins have been
reported to induce secretion of proinflammatory cytokines and mediate
enhancement of apoptosis (reviewed in Ref. 62). The underlying basis
for the observed biological functions of such virus proteins is thought
to involve the interaction of the HIV-1 envelope with the CD4
glycoprotein, the primary cellular receptor for this retrovirus. Upon
the discovery of CCR5 as a major coreceptor for HIV-1, many
laboratories have demonstrated that HIV-1 envelope can transduce
intracellular signals through CCR5 in a manner analogous to that of
Our studies were focused on the functionality of some specific virally
embedded host proteins, namely CD28, ICAM-1, B7-1, and B7-2. These
cells surface constituents were deliberately selected for the following
reasons. First, the primary cellular reservoirs of HIV-1 in infected
persons, i.e. T helper cells and macrophages, express these
surface proteins either in an inducible or constitutive manner. Indeed,
CD28 is a homodimeric cell surface glycoprotein that is largely
restricted to the T cell lineage. Ninety-five percentage of CD4+ T
cells express CD28 and activation of T cells leads to enhanced CD28
expression (75). CD54 (ICAM-1) is widely distributed, and its
expression can be induced by a variety of inflammatory cytokines (76).
Thus, surface expression of ICAM-1 is high on activated T helper cells
and macrophages, two different cell types known to be active producer
of virions in infected individuals. B7-1 and B7-2 are expressed on
activated macrophages and T cells (75). Enhanced levels of B7-1 were
detected upon stimulation in vitro of T cells from
HIV-1-infected individuals and also on viral p24-expressing T cells
inoculated in vitro with HIV-1 (77, 78). Circulating
monocytes from HIV-1-infected patients were also found to express
significant amounts of surface B7-1 (79). Second, some of these cell
surface markers have been shown to be acquired by field isolates of
HIV-1 expanded in mitogen-stimulated peripheral blood mononuclear cells
(reviewed in Ref. 14). Moreover, our findings indicate that a
laboratory isolate of HIV-1 (NL4-3) expanded in a physiological tissue
culture system (i.e. histocultures of human lymphoid tissue
(tonsils)) can acquire amounts of foreign B7-2 proteins that are
comparable with virions produced by 293T cells. This last observation
provides a physiological significance to the current work. Third, the
normal counterligands for CD28, ICAM-1, B7-1, and B7-2 proteins are all
known to provide signaling cascades upon ligation. For example,
antibodies against CD28, B7-1, and B7-2 in T cells elicit different
biochemical signals, including phospholipase C- Although signaling through the TCR/CD3 complex alone is essential for
the initial stages of T cell activation, it is not sufficient to induce
all events that accompany activation of freshly isolated resting T
cells or T cell clones (84). A second costimulatory signal provided by
ligand engagement of cell-surface receptor molecules such as CD28 is
required (85-87). Interestingly, optimal activation of gene expression
directed by the HIV-1 LTR was also found to be exerted by ligands to
both TCR/CD3 and CD28 (51, 52). Our results are perfectly in line with
such findings because binding of HIV-1 particles bearing B7-2, one of
the natural cognate ligands of CD28, is not sufficient per
se to up-regulate HIV-1 LTR-driven transcription. Indeed, a
concomitant ligation of the TCR/CD3 complex by specific monoclonal
antibodies is necessary to achieve activation of virus promoter with
B7-2-bearing virions in human T cells carrying an HIV-1 LTR construct
either in a transient or stable form. Our results are thus indicating
that virally embedded foreign B7-2 proteins are functional even when
located within the HIV-1 envelope and that such host-derived proteins
can engage with their natural ligand CD28 and mediate
CD28-dependent signaling cascades. The observation that
NL4-3/Null (i.e. virions devoid of studied host cell
surface constituents) and isogenic viruses bearing host-derived B7-1,
CD28, or ICAM-1 proteins do not modulate LTR activity even in the
presence of anti-CD3 antibodies suggests that the observed phenomenon
is really due to the additional interaction between virion-anchored
host B7-2 and cell surface CD28 found on the target cell and not to
soluble factors present in virus preparations. In addition, a Tat
antagonist (i.e. Ro 24-7429) further confirmed that the
positive effect on HIV-1 LTR activity is really due to signal
transduction pathways engaged upon binding of viron-anchored foreign
B7-2 with its CD28 ligand and not to virus-associated Tat.
We were able to demonstrate that transcription factor NF- Although B7-1 and B7-2 proteins can both interact with CD28 with
similar low affinities (89) and provide the costimulatory signal
necessary to prevent the induction of T cell anergy and enhance
cytokine production (90), only a limited number of studies have
compared the signals generated following B7-1 or B7-2 engagement of
CD28. Nonetheless, a growing body of evidence suggests that there are
different functional outcomes of CD28 engagement by B7-1 and B7-2
(91-93). Our results support this concept since an increase in HIV-1
LTR-driven gene activity was observed only when anti-CD3 antibodies
were coupled with B7-2-bearing virions but not with isogenic viruses
having incorporated foreign B7-1 proteins.
Based on results from this work and previous studies, we are proposing
that intracellular signaling events transduced by HIV-1 envelope and
virion-anchored host proteins may directly contribute to the
pathogenesis of this retroviral infection. For example, induction of
such intracellular biochemical events can modulate the early events in
the viral life cycle in uninfected cells and/or, as demonstrated in the
present study, affect proviral DNA in already infected cells. In the
first scenario, it is known that the replicative cycle of HIV-1 is
greatly influenced by the stage of the cell cycle at the time of
infection. Successful infection of CD4+ T lymphocytes by HIV-1 requires
the activation of target cells whereas infection of quiescent CD4+ T
lymphocytes leads to incomplete, labile, reverse transcripts (94-96).
Complete HIV-1 reverse transcription was observed only when quiescent
peripheral CD4+ T lymphocytes were induced to switch from the
G1a phase of the cell cycle to the G1b phase by
costimulation through the T cell receptor and CD28 (97). Therefore,
depending of the virion-bound cell surface protein, it is quite
possible that the initial contact between the viral entity and its
target will render the intracellular milieu more favorable for HIV-1
reverse transcription and integration process. The second scenario is
based on published work showing that HIV-1 replication is very
intimately linked to T cell activation, due to the overlapping of the
signal transduction requirement between T cell lymphokine gene
expression and HIV-1 LTR transactivation (98). This overlapping is a
consequence of the HIV-1 LTR architecture, composed of many different
motifs found in regulatory regions of gene induced following T cell
activation (53). Our findings clearly suggest that the exact nature of
host-encoded protein present on the virion's surface can influence
activation of transcription factors recognized as potent inducers of
HIV-1 replication (i.e. NF- Taken together, our results suggest that attachment of HIV-1 to its
target should be considered as an event that, depending of the nature
of virion-anchored host proteins, can influence several steps in the
virus replicative cycle.
B (NF-
B)
and nuclear factor of activated T cells (NFAT) as revealed by the use
of vectors coding for dominant negative versions of both transcription
factors (i.e. I
B
S32A/36A and dnNFAT) and band shift
assays. The increase of NF-
B activity was abolished when infection
with B7-2-bearing HIV-1 particles was performed in the presence of the
fusion protein CTLA-4 Ig suggesting that the interaction between
virally embedded B7-2 and CD28 on the target cell is responsible for
the observed NF-
B induction. The findings presented here provide the
first demonstration that host-encoded proteins acquired by HIV-1 can
mediate signal transduction events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-microglobulin, CD3, CD43, CD44, CD55,
CD59, CD63, and the transferrin receptor (CD71) (reviewed in Ref. 14).
It should be stated that incorporation of selected host cell molecules
was found to be conserved among different HIV-1 subtypes and strains
that were expanded on phytohemagglutinin-activated peripheral blood
mononuclear cells (15). An initial report has shown that incorporation
of MHC-I molecules is not essential for HIV-1 infectivity (16), but
data from recent studies clearly indicate that several virion-acquired
host proteins have functional effects on the biology of HIV-1 (reviewed
in Ref. 14).
B (NF-
B) and nuclear
factor of activated T cells (NFAT), two transcription factors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
BLTR-Luc plasmids, which were kindly provided by Dr. K. L. Calame (Columbia University, New York, NY). Such molecular
constructs contain the luciferase reporter gene under the control of
the wild-type (GGGACTTTCC) or the NF-
B-mutated
(CTCACTTTCC) HIV-1 LTR domain (
453 to +80)
(32). The p
B-TATA-LUC plasmid only contains the HIV-1 enhancer
region (
105/
70) and a TATA box placed upstream of the luciferase
gene (33). The dominant negative I
B
-expressing vector
pCMV-I
B
S32A/36A has been described previously (33).
These two latter molecular constructs were generous gifts from Dr. W.C.
Greene (J. Gladstone Institutes, San Francisco, CA). The
pCDNA3-dnNFAT vector codes for a dominant negative NFAT mutant and
was supplied by Dr. R. J. Davis (Howard Hughes Medical Institute,
Worcester, MA) (34). pNFAT-Luc, containing the minimal IL-2 promoter
with three tandem copies of the NFAT1-binding site, was kindly provided
by Dr. G. Crabtree (Howard Hughes Medical Institute, Stanford, CA)
(35). The commercial pNF
B-Luc molecular construct contains five
consensus NF-
B-binding sequences placed in front of the luciferase
gene along with a minimal promoter (Stratagene). pNL4-3 is a
full-length infectious molecular clone of HIV-1 (provided by the AIDS
Research and Reference Reagent Program) (36). pCD1.8 is an eukaryotic
expression vector containing the entire human ICAM-1 cDNA and was
obtained from Dr. T. A. Springer (Center for Blood Research,
Boston, MA) (37). pH
Apr-1-neo is a human expression
vector containing the entire human CD28 cDNA and has been described
previously (a generous gift from Dr. D. Olive, INSERM U119, Marseille,
France) (38). Mammalian expression vectors coding for human B7-1
(pCDL-SR
-B7-1) and B7-2 (pCN-B7-2) were obtained from Dr. A. Truneh
(SmithKline Beecham Pharmaceuticals, King of Prussia, PA) (39).
chain of the CD3 complex) was obtained from the
American Type Culture Collection (Rockville, MD). Antibodies from this
hybridoma were purified with mAbTrap protein G affinity columns
according to manufacturer's instructions (Pharmacia Biotechnology AB,
Uppsala, Sweden). The monoclonal antibody BU-63 (IgG2a) is
specific for human B7-2 (CD86) (40) and has been supplied by Dr.
D. L. Hardie (University of Birmingham, Birmingham, United
Kingdom). UCHL-1 (IgG2a) is a monoclonal anti-human CD45RO
antibody that was used as a control to semiquantitatively estimate the
incorporation rate of foreign B7-2 proteins in virus preparations.
CTLA-4 Ig is constituted of the extracellular domain of CTLA-4 fused to
the Fc fragment of the immunoglobulin G1 (IgG1). Previous studies have
indicated that this fusion protein demonstrates a strong affinity for
B7-1 and B7-2 molecules and blocks the interaction between CD28 and
B7-1/B7-2 (41). Fluorescein isothiocyanate-conjugated goat anti-mouse
immunoglobulin G (IgG) was purchased from Jackson Immunoresearch
Laboratories, Inc. (West Grove, PA).
-B7-1 (virus stock called
NL4-3/B7-1), pCN-B7-2 (virus stock called NL4-3/B7-2),
pH
Apr-1-neo (virus stock called NL4-3/CD28), or pCD1.8
(virus stock called NL4-3/ICAM-1). At 16 h after transfection,
cells were washed twice with 3 ml of PBS and were incubated for an
additional 24 h with 3 ml of Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Virion-containing
supernatants were filtered through a 0.45-µm cellulose acetate
membrane (Millipore, MA), aliquoted in 200-µl fractions, and were
finally frozen at
85 °C until needed. Virus stocks were normalized
for virion content using a commercial available enzyme-linked
immunosorbent assay for the viral major core protein p24 (Organon
Teknika, Durham, NC). The standardization on p24 contents is based on
our previous observation indicating that virus preparations harvested
from transfected 293T cells contain minimal amounts of p24 that are not
associated with infectious virions (44). All virus stocks underwent one
freeze-thaw cycle prior to initiation of infection studies. It should
be stated that virus preparations were made from 293T cells expressing
comparable levels of studied host cell surface constituents
(i.e. B7-1, B7-2, CD28, and ICAM-1) and the physical
presence of foreign proteins on the exterior of HIV-1 particles was
assessed using our previously described immunomagnetic-based virus
capture assay (see below) (45). Virus preparations were also made by
infecting human tonsil histocultures as described previously (46).
Briefly, human tonsillar tissue removed during a routine tonsillectomy
and not required for clinical purposes was received within 5 h of
excision. The tonsils were washed thoroughly with medium containing
antibiotics and then sectioned into 2-3-mm blocks. These tissue blocks
were placed on top of collagen sponge gels in the culture medium at the
air-liquid interface and infected for 10 days with NL4-3 (2.5 ng of
p24) added to the top of each tissue block (3-5 µl of clarified virus). Productive HIV-1 infection was assessed by measuring p24 in the
culture medium with a commercial HIV-1 p24 antigen enzyme-linked immunosorbent assay.
-32P-5'-end-labeled, double-stranded (dsDNA)
oligonucleotide. This mixture was incubated for 30 min at 37 °C, and
the reaction was stopped with 5 µl of 0.2 M EDTA. The
labeled oligonucleotide was extracted with phenol/chloroform and passed
through a G-50 spin column. The dsDNA oligonucleotide, which was used
as a probe, contains the consensus NF-
B-binding site corresponding
to the sequence 5'-ATGTGAGGGGACTTTCCCAGGC-3' (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). DNA-NF-
B complexes were resolved from free
labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels
and 0.5× TBE buffer. The gels were subsequently dried and autoradiographed. Cold competitor assays were carried out by adding a
100-fold molar excess of homologous unlabeled dsDNA NF-
B
oligonucleotide simultaneously with the labeled probe. Supershift
assays were performed by preincubation of nuclear extracts with 1 µl
of either specific anti-p50 or anti-p65 polyclonal antibodies (Dr.
Nancy Rice, NCI, National Institutes of Health, Frederick, MD) in the presence of all the components of the binding reaction described above
for 20 min at room temperature prior to the addition of the probe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of HIV-1 LTR-driven gene activity
is seen following incubation with anti-CD3 antibody and B7-2-bearing
HIV-1NL4-3. A, at 18 h following
transient transfection of Jurkat with pLTR-Luc, cells were incubated
for 7 h with soluble anti-CD3 antibody, DT30 cells, a combination
of anti-CD3 and DT30 cells, or 150 ng of p24 for each isogenic virus
stocks tested (i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/B7-2,
NL4-3/CD28, and NL4-3/ICAM-1). B, transiently transfected
Jurkat cells were also incubated for 7 h with virions bearing or
not bearing host-derived B7-1, B7-2, CD28, and ICAM-1 proteins in the
presence of anti-CD3 antibody. C, 1G5 cells were incubated
for 7 h in the presence of soluble anti-CD3 antibody, DT30 cells,
or a combination of anti-CD3 and DT30 cells. 1G5 cells were also
incubated for 7 h with anti-CD3 antibody and NL4-3/Null or NL4-3/B7-2 viruses (130 ng
of p24). Cells were subsequently lysed, and the lysates were then
assayed for luciferase activity with a microplate luminometer. Results
shown are the means (± S.D.) of quadruplicate samples and are
expressed as -fold induction relative to basal luciferase activity in
untreated/uninfected control cells. These results are representative of
three independent experiments.
-CD3) and CD28 (DT30) resulted in a strong activation of HIV-1 gene
expression (Fig. 1B). Interestingly, incubation of
transiently transfected Jurkat cells with ligand to CD3 and virions
bearing host-derived B7-2 proteins resulted in a greater induction of HIV-1 LTR as compared with isogenic HIV-1NL4-3 particles devoid of this host cell surface constituent (i.e.
NL4-3/Null). In an attempt to define whether such a virus-mediated
modulation of HIV-1 LTR activity can still be observed in the context
of an integrated provirus, 1G5 cells were incubated with anti-CD3 antibodies and B7-2-bearing virus particles. As depicted in Fig. 1C, HIV-1 LTR-driven gene activity was still up-regulated
with B7-2-bearing virions and not with isogenic viral entities devoid of foreign B7-2 proteins, hence confirming the results obtained in
transient transfection experiments.
B-binding sites separated by an AP-2 site that has recently been
demonstrated to be important for the binding of another transcription
factor, NFAT (54). To determine whether the enhancer domain is the
target for the observed increase in LTR activity, p
B-TATA-Luc, which
only contains the HIV-1 enhancer region (
105/
70) upstream of a TATA
box, was transfected into Jurkat cells. The combined action of anti-CD3 and DT30 cells resulted in a significant increase in p
B-TATA-Luc gene expression (Fig. 2A).
Anti-CD3 antibodies and progeny viruses devoid of foreign B7-2 proteins
(i.e. NL4-3/Null) had no detectable effect on HIV-1
promoter-driven gene activity. However, a dose-dependent increase in p
B-TATA-Luc gene expression was noticed when cells were
incubated with anti-CD3 antibodies along with isogenic B7-2-bearing HIV-1 particles. For example, addition of 150 ng of p24 of NL4-3/B7-2 resulted in a significant 95-fold increase in HIV-1
enhancer-dependent luciferase activity. The direct
involvement of the interaction between virion-bound B7-2 and CD28 on
the target cell surface in the observed activation of HIV-1 enhancer
was assessed by inhibiting interaction between B7-2 and CD28. This goal
was achieved by adding a soluble form of CTLA-4 (CTLA-4 Ig) to the
mixture composed of NL4-3/B7-2 virions, anti-CD3 antibodies, and
Jurkat cells transiently transfected with p
B-TATA-Luc vector. Data
from Fig. 2B indicate that CTLA-4 can totally abrogate the
significant up-regulation of HIV-1 enhancer-driven luciferase activity
mediated by anti-CD3 and isogenic B7-2-bearing virions. It should be
noted that incubation of transfected Jurkat cells with B7-2-bearing
progeny viruses in the absence of anti-CD3 could not induce
up-regulation of HIV-1 enhancer region.
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Fig. 2.
Interaction between virion-bound host B7-2
and cell surface CD28 is playing a key role in the observed
up-regulation of LTR activity. A, Jurkat cells were
transiently transfected with a construct bearing the HIV-1 enhancer
placed in front of the luciferase reporter gene (p B-TATA-Luc) and
were incubated 18 h later with anti-CD3, DT30 cells, the
combination of anti-CD3 antibody and DT30 cells, or anti-CD3 antibody
with increasing concentrations of virons bearing or not bearing
host-encoded B7-2 proteins. B, in some experiments, the
CTLA-4 Ig fusion protein was added before the incubation period.
C, 1G5 cells were infected for 48 h with 130 ng of p24
of NL4-3/Null (
) or NL4-3/B7-2 (
) virus preparations that
previously underwent three freeze-thaw cycles. D, Jurkat
cells transiently transfected with p
B-TATA-Luc were treated with
anti-CD3 antibody and NL4-3/Null virions or NL4-3/B7-2 (130 ng of
p24) that previously underwent three freeze-thaw cycles. E,
1G5 cells were treated with increasing concentrations of the Tat
inhibitor Ro 24-7429 for 45 min and cells were then infected with
NL4-3/Null (100 ng of p24). Luciferase activity was assayed 48 h
after virus inoculation. F, Jurkat cells transiently
transfected with p
B-TATA-Luc were treated with the Tat inhibitor Ro
24-7429 for 45 min and were next incubated for 7 h in the presence
of anti-CD3 antibody and NL4-3/Null or NL4-3/B7-2 virions (100 ng of
p24). Next, cells were lysed and the lysates were assayed for
luciferase activity with a microplate luminometer. Results shown are
the means (± S.D.) of quadruplicate samples and are expressed as -fold
induction relative to basal luciferase activity in untreated/uninfected
control cells. These results are representative of three independent
experiments.
B-TATA-Luc were incubated with anti-CD3 antibodies and B7-2-bearing
virions that initially underwent three freeze-thaw cycles. This
treatment results in the production of viruses denuded of their
external gp120 envelope proteins and renders such viral entities thus
unable to enter target cells. Data from Fig. 2C confirm that
such treated virions are no longer infectious for susceptible human T
lymphoid cells. Interestingly, B7-2-bearing virions are still able to
lead to an increase in HIV-1 LTR-dependent gene activity
(30-fold increase) that is not seen with isogenic viruses devoid of
foreign B7-2 proteins (Fig. 2D). To further confirm that the
observed effect is due to signaling events mediated by virion-anchored
B7-2 and not by, for example, virion-associated Tat protein, we carried
out a series of experiments with the benzodiazepine Ro 24-7429 Tat
inhibitor (55-57). First, 1G5 cells were pretreated with various
concentrations of Ro 24-7429 before being inoculated with HIV-1. Data
presented in Fig. 2E demonstrate that Ro 24-7429 can
potently inhibit the process of HIV-1 infection in a
dose-dependent manner. Next, Jurkat cells transiently
transfected with p
B-TATA-Luc were pretreated with increasing
subcytotoxic concentrations of Ro 24-7429 before being incubated with a
similar amount of isogenic virions bearing or not on their surface
host-encoded B7-2. Interestingly, B7-2-bearing viruses can still
up-regulate HIV-1 LTR-driven gene expression (Fig. 2F),
therefore eliminating the possibility that the observed effect is due
to soluble virion-associated Tat protein. We are now providing
conclusive evidence that HIV-1 LTR activation mediated by B7-2-bearing
viruses is caused by signaling from the cell surface as opposed to an
intracellular Tat-mediated effect.
B
and NFAT--
To define the cis-acting sequences required
for the noticed effects on the HIV-1 enhancer, we transfected a number
of plasmids into Jurkat cells, which were then incubated with anti-CD3,
DT30, anti-CD30 and DT30, and, finally, anti-CD3 and isogenic virions bearing or not bearing studied host proteins. We initially tested a
molecular construct containing the complete regulatory elements of
HIV-1 (
453 to +80) mutated at the two NF-
B-binding sites (pm
BLTR-Luc). When both
B-binding sites were mutated, the
combined action of anti-CD3 antibody and B7-2-bearing virions was no
longer able to up-regulate HIV-1 LTR-driven luciferase activity (Fig. 3A). However, as expected, the
engagement of the TCR/CD3 complex in the presence of CD28 costimulatory
signal resulted in weak but detectable response of the entire mutated
LTR (compare 5.4-fold increase in Fig. 3A and 20-fold
increase in Fig. 1A). Data from experiments performed with
pm
BLTR-Luc permit to conclude that the NF-
B transcription factor
is an essential factor for the response of the HIV-1 LTR to soluble
anti-CD3 antibody and progeny virions bearing host-encoded B7-2
proteins.
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Fig. 3.
Treatment of Jurkat cells with anti-CD3 and
B7-2-bearing virions leads to
NF- B-dependent activation of HIV-1
enhancer. A, Jurkat cells were transiently transfected
with pm
BLTR-Luc, a luciferase-encoding vector carrying
NF-
B-mutated HIV-1 LTR domain, and were incubated 18 h later
with soluble anti-CD3 antibody, DT30 cells, a combination of anti-CD3
and DT30 cells, or 150 ng of p24 for each isogenic virus stocks tested
(i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/B7-2, NL4-3/CD28,
and NL4-3/ICAM-1). B, Jurkat cells were also transfected
with a
B-driven reporter gene construct (pNF
B-Luc) and incubated
18 h later with soluble anti-CD3 antibody, DT30 cells, a
combination of anti-CD3 and DT30 cells, or two different concentrations
of each isogenic virus stocks tested (100 ng of p24,
; 200 ng of
p24,
). After 7 h of incubation, cells were lysed and assayed
for luciferase activity. Results shown are the means (± S.D.) of
quadruplicate samples and are expressed as -fold induction relative to
basal luciferase activity in untreated/uninfected control cells. These
results are representative of three independent experiments.
B activation mediated by anti-CD3 antibody and
B7-2-bearing virions, we transiently transfected Jurkat cells with a
cis-reporter plasmid made of the luciferase reporter gene driven by a basic promoter element (TATA box) joined to five tandem repeats of NF-
B binding elements (pNF
B-Luc). Exposure of Jurkat cells to anti-CD3 antibody and the highest concentration of NL4-3/B7-2 (i.e. 200 ng of p24) was found to lead to a significant
135-fold increase in NF-
B-dependent gene activity (Fig.
3B). Similar levels of NF-
B-driven reporter gene
expression were seen following treatment with anti-CD3 alone and
anti-CD3 antibody in combination with other isogenic virus stocks
tested (i.e. NL4-3/Null, NL4-3/B7-1, NL4-3/CD28, and
NL4-3/ICAM-1). To directly demonstrate nuclear translocation of
NF-
B following multivalent occupancy of TCR/CD3 complex and
NL4-3/B7-2 attachment to target cells, we performed DNA mobility shift
assays. In this series of experiments, the positive control consisted
of Jurkat cells treated with TNF-
. This proinflammatory cytokine was
able to induce the appearance of a specific band corresponding to
NF-
B as competition assays performed with 100-fold excess of the
cold NF-
B oligonucleotide led to a complete disappearance of this
signal (Fig. 4, compare lanes
4 and 12). Treatment of Jurkat cells with either
NL4-3/Null or NL4-3/B7-2 alone did not induce nuclear translocation
of NF-
B (lanes 5 and 6,
respectively). Incubation of Jurkat cells with anti-CD3 antibody in
combination with isogenic B7-2-bearing HIV-1 particles resulted in the
appearance of a band corresponding to the NF-
B factor
(lane 8), which was specifically outcompeted by
cold excess of the NF-
B oligonucleotide (lane
11). The addition of the specific inhibitor of B7-2/CD28
interaction, i.e. CTLA-4 Ig, was found to eliminate the
NF-
B-specific band (lane 10). We next
investigated the identity of the subunit(s) composing the NF-
B
complex by performing supershift assays with polyclonal anti-p50 and
anti-p65 antibodies. Both antibodies led to the complete disappearance
of NF-
B-specific band (lane 13 for anti-p50
and lane 14 for anti-p65), which was seen
following incubation of Jurkat cells with anti-CD3 antibody and
B7-2-bearing virions.
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Fig. 4.
Nuclear translocation and activation of
NF- B is confirmed by mobility shift
assays. Jurkat cells were either left untreated (lane
1) or were incubated for 1 h with anti-CD3
(lane 2), 100 µM forskolin
(lane 3, used as a negative control), 10 ng/ml
TNF-
(lane 4, used as a positive control),
NL4-3/Null (lane 5, 150 ng of p24), NL4-3/B7-2
(lane 6, 150 ng of p24), anti-CD3 and NL4-3/Null
viruses (lane 7, 150 ng of p24), anti-CD3 and
NL4-3/B7-2 viruses (lane 8, 150 ng of p24),
anti-CD3 + NL4-3/Null viruses (150 ng of p24) + CTLA-4 Ig
(lane 9), and anti-CD3 + NL4-3/B7-2 viruses (150 ng of p24) + CTLA-4 Ig (lane 10).
Lanes 11 and 12 represent a 100×
competition with the unlabeled probe for NF-
B for transiently
transfected Jurkat cells incubated with anti-CD3 + NL4-3/B7-2 viruses
(150 ng of p24) and TNF-
, respectively. Lanes
13 and 14 represent a supershift with anti-p50
and anti-p65 antibody, respectively. The nuclear extracts were next
incubated with a 32P-end-labeled synthetic double-stranded
NF-
B probe. The position of the specific complex bound by the
B
site probe is indicated by an arrow on the left
side.
B in
transcriptional activation of HIV-1 through its action on the virus
enhancer region (54). We therefore evaluated whether NFAT activation
could be achieved by the combined action of anti-CD3 antibody and HIV-1
particles having incorporated host B7-2 proteins in their envelope.
This task was accomplished by using a vector composed of the luciferase
reporter gene placed under the control of the minimal IL-2 promoter
containing three tandem copies of the NFAT-binding site (Fig.
5). Treatment of transiently transfected Jurkat cells with anti-CD3 antibody alone led to a 2.9-fold increase in
NFAT-dependent gene activity, a value comparable to the one seen when cells are incubated with anti-CD3 and virions devoid of
host-encoded B7-2 proteins (i.e. NL4-3/Null).
Interestingly, a higher increase in reporter gene activity was seen
when cells are incubated with anti-CD3 and isogenic B7-2-bearing viral
entities (7-fold activation). Thus, the combined action of
TCR/CD3-mediated biochemical events and binding of viruses bearing
foreign B7-2 proteins in their envelope enhance activation of the
transcription factor NFAT.
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Fig. 5.
Enhancement of NFAT activation by the
combined action of anti-CD3 and B7-2-bearing HIV-1 particles.
Jurkat cells were transfected with a NFAT-driven vector (pNFAT-Luc) and
were incubated for 7 h with soluble anti-CD3 antibody, DT30 cells,
a combination of anti-CD3 and DT30 cells, anti-CD3 and NL4-3/Null
viruses (150 ng of p24), or anti-CD3 and NL4-3/B7-2 viruses (150 ng of
p24). Next, cell lysates were assayed for luciferase activity with a
microplate luminometer. Results shown are the means (± S.D.) of
quadruplicate samples and are expressed as -fold induction relative to
basal luciferase activity in untreated/uninfected control cells. These
results are representative of three independent experiments.
B
and NFAT Block
Activation of HIV-1 Enhancer by Anti-CD3 Antibody and B7-2-bearing
HIV-1--
Previous studies have demonstrated that nuclear
translocation and activation of NF-
B is mainly mediated by the
degradation of the repressor I
B
, which sequesters the NF-
B
complex in the cytoplasm (58, 59). This degradation is known to be
highly dependent on the phosphorylation of the two serine residues 32 and 36 (60, 61). To determine the contribution of both NF-
B and NFAT
in the noticed increase in HIV-1 enhancer-mediated activity, we used
trans-dominant negative mutants of each transcription factor.
Initially, we transfected Jurkat cells with a vector coding for a
modified version of I
B
carrying alanine on both serine 32 and 36 residues. This protein is unable to be serine-phosphorylated but
retains its ability to bind to NF-
B and can thus act as a dominant
negative mutant of wild-type NF-
B. Cotransfection of Jurkat cells
with p
B-TATA-Luc and pCMV-I
B
S32A/36A resulted in
an almost complete inhibition of the activation of HIV-1
enhancer-driven gene activity mediated by anti-CD3 antibody and
B7-2-bearing viruses (Fig.
6A). The positive control made
of anti-CD3 antibody and DT30 cells was also dramatically affected by
the introduction of I
B
S32A/36A. The involvement of
NFAT and a possible synergistic effect with NF-
B was next
investigated with dnNFAT, a dominant negative NFAT mutant that
suppresses activation-induced nuclear translocation of all NFAT members
(34). The increase in HIV-1 enhancer-dependent luciferase
activity mediated by the combination of anti-CD3 and B7-2-bearing
virions was diminished by dnNFAT but to a lesser extent than by
pCMV-I
B
S32A/36A (Fig. 6B). It is of
interest to note that such a diminution was further augmented when both
negative mutants were introduced into Jurkat cells. Taken together,
these results suggest that NF-
B and NFAT are both active players in
the up-regulation of HIV-1 enhancer activity, which is seen following
treatment with anti-CD3 antibody and HIV-1 particles bearing
host-derived B7-2 proteins on their surface.
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Fig. 6.
Trans-dominant negative mutant of
I B
and NFAT inhibit
HIV-1 LTR activity mediated by anti-CD3 and B7-2-bearing virions.
A, Jurkat cells were cotransfected with p
B-TATA-Luc (15 µg) and an empty vector control (empty bars, 30 µg) or pCMV-I
B
S32A/36A (filled
bars, 15 µg; striped bars, 30 µg).
B, Jurkat cells were also cotransfected with with
p
B-TATA-Luc (15 µg)/empty vector control (empty
bars, 30 µg), p
B-TATA-Luc (15 µg)/pCMV-I
B
S32A/36A (filled
bars, 15 µg), p
B-TATA-Luc (15 µg)/pCDNA3-dnNFAT
(striped bars, 7.5 µg) or p
B-TATA-Luc (15 µg)/pCMV-I
B
S32A/36A (15 µg)/pCDNA3-dnNFAT
(dotted bars, 7.5 µg). After 18 h, cells
were next incubated for 7 h with soluble anti-CD3 antibody, DT30
cells, a combination of anti-CD3 and DT30 cells, anti-CD3 and
NL4-3/Null viruses (150 ng of p24), or anti-CD3 and NL4-3/B7-2
viruses (150 ng of p24). The lysates were next assayed for luciferase
activity with a microplate luminometer. Results shown are the means (± S.D.) of quadruplicate samples and are expressed as -fold induction
relative to basal luciferase activity in untreated/uninfected control
cells. These results are representative of three independent
experiments.
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Fig. 7.
Comparative analysis of the degree of
incorporation of B7-2 in HIV-1NL4-3 produced either in
293T cells or human lymphoid tissue. A, 293T cells were
cotransfected with pNL4-3 and increasing concentrations of the
pCN-B7-2 expression vector (i.e. 0, 0.1, 0.5, 1, 2.5, and 5 µg). Similar amounts of virus stocks (2.5 ng) were next incubated
with magnetic beads coated with antibodies specific for human B7-2
(clone BU-63) and CD45RO (clone UCHL-1). B, viruses (2.5 ng
of p24) produced either in transiently transfected 293T cells or
histocultures of human lymphoid tissue (i.e. tonsils) were
incubated with magnetic beads coated with antibodies specific for human
B7-2 (clone BU-63) and CD45RO (clone UCHL-1). The levels of captured
virions were quantified by using a p24 enzymatic assay. Magnetic beads
coated with the anti-CD45RO antibody served as controls to determine
background levels of captured viruses. Data shown represent ratio of
captured virions with antibody reactive with human B7-2 and CD45RO,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chemokines (21, 63). The signaling events transduced by the
interaction of HIV-1 envelope with CCR5 result in chemotaxis of
CD4-expressing T cells, raising the possibility that such response may
promote the recruitment of uninfected cells to sites of active viral
replication (63). We have previously shown that binding of HIV-1 to its
target leads to phosphorylation of phosphatidylinositol 3-kinase (17),
as well as to a decrease in HIV-1 transcription and virion production (64, 65). Conflicting data were reported by Deveaux's group (66, 67),
who have demonstrated that HIV-1 attachment mediates activation of
virus transcription via an NF-
B-dependent pathway. Given
that numerous functional foreign cell membrane proteins have been found
to be acquired by HIV-1, we were interested in defining whether the
exact nature of virally embedded host proteins might explain such
contrasting findings as we suggested previously (14). This postulate is
founded on the observation that the number and type of cell membrane
surface proteins acquired by HIV-1 particles can have profound effects
on the attachment of virions to target cells, the host cell range, the
binding avidity between virus and cell, and the neutralization
sensitivity of virions (6, 7, 42, 43, 68-74). It is therefore
legitimate to propose that binding of some virally incorporated host
proteins with their normal counter-receptors found on the cell surface can lead to signal transduction into target cell. In this work, we
report that virions bearing host-derived B7-2 (CD86) proteins can, in
association with engagement of the TCR/CD3 complex, activate HIV-1
LTR-driven gene expression through the induction of NF-
B and
NFAT-dependent second-message pathways.
phosphorylation,
calcium influx, PI 3-kinase activation, and activation of
p21ras, Raf-1, and extracellular
signal-regulated kinase/c-Jun N-terminal kinase (80). Although CD28 is
primarily seen as a costimulus to the T cell receptor, a recent study
indicates that engagement of CD28 alone is sufficient to lead to
NF-
B activation in human T cells (81). Ligation of CD28 has also
been reported to generate tyrosine phosphorylation of the CD28
cytoplasmic tail and the formation of a complex with PI 3-kinase (38).
ICAM-1 binding to LFA-1, its cognate ligand, has been demonstrated to
up-regulate the activities of PI 3-kinase, sphingomyelinase, and c-Jun
N-terminal kinase (83).
B is
playing a crucial role in the increase of HIV-1 LTR activity, which is
seen following ligation of cell surface TCR/CD3 complex and CD28 with
specific antibodies and B7-2-bearing virions, respectively. Earlier
studies have demonstrated that NFAT is an immediate-early activation
factor that plays a crucial role in T cell activation and commitment
processes through its control of interleukin-2 gene activation (88).
The previously demonstrated synergistic effect between NF-
B and
NFAT, with respect to activation of HIV-1 transcription (54), prompted
us to also investigate the implication of NFAT. We found that NFAT was
indeed acting in a synergistic mode with NF-
B and that both
transcription factors were responsible for the up-regulation of LTR
activity mediated by anti-CD3 antibody and B7-2-bearing HIV-1 particles.
B and NFAT). Although
antibody-mediated engagement of TCR/CD3 complex was found to be
necessary to achieve activation of HIV-1 LTR-dependent gene
expression by B7-2-bearing progeny virus, it should be kept in mind
that substantial amounts of foreign MHC-II proteins are acquired by
HIV-1 (45, 82). Therefore, assuming that the nominal antigen occupies
the peptide binding groove, interaction with the TCR/CD3 complex can be
achieved with virally embedded host MHC-II proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. B. Barbeau for critical reading of the manuscript and Dr. M. Dufour for technical assistance in flow cytometry studies.
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FOOTNOTES |
---|
* This work was supported in part by Canadian Institutes of Health Research HIV/AIDS Research Program Grant HOP-14438 and by a Fonds de la Recherche en Santé du Québec (Réseau FRSQ SIDA et Maladies Infectieuses) grant (both to M. J. T.).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 Ph.D. fellowship from the Fonds de la Recherche en
Santé du Québec/Fonds pour la Formation de chercheurs et
l'Aide à la Recherche-Program Santé. This work was
performed in partial fulfillment of the requirements for a Ph.D. degree at the Faculty of Graduate Studies, Department of Medical Biology, Faculty of Medicine, Laval University.
§ Holder of a Canada Research Chair in Human Immuno Retrovirology. To whom correspondence should be addressed: Laboratoire d'ImmunoRétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Tel.: 418-654-2705; Fax: 418-654-2212; E-mail: michel.j.tremblay@crchul.ulaval.ca.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M002198200
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ABBREVIATIONS |
---|
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1;
LTR, long terminal repeat;
MHC, major
histocompatibility complex;
PBS, phosphate-buffered saline;
NF-B, nuclear factor-
B;
NFAT, nuclear factor of activated T cells;
TNF-
, tumor necrosis factor-
;
TCR, T cell receptor;
dsDNA, double-stranded DNA;
PI 3-kinase, phosphatidylinositol 3-kinase;
Tricine, N-tris(hydroxymethyl)methylglycine;
ICAM-1, intercellular adhesion molecule 1;
LFA-1, lymphocyte
function-associated antigen-1.
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REFERENCES |
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1. | Horuk, R. (1999) Immunol. Today 20, 89-94[CrossRef][Medline] [Order article via Infotrieve] |
2. | Fenouillet, E., Clerget-Raslain, B., Gluckman, J. C., Guetard, D., Montagnier, L., and Bahraoui, E. (1989) J. Exp. Med. 169, 807-822[Abstract] |
3. | McKeating, J. A., McKnight, A., and Moore, J. P. (1991) J. Virol. 65, 852-860[Medline] [Order article via Infotrieve] |
4. | Gelderblom, H. R., Reupke, H., and Pauli, G. (1985) Lancet ii, 1016-1017 |
5. | Dimitrov, D. S., Willey, R. L., Sato, H., Chang, L.-J., Blumenthal, R., and Martin, M. A. (1993) J. Virol. 67, 2182-2190[Abstract] |
6. | Cantin, R., Fortin, J.-F., Lamontagne, G., and Tremblay, M. (1997) J. Virol. 71, 1922-1930[Abstract] |
7. | Guo, M. M. L., and Hildreth, J. E. K. (1995) AIDS Res. Hum. Retroviruses 11, 1007-1013[Medline] [Order article via Infotrieve] |
8. | Ugolini, S., Mondor, I., and Sattentau, Q. J. (1999) Trends Microbiol. 7, 144-149[CrossRef][Medline] [Order article via Infotrieve] |
9. | Azocar, J., and Essex, M. (1979) Cancer Res. 39, 3388-3391[Abstract] |
10. | Bubbers, J. E., and Lilly, F. (1977) Nature 266, 458-459[Medline] [Order article via Infotrieve] |
11. | deThé, G., Becker, C., and Beard, J. W. (1964) J. Natl. Cancer Inst. 32, 201-235[Medline] [Order article via Infotrieve] |
12. | Lando, Z., Sarin, P., Megson, M., Greene, W. C., Waldman, T. A., Gallo, R. C., and Broder, S. (1983) Nature 305, 733-736[Medline] [Order article via Infotrieve] |
13. | Lee, T. H., Essex, M., De Noronha, F., and Azocar, J. (1982) Cancer Res. 42, 3995-4002[Abstract] |
14. | Tremblay, M. J., Fortin, J.-F., and Cantin, R. (1998) Immunol. Today 19, 346-351[CrossRef][Medline] [Order article via Infotrieve] |
15. | Roberts, B. D., and Butera, S. T. (1999) AIDS 13, 425-427[CrossRef][Medline] [Order article via Infotrieve] |
16. | Benkirane, M., Blanc-Zouaoui, D., Hirn, M., and Deveaux, C. (1994) J. Virol. 68, 6332-6339[Abstract] |
17. | Briand, G., Barbeau, B., and Tremblay, M. (1997) Virology 228, 171-179[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Chirmurle, N.,
Goonewardena, H.,
Pahwa, S.,
Pasieka, R.,
Kalyanaraman, V. S.,
and Pahwa, S.
(1995)
J. Biol. Chem.
270,
19364-19369 |
19. |
Cicala, C.,
Arthos, J.,
Ruiz, M.,
Vaccarezza, M.,
Rubbert, A.,
Riva, A.,
Wildt, K.,
Cohen, O.,
and Fauci, A. S.
(1999)
J. Immunol.
163,
420-426 |
20. | Cruikshank, W. W., Center, D. M., Pyle, S. W., and Kornfeld, H. (1990) Biomed. Pharmacother. 44, 5-11[Medline] [Order article via Infotrieve] |
21. |
Davis, C. B.,
Dikic, I.,
Unutmaz, D.,
Hill, C. M.,
Arthos, J.,
Siani, M. A.,
Thompson, D. A.,
Schlessinger, J.,
and Littman, D. R.
(1997)
J. Exp. Med.
186,
1793-1798 |
22. | Hivroz, C., Mazerolles, F., Soula, M., Fagard, R., Gratton, S., Meloche, S., Sékaly, R. P., and Fischer, A. (1993) Eur. J. Immunol. 23, 600-607[Medline] [Order article via Infotrieve] |
23. | Kornfeld, H., Cruikshank, W. W., Pyle, S. W., Berman, J. S., and Center, D. M. (1988) Nature 335, 445-448[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Misse, D.,
Cerutti, M.,
Noraz, N.,
Jourdan, P.,
Favero, J.,
Devauchelle, G.,
Yssel, H.,
Taylor, N.,
and Veas, F.
(1999)
Blood
93,
2454-2462 |
25. | Popik, W., and Pitha, P. M. (1996) Mol. Cell. Biol. 16, 6532-6541[Abstract] |
26. |
Popik, W.,
Hesselgesser, J. E.,
and Pitha, P. M.
(1998)
J. Virol.
72,
6406-6413 |
27. | Popik, W., and Pitha, P. M. (1998) Virology 252, 210-217[CrossRef][Medline] [Order article via Infotrieve] |
28. | Schmid-Antomarchi, H., Benkirane, M., Breittmayer, V., Husson, H., Ticchioni, M., Devaux, C., and Rossi, B. (1996) Eur. J. Immunol. 26, 717-720[Medline] [Order article via Infotrieve] |
29. | Aguilar-Cordova, E., Chinen, J., Donehower, L., Lewis, D. E., and Belmont, J. W. (1994) AIDS Res. Hum. Retroviruses 10, 295-301[Medline] [Order article via Infotrieve] |
30. | Straus, D. B., and Weiss, A. (1992) Cell 70, 585-593[Medline] [Order article via Infotrieve] |
31. | Azuma, M., Cayabyab, M., Buck, D., Phillips, J. H., and Lanier, L. L. (1992) J. Exp. Med. 175, 353-360[Abstract] |
32. | Henderson, A. J., Zou, X., and Calame, K. L. (1995) J. Virol. 69, 5337-5344[Abstract] |
33. | Sun, S.-C., Elwood, J., and Greene, W. C. (1996) Mol. Cell. Biol. 16, 1058-1065[Abstract] |
34. |
Chow, C.-W.,
Rincón, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
2300-2307 |
35. | Timmerman, L. A., Clipstone, N. A., Ho, S. N., Northrop, J. P., and Crabtree, G. R. (1996) Nature 383, 837-840[CrossRef][Medline] [Order article via Infotrieve] |
36. | Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986) J. Virol. 59, 284-291[Medline] [Order article via Infotrieve] |
37. | Staunton, D. E., Merluzzi, V. J., Rothlein, R., Barton, R., Marlin, S. D., and Springer, T. A. (1989) Cell 56, 849-853[Medline] [Order article via Infotrieve] |
38. | Pagès, F., Ragueneau, M., Rottapel, R., Truneh, A., Nunes, J., Imbert, J., and Olive, D. (1994) Nature 369, 327-329[CrossRef][Medline] [Order article via Infotrieve] |
39. | Truneh, A., Reddy, M., Ryan, P., Lyn, S. D., Eichman, C., Couez, D., Hurle, M. R., Sekaly, R. P., Olive, D., and Sweet, R. (1996) Mol. Immunol. 33, 321-334[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Engel, P.,
Gribben, J. G.,
Freeman, G. J.,
Zhou, L. J.,
Nozawa, Y.,
Abe, M.,
Nadler, L. M.,
Wakasa, H.,
and Tedder, T. F.
(1994)
Blood
84,
1402-1407 |
41. | Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L. S., Damle, N. K., and Ledbetter, J. A. (1991) J. Exp. Med. 174, 561-569[Abstract] |
42. | Fortin, J.-F., Cantin, R., Lamontagne, G., and Tremblay, M. (1997) J. Virol. 71, 3588-3596[Abstract] |
43. |
Fortin, J.-F.,
Cantin, R.,
and Tremblay, M.
(1998)
J. Virol.
72,
2105-2112 |
44. |
Paquette, J. S.,
Fortin, J. F.,
Blanchard, L.,
and Tremblay, M. J.
(1998)
J. Virol.
72,
9329-9336 |
45. | Cantin, R., Fortin, J.-F., and Tremblay, M. (1996) Virology 218, 372-381[CrossRef][Medline] [Order article via Infotrieve] |
46. | Glushakova, S., Baibakov, B., Margolis, L. B., and Zimmerberg, J. (1995) Nat. Med 1, 1320-1322[Medline] [Order article via Infotrieve] |
47. | Orentas, R. J., and Hildreth, J. E. K. (1993) AIDS Res. Hum. Retroviruses 9, 1157-1165[Medline] [Order article via Infotrieve] |
48. | Schreiber, E., Matthias, P., Müller, M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve] |
49. |
Dumais, N.,
Barbeau, B.,
Olivier, M.,
and Tremblay, M. J.
(1998)
J. Biol. Chem.
273,
27306-27314 |
50. | Lenschow, D. J., Walunas, T. L., and Bluestone, J. A. (1996) Annu. Rev. Immunol. 14, 233-258[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Tong-Starksen, S. E.,
Luciw, P. A.,
and Peterlin, M. B.
(1989)
J. Immunol.
142,
702-707 |
52. | Smithgall, M. D., Wong, J. G. P., Linsley, P. S., and Haffar, O. K. (1995) AIDS Res. Hum. Retroviruses 11, 885-892[Medline] [Order article via Infotrieve] |
53. | Gaynor, R. (1992) AIDS 6, 347-363[Medline] [Order article via Infotrieve] |
54. | Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., and Nolan, G. P. (1997) Immunity 6, 235-244[Medline] [Order article via Infotrieve] |
55. | Hsu, M. C., Dhingra, U., Earley, J. V., Holly, M., Keith, D., Nalin, C. M., Richou, A. R., Schutt, A. D., Tam, S. Y., Potash, M. J., Volsky, M. J., Richman, D. J., and Douglas, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6395-6399[Abstract] |
56. | Braddock, M., Cannon, P., Muckenthaler, M., Kingsman, A. J., and Kingsman, S. M. (1994) J. Virol. 68, 25-33[Abstract] |
57. | Ehret, A., Westendorp, M. O., Herr, I., Debatin, K. M., Heeney, J. L., Frank, R., and Krammer, P. H. (1996) J. Virol. 70, 6502-7[Abstract] |
58. |
Finco, T.,
Beg, A.,
and Baldwin, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11884-1188 |
59. | Traenckner, E., Wilk, E., and Baeuerle, P. (1994) EMBO J. 13, 5433-5441[Abstract] |
60. | Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve] |
61. | Koong, A. C., Chen, E. Y., and Giaccia, A. J. (1994) Cancer Res. 54, 1425-1430[Abstract] |
62. | Chirmule, N., and Pahwa, S. (1996) Microbiol. Rev. 60, 386-406[Abstract] |
63. | Weissman, D., Rabin, R. L., Arthos, J., Rubbert, A., Dybul, M., Swofford, R., Venkatesan, S., Farber, J. M., and Fauci, A. S. (1997) Nature 389, 981-985[CrossRef][Medline] [Order article via Infotrieve] |
64. | Bérubé, P., Barbeau, B., Cantin, R., Sékaly, R.-P., and Tremblay, M. (1996) J. Virol. 70, 4009-4016[Abstract] |
65. | Tremblay, M., Meloche, S., Gratton, S., Wainberg, M. A., and Sekaly, R. P. (1994) EMBO J. 13, 774-783[Abstract] |
66. | Briant, L., Coudronnière, N., Robert-Hebmann, V., Benkirane, M., and Deveaux, C. (1996) J. Immunol. 156, 3994-4004[Abstract] |
67. | Benkirane, M., Jeang, K.-T., and Devaux, C. (1994) EMBO J. 13, 5559-5569[Abstract] |
68. | Saifuddin, M., Ghassemi, M., Patki, C., Parker, C. J., and Spear, G. T. (1994) AIDS Res. Hum. Retroviruses 10, 829-837[Medline] [Order article via Infotrieve] |
69. | Saifuddin, M., Parker, C. J., Peeples, M. E., Gorny, M. K., Zolla-Pazner, S., Ghassemi, M., Rooney, I. A., Atkinson, J. P., and Spear, G. T. (1995) J. Exp. Med. 182, 501-509[Abstract] |
70. | Rossio, J. L., Bess, J., Henderson, L. E., Cresswell, P., and Arthur, L. O. (1995) AIDS Res. Hum. Retroviruses 11, 1433-1439[Medline] [Order article via Infotrieve] |
71. |
Fujiwara, M.,
Tsunoda, R.,
Shigeta, S.,
Yokota, T.,
and Baba, M.
(1999)
J. Virol.
73,
3603-3607 |
72. |
Cantin, R.,
Fortin, J.-F.,
Lamontagne, G.,
and Tremblay, M.
(1997)
Blood
90,
1091-1100 |
73. | Rizzuto, C. D., and Sodroski, J. G. (1997) J. Virol. 71, 4847-4851[Abstract] |
74. | Cosma, A., Blanc, D., Braun, J., Quillent, C., Barassi, C., Moog, C., Klasen, S., Spire, B., Scarlatti, G., Pesenti, E., Siccardi, A. G., and Beretta, A. (1999) AIDS 13, 2033-2042[CrossRef][Medline] [Order article via Infotrieve] |
75. | June, C. H., Bluestone, J. A., Nadler, L. M., and Thompson, C. B. (1994) Immunol. Today 15, 321-331[CrossRef][Medline] [Order article via Infotrieve] |
76. | Springer, T. A. (1990) Nature 346, 425-434[CrossRef][Medline] [Order article via Infotrieve] |
77. | Jason, J., and Inge, K. L. (1999) AIDS Res. Hum. Retroviruses 15, 173-181[CrossRef][Medline] [Order article via Infotrieve] |
78. | Wolthers, K. C., Otto, S. A., Lens, S. M., Kolbach, D. N., van Lier, R. A., Miedema, F., and Meyaard, L. (1996) Eur. J. Immunol. 26, 1700-1706[Medline] [Order article via Infotrieve] |
79. | Low, P., Weber, C., Harrer, E., Rohwer, P., Kalden, J. R., and Harrer, T. (1997) J. AIDS Hum. Retrovirol. 15, 264-268[Medline] [Order article via Infotrieve] |
80. | Ward, S. G. (1996) Biochem. J. 318, 361-377[Medline] [Order article via Infotrieve] |
81. |
Siefken, R.,
Klein-Hessling, S.,
Serfling, E.,
Kurrle, R.,
and Schwinzer, R.
(1998)
J. Immunol.
161,
1645-1651 |
82. | Arthur, L. O., Bess, J. W. J., Sowder, R. C., II, Benveniste, R. E., Mann, D. L., Cherman, J.-C., and Henderson, L. E. (1992) Science 258, 1935-1938[Medline] [Order article via Infotrieve] |
83. |
Ni, H. T.,
Deeths, M. J.,
Li, W.,
Mueller, D. L.,
and Mescher, M. F.
(1999)
J. Immunol.
162,
5183-5189 |
84. | Schwartz, R. H. (1990) Science 248, 1349-1356[Medline] [Order article via Infotrieve] |
85. | June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T., and Thompson, C. B. (1987) Mol. Cell. Biol. 7, 4472-4481[Medline] [Order article via Infotrieve] |
86. |
Weiss, A.,
Manger, B.,
and Imboden, J.
(1986)
J. Immunol.
137,
819-825 |
87. |
Martin, P. J.,
Ledbetter, J. A.,
Morishita, Y.,
June, C. H.,
Beatty, P. G.,
and Hansen, J. A.
(1986)
J. Immunol.
136,
3282-3287 |
88. | Crabtree, G. R. (1989) Science 243, 355-361[Medline] [Order article via Infotrieve] |
89. | Linsley, P. S., Greene, J. L., Brady, W., Bajorath, J., Ledbetter, J. A., and Peach, R. (1994) Immunity 9, 793-801 |
90. |
Lanier, L. L.,
O'Fallon, S.,
Somoza, C.,
Phillips, J. H.,
Linsley, P. S.,
Okumura, K.,
Ito, D.,
and Azuma, M.
(1995)
J. Immunol.
154,
97-105 |
91. | Gajewski, T. F. (1996) J. Immunol. 156, 465-472[Abstract] |
92. | Eck, S. C., Chang, D., Wells, A. D., and Turka, L. A. (1997) Transplantation 64, 1497-1499[CrossRef][Medline] [Order article via Infotrieve] |
93. | Freeman, G. J., Boussiotis, V. A., Anumanthan, A., Bernstein, G. M., Ke, X. Y., Rennert, P. D., Gray, G. S., Gribben, J. G., and Nadler, L. M. (1995) Immunity 2, 523-532[Medline] [Order article via Infotrieve] |
94. | Spina, C. A., Guatelli, J. C., and Richman, D. D. (1995) J. Virol. 69, 2977-2988[Abstract] |
95. | Stevenson, M., Stanwick, T. L., Dempsey, M. P., and Lamonica, C. A. (1990) EMBO J. 9, 1551-1560[Abstract] |
96. | Zack, J. A., Haislip, A. M., Krogstad, P., and Chen, I. S. (1992) J. Virol. 66, 1717-1725[Abstract] |
97. |
Korin, Y. D.,
and Zack, J. A.
(1998)
J. Virol.
72,
3161-3168 |
98. | Siekevitz, M., Joseph, S. F., Dokovich, M., Peffer, N., Wong-Staal, F., and Greene, W. C. (1987) Science 238, 1575-1578[Medline] [Order article via Infotrieve] |