By
From the * Medical Research Council Human Immunology Unit, Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; the Institute of Clinical and
Molecular Virology, University of Erlangen/Nürnberg, 91054 Erlangen, Germany; and § Genzentrum, Ludwig-Maximilians-Universität München, 81377 München, Germany
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Abstract |
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During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand
(FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression
and apoptotic cell death of both CD4+ and CD8+ T cells by SIV is dependent on a functional
nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL
remained poorly understood. Here we report a direct association of HIV-1 Nef with the chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of
the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR
chain. Conformation for the importance of
for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of
but not CD3
was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef
can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may
provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein
Nef contributes to the pathogenesis of HIV.
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Introduction |
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In HIV/simian immunodeficiency virus (SIV)1 infection, the nef gene plays a key role in viral replication and progression of disease. This is based on studies in macaques and humans, who remain asymptomatic or long-term nonprogressing when infected with an SIV mutant lacking a nef gene or HIV with multiple nef deletions, respectively (1). More recently, a study using a transgenic mouse model has demonstrated that Nef harbors a major determinant for HIV-induced pathogenicity (4). Despite the considerable importance of Nef for HIV/SIV pathogenesis, its function at the molecular level is poorly understood. At least three in vitro effects of Nef have been described. Nef downregulates the surface receptors CD4 and MHC I (5, 6), increases viral infectivity (7), and stimulates T cell signaling pathways (8).
The major consequence of HIV infection is the depletion of T cells leading to immunoparesis characteristic of AIDS. This is likely due to the widespread programmed cell death (apoptosis) induced by HIV (11). Several different apoptotic pathways have been proposed in HIV infection, including Fas/FasL (14), TNF/TNFR (15), and an interaction between TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (16). In HIV-infected patients, there is upregulation of both Fas and FasL as well as an increased susceptibility of CD4+ and CD8+ cells to Fas-mediated killing (17). Recently, we have demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4+ and CD8+ T cells by SIV is dependent on a functional nef gene (22). However, a molecular mechanism integrating this observation into other documented effects of Nef is lacking.
The concept of Nef interfering with early events emanating from the TCR could explain its dual effects on T cell
activation and FasL expression, since both functions are
regulated by the TCR complex (23). Here we report
that HIV-mediated upregulation of FasL in T cells is dependent on the association of Nef with the TCR chain.
By demonstrating that Nef directly targets the TCR of the
infected cell, we provide novel insight into the molecular function of Nef in HIV infection.
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Materials and Methods |
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Cell Lines and Antibodies.
Generation of Jurkat cell lines constitutively expressing CD8 tag or CD8-Nef chimeras was described recently (8, 26). Jurkat, J.CaM.1 (LckPlasmid Constructions.
Generation of the CD8-Nef (SF2) and CD16Protein Expression Assays.
Transfections into 293T cells, metabolic labeling with 35S-Translabel, immunoprecipitation, Western blot, and in vitro kinase assays were performed as described previously (8, 26). The immunoprecipitates were washed three times (wash buffer: 1% NP-40, 450 mM NaCl, 50 mM Tris-HCl [pH 8], 1 mM EDTA). To show an interaction between Nef andIn Vitro HIV Infection of Jurkat or Jurkat Mutant Cell Lines.
Cells (5 × 106) were superinfected with 1 ml of HIV IIIB (1.6 × 104 cpm/ml, reverse transcriptase [RT] activity), NL4-3.SF2.Nef (2.4 × 104 cpm/ml, RT activity), or NL4-3Analysis of FasL Expression by Flow Cytometry and Immunoprecipitation.
To assess cell surface FasL expression on HIV-infected cells or transiently transfected Jurkat TAg cells, the metalloprotease inhibitor BB2116 (British Biotech [30]) was added to the medium 4-6 h before the assay to enhance cell surface FasL expression. In brief, cells were stained with 20 µl of biotin-conjugated anti-human FasL mAb (NOK-1; PharMingen) followed by 5 µl of PE-conjugated streptavidin (Sigma). Labeled cells were analyzed on a FACScanTM (Becton Dickinson). Isotype-specific mAbs of irrelevant specificity were used as negative controls (Dako Diagnostics). To assess expression of whole FasL protein, 35S-labeled cells (5 × 106) were immunopreciptatied for FasL using anti-FasL-specific mAb (NOK-1) as described previously (30). ![]() |
Results |
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As shown previously, Nef associates
with a serine kinase, termed p62 or Nef-associated kinase
(NAK [31]). The Nef-NAK interaction is complex: Nef
stimulates the phosphorylation/activation of NAK, and it is
only in this activated form that NAK can bind Nef (32). This suggests that Nef must act upstream of NAK to promote NAK activation. Our previous results showing that
Nef interfered with early signals emanating from the TCR
suggested it may interact with a component of the TCR
signaling complex. This prompted us to study Nef-mediated NAK/p62 activation in cell lines with TCR signaling
defects. CD8-Nef chimeras (CD8-Nef), containing the extracellular domain of CD8 fused to Nef, were stably transfected into wild-type Jurkat and a variety of Jurkat mutant
cell lines lacking either Lck (J.CaM.1), CD45 (CD45
), or
the entire TCR signaling complex (RT3.T3.5). Expression of CD8-Nef in these cell lines was verified by metabolic labeling and immunoprecipitation (Fig. 1 B). The Nef chimeras from these transfectants were immunoprecipitated
and subjected to an in vitro kinase assay. NAK/p62 association was observed in all cell lines except the TCR
cells
(Fig. 1 A). The latter result was confirmed in a second, independently transfected cell clone (data not shown).
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Next we asked whether NAK binding could be restored
in cells lacking the TCR complex by stable transfection
with TCR- or CD3
fused to the extracellular domain of
CD16 (CD16
and CD16
). These TCR subcomponents
contain signaling motifs (immunoreceptor tyrosine-based T
cell activation motifs [ITAMs]), which are required and
sufficient for T cell activation (28, 33). After obtaining single cell clones, expression of the chimeras was verified by metabolic protein labeling and FACS® analysis (data not
shown). In several attempts, we were unable to coexpress
CD8-Nef (CN) with CD16
in TCR
cells. Cell clones
that were obtained either showed no detectable CN or
CD16 expression or died rapidly. The effect resembled activation-induced cell death (AICD) by Nef as reported previously (8). Coexpression of CN and CD16
was achieved;
however, the obtained cell clones had a low CN as well
as CD16 surface expression (Fig. 1 D, lane 3). Therefore,
we constructed
chimeras containing the three individual
ITAMs in isolation (CD16
1, 2, or 3; see Materials and
Methods for details). In a seperate construct, the tyrosine residues in all three YXXL motifs of CD16
were mutated
to alanines (CD16
mu). We failed to coexpress the first
ITAM with Nef. However, NAK/p62 binding to Nef
was reestablished in the TCR
cells by coexpression with
the second or third ITAM of
(Fig. 1 C, lanes 4 and 5). In
these latter cell lines, expression of CD16
1 and 2 as well as
CD8-Nef decreased significantly over time (data not shown),
indicating that coexpression of both proteins was not favorable. The difficulties regarding the coexpression of the individual
ITAMs with CD8-Nef may be explained by
studies published by Combadiere et al. (34) showing that in
particular the first
ITAM but much less the second and
third are capable of inducing apoptosis when activated. The
signaling-defective
chain (CD16
mu) expressed well, but
NAK binding to Nef was greatly reduced (lane 6). No
NAK/p62 binding was observed by coexpression of CD3
(lane 3). Since NAK binding to Nef was not completely negative with CD16
mu, the Nef-
complex may recruit
additional signaling molecules to the plasma membrane which
are important for NAK activation. Assuming that the effects of the first
ITAM would be similar to ITAM 2 and 3, it appeared that at least one functional ITAM of the CD3
chain was required for binding of p62/NAK to Nef.
The functional link between Nef, , and NAK was confirmed by transient transfection assays in a heterologous system. As shown in Fig. 1 E, cotransfection of CD16
(lanes 4 and 5) but not CD16
(lanes 2 and 3) significantly increased
binding of p62/NAK to Nef. A minimal increase was seen
after cotransfection of CD16
mu (lanes 6 and 7), which
paralleled the small effect seen in Fig. 1 C, lane 6. Thus, no
other T cell-specific components except the functional
ITAM(s) from the TCR
chain, were required for NAK
activation and NAK/Nef association in 293T cells.
Full-length
CD8-Nef when expressed at the cell membrane promotes
AICD. Upon stable transfection, cell clones are preferentially selected in which Nef is predominantly expressed in
the cytoplasm, where it does not exert such a detrimental
effect on cell survival. In contrast, NH2-terminal fragments
of Nef are expressed at high levels at the plasma membrane
where TCR- is located (8). These NH2-terminal fragments can recruit a complex of proteins to form the NH2-terminal kinase complex, which binds between amino acids 20 and 35, and may contribute to T cell activation (26).
The NH2 terminus also has a conserved domain containing
a proline-rich motif (PxxP, aa 73-82) known to associate
with SH3 domains of tyrosine kinases (35). We reasoned
that these domains/motifs could interact with and bind
TCR-
. To prove a direct interaction, coimmunoprecipitation experiments were performed using stable cell lines
with different CD8-Nef chimeras (Fig. 2, A and B). The
only interaction was seen with a construct expressing the NH2-terminal 94 amino acids of Nef containing the PxxP
motif (lane 4). Underscoring the importance of the PxxP motif for
binding, we found that point mutations in the PxxP
motif of the 94-amino acid Nef construct (CN.94.PXmu)
almost completely abolished
binding (lane 5).
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Further evidence for an interaction between Nef and was obtained by coimmunoprecipitation of Nef and
from
Hi5 insect cells coinfected with recombinant baculoviruses
(Fig. 2 D). CD16
was found to coprecipitate with Nef
(Fig. 2 D, lane 3), but not with Nef.PXmu (lane 6). To
confirm the specificity of the interaction, aliquots of the
anti-
immunoprecipitates were incubated with Jurkat
(
-containing) or the TCR
(
-lacking) cytoplasmic lysates.
Wild-type Jurkat competed for Nef binding (lane 4),
whereas the TCR-
-negative cytoplasmic lysates did not
(lane 5). The reduced Nef signal in lane 5 may be explained by the reduced amount of immunoprecipitated
(Fig. 2 E,
lane 5). Additional evidence for the interaction of both
proteins was obtained by coimmunoprecipitation after
transient transfection into COS cells and subsequent in
vitro kinase assay (data not presented).
We have previously shown that
the upregulation of FasL in SIV infection requires an intact
nef gene (22). In general, the level of cell surface FasL expression is quite low when analyzed by FACS® even when metalloproteinase inhibitors are used which block cleavage of
FasL from the cell surface. In view of this difficulty, we used
additional experimental approaches to analyze Nef-mediated FasL expression (see below). Since stimulation of TCR- effectively upregulates FasL expression (34, 36), we speculated
that the interaction of Nef with TCR-
would lead to a similar effect. First, to show that HIV-Nef is required for FasL
upregulation, we infected Jurkat with wild-type HIV (NL4-3.SF2Nef) or a mutant lacking the nef gene (NL4-3
Nef)
(Fig. 3). Little if any FasL is seen on cells infected with Nef-deleted HIV, thus confirming our previous results with SIV.
The level of viral replication in Jurkat cells was comparable,
as determined by RT activity (NL4-3.SF2Nef, 4.6 × 103
cpm; NL4-3
Nef, 5.8 × 103 cpm). Upregulation of FasL by
HIV is also lost in mutant Jurkat cells lacking the TCR complex, whereas cells reconstituted with
but not with the
mutant restored the FasL expression upon HIV infection as
determined by both immunoprecipitation (Fig. 4 A) and
FACS® analysis (Fig. 4 B). Viral replication assessed by p24
assay indicated that these cell lines were comparably infected
(wild-type, 3.5 ± 0.5; TCR
, 3.8 ± 1.0; CD16
, 3.8 ± 0.5;
CD16
mu, 3.1 ± 0.25 ng/ml).
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To investigate a direct upregulation of FasL by Nef, a
CD8-Nef construct not capable of binding (Nef.PXmu;
see Fig. 2 B) and CD8-Nef were transiently expressed in
Jurkat cells and analyzed for FasL upregulation. CD8-Nef
but not the Nef mutant led to a significant cell surface expression of FasL (Fig. 5). Next, FasL upregulation was studied in Jurkat and TCR mutant cell lines using a FasL promotor/luciferase reporter construct. The latter has been
shown to be stimulated in transient assays by the HTLV I Tax protein (29). Nef stimulated the FasL promotor in Jurkat and TCR
mutant cells reconstituted with the TCR
chain. No effect was seen using the Nef.PXmu construct or
the TCR
Jurkat cell line (Fig. 6). These assays confirmed
that a functional Nef protein and the TCR
chain were
both required and sufficient to upregulate FasL in T cells.
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Discussion |
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In general, the interaction between Fas and FasL plays a
important role in the homeostatic regulation of normal immune responses (37). Stimulation of the TCR-CD3 complex
in T cells causes upregulation of FasL and eventually leads to
AICD or apoptosis (23). A key molecule in this process is
the TCR chain and the three ITAMs contained therein.
Cross-linking of the
chain or constructs containing individual
ITAMs alone were found to be sufficient to induce
T cell activation and Fas-mediated apoptosis (28, 35, 36). In
agreement with these findings, we have shown here that
TCR-
as well as the functional integrity of the ITAM signaling motifs of
were required for HIV-mediated upregulation of FasL. However, these findings further implied that
HIV targets the TCR
chain directly through a viral protein.
To date, several lines of evidence indicated that the Nef
protein exerted such a role. First, Nef-mediated activation
of T cells has been demonstrated in a number of reports (8-
10). Second, expression of Nef in the cytoplasm of T cells
interferes with early T cell signaling events emanating from
the TCR-CD3 complex, including hypophosphorylation
of TCR-, whereas expression of a plasma membrane-
associated form of Nef causes AICD in Jurkat cells (8).
Third, a very aggressive form of Nef from SIV, SIV-YE-Nef, basically functions like an ITAM domain of TCR-
(38). Finally, SIV-induced upregulation of FasL in T cells
depends on the expression of an intact Nef protein (22), and
Nef from a lethal SIV strain (smmPBj14) alone can directly
cause FasL upregulation (39). Thus, it appeared very likely
that Nef acted at the level of the TCR. Indeed, our study
confirms this assumption by showing that Nef can directly
interact with the TCR
chain.
Strong evidence for the interaction of Nef with came
from a second, surprising finding. In Jurkat cells lacking the
TCR, binding of the Nef-associated serine kinase p62/NAK
was abolished. Conversely, reconstitution of these cells with
the
ITAM 2 and 3 restored binding of p62/NAK with Nef.
Furthermore, the integrity of the ITAM motif appeared to be
important, since mutation of the
ITAMs greatly reduced the
effect. As shown previously, the p62/NAK kinase has to be
activated in order to bind to Nef (32). These results suggest a
dynamic interaction of Nef with the
ITAMs, ultimately resulting in the activation of p62/NAK, which in turn binds to
Nef. In view of our and other studies, it is likely that activation
of p62/NAK is part of Nef-mediated stimulation of T cell signaling pathways; however, at this point it is not clear whether
p62/NAK has a role in the Nef-mediated upregulation of
FasL. Notably, Nef binds to p62/NAK in cells lacking a TCR (31; e.g., COS cells). In these cells, the TCR
chain may be functionally replaced by other receptors, possibly containing ITAMs. This would explain why Nef has effects in cells usually not infected by HIV (40; e.g., NIH 3T3 cells).
More recently, Howe et al. showed that Nef from SIV
or HIV-2 associated with the TCR chain but failed to
show an interaction with HIV-1 Nef (41). Our study differs from that of Howe et al. in at least two respects. First
we made constructs to target Nef to the plasma membrane
where the TCR is located. Second, we have established
functional consequence of the Nef-
interation which may
have relevence to the pathogenesis of HIV interaction.
Induction of cell death by HIV could be mediated by different viral proteins. Cross-linking of CD4 by HIVgp120 in
the presence of Tat protein can induce FasL expression and
apoptosis of uninfected T cells (42). Additionally, interaction of HIVgp120 with chemokine receptor CXCR4 on
macrophages leads to death of CD8+ T cells mediated by
TNF-TNFRII interaction (15). In this study, we report an
additional important mechanism of HIV-mediated apoptosis by demonstrating that Nef directly interacts with TCR-
and that both molecules are required for HIV-mediated upregulation of FasL. The interaction between Nef and TCR-
forms a signaling complex, bypassing the requirement for
TCR ligation by antigen, and allowing HIV/SIV to activate
T cells and upregulate FasL expression on the infected cells.
Thus, upregulation of FasL by Nef on HIV- or SIV- infected cells may, like FasL expression at sites of immune privilege and on some tumors, allow infected cells to evade the immune response. In addition, the effect of immune evasion is enhanced by Nef-mediated downregulation of surface MHC class I and CD4 expression (5, 6, 22, 43-46; see Fig. 7). Taken together, our results provide additional insights into the molecular mechanism whereby the HIV accessory protein Nef regulates T cell activity and contributes to the pathogenesis of HIV.
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Footnotes |
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Address correspondence to Andreas S. Baur, Institute of Clinical and Molecular Virology, University of Erlangen/Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany. Phone: 49-9131-852-6784; Fax: 49-9131-852-101; E-mail: asbaur{at}viro.med.uni-erlangen.de
Received for publication 8 February 1999.
X.-N. Xu, B. Laffert, and G.R. Screaton contributed equally to this work.We thank Drs. Tao Dong for help in virus infection and p24 assay, Xiangdong Liu for the pFasL-luc construct, Ralph Grassman for the pCTax construct, and Arthur Weiss for Jurkat mutant cell lines.
This study was supported in part by the Commonwealth AIDS grant (Australia), the Medical Research Council and the Wellcome Trust (UK), and the Deutsche Forschungsgemeinschaft (Germany).
Abbreviations used in this paper aa, amino acid(s); AICD, activation- induced cell death; ITAM, immunoreceptor tyrosine-based activation motif; mu, mutant; NAK, nef-associated kinase; RT, reverse transcriptase; SIV, simian immunodeficiency virus.
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