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Volume 271, Number 11, Issue of March 15, 1996 pp. 6333-6341
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Physical and Functional Interaction of Nef with Lck
HIV-1 Nef-INDUCED T-CELL SIGNALING DEFECTS (*)

(Received for publication, July 5, 1995; and in revised form, November 8, 1995)

Y. Collette (1), (§),  H. Dutartre (1), (¶),  A. Benziane (1),  F. Ramos-Morales (2),  R. Benarous (3),  M. Harris (4),  D. Olive (1)(**)

From the  (1)From INSERM U119, 27 Boulevard Leï Roure, 13009 Marseille, France, (2)Institut Cochin de Genetique Moleculaire (ICGM), U363 INSERM, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France, (3)ICGM, U332 INSERM, 22 Rue Mechain, 75014 Paris, France, and (4)University of Glasgow, Department of Veterinary Pathology, Bearsden, G61 1QH Glasgow, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nef gene is unique to the primate lentiviruses and encodes a cytoplasmic membrane-associated protein that affects T-cell signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell signaling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lck tyrosine kinase both in vitro and in vivo. The molecular basis for this interaction was analyzed. We show that cell-derived Nef is precipitated in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely participants in this interaction. The precipitation of Nef by the Lck recombinant proteins was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase nor phospholipase C SH2 domains coprecipitated Nef from T-cells. Finally, depressed Lck kinase activity resulted from the presence of Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal Lck-mediated signaling events. These results provide a molecular basis for the Nef-induced T-cell signaling defect and its role in AIDS pathogenesis.

INTRODUCTION

HIV infection is associated with a severe CD4+ T-cell depletion. This quantitative defect is preceded by immune qualitative dysfunctions resulting in a profound disturbance of the complex network of cytokines that maintains immune homeostasis, in particular within the Th1-produced cytokines (IL-2, (^1)interferon-). Three HIV-1-encoded proteins exhibit immunosuppressive effects that may, at least partly, account for these T-cell dysfunctions. First, soluble gp120 envelope gene product added to T-cells affects their activation(1) . Second, immunosuppressive effects of Tat have also been reported (2) but may not play an important role in vivo, since these in vitro immunosuppressive functions of Tat are not observed in the presence of accessory cells and Tat did not affect recall antigen-mediated T-cell proliferation(3) . Finally, the altered T-cell activation and development associated with nef transgene expression in mice (4, 5, 6) make this viral gene a candidate for the severe immunodeficiency induced by HIV-1 infection.

nef is an early and abundantly (7) transcribed viral gene conserved in human (HIV-1 and HIV-2) and in simian (SIV) immunodeficiency viruses. nef encodes a 25-32-kDa myristoylated and membrane-associated protein in infected cells(8) . Although its function remains to be defined, in vivo experiments indicate that nef is required for both viral replication and full development of the pathogenesis associated with SIV infection of rhesus monkeys (9) or HIV-1 infection in SCID-Hu mouse (10) . In contrast to these in vivo data, nef was initially described as a negative regulator of the viral replication (11, 12, 13, 14, 15) , but this observation remains controversial as other reports demonstrated a lack of effect (16, 17, 18) or a moderate positive effect (19, 20) on rates of viral transcription or replication. More recently, nef was shown to contribute to the induction of viral replication in primary quiescent T-cells, its positive role being readily discernible in the primary cell setting of virus induction through T-cell activation(21, 22) . A known consequence of nef expression is CD4 membrane down-regulation(23, 24, 25, 26) , which has been proposed to prevent cell superinfection(27) . This down-regulation occurs at the post-translational level (23, 25) and might involve either a critical dileucine motif (28) or the Src family protein-tyrosine kinase Lck binding site in CD4(29) . However, the precise mechanism by which Nef down-modulates CD4 remains unclear. Recent studies provide further evidence that HIV-1 nef gene function is closely related to T-cell signaling pathways as demonstrated by its ability to affect T-cell activation in nef-transgenic mice(4, 5, 6) , to inhibit growth of CD4+ T lymphocytes(30) , and to down-regulate both IL-2 induction (31) (^2)and activation of NF-kappaB and AP-1 (32, 33, 34) transcription factors in human T-cells. Nef appears to exert different effects on T-cell signaling, depending on its intracellular localization(29) , and has been reported to associate with various cellular proteins from T-cell lysates(35) , notably serine/threonine protein kinases (36) and beta-COP(37) , an essential component of the molecular machinery of the membrane trafficking. These molecular interactions of Nef with different families of cellular proteins indicate that Nef contains at least one domain that can mediate protein-protein interactions. Indeed, a proline-rich motif has been identified within Nef (38) that allows binding to the SH3 domain from the Src family protein-tyrosine kinases Hck and Lyn and is required for enhanced growth of nef+ viruses in monocytes(39) . However, as the expression of these proteins is mainly restricted to monocytes, the Nef-Hck interaction does not account for the Nef-induced T-cell signaling defects.

The Src family of protein-tyrosine kinases comprises nine identified members (Src, Lck, Fyn, Yes, Blk, Fgr, Lyn, Hck, and Yrk), defined by the presence of the catalytic domain, Src homology domain 1 (SH1), a specific phosphotyrosine residue binding domain (SH2), a proline-rich binding region (SH3), and a myristoylated membrane-targeting domain (SH4)(40, 41, 42) . Importantly, the T-cell-specific Lck product binds to the CD4 cytoplasmic tail(43, 44) , hence regulating its cell surface expression by a post-translational mechanism(45) , and is also involved in T-cell activation(46, 47, 48) , IL-2 induction(49) , thymic development (50) , and HIV-1 expression(51) .

Here, we describe the physical and functional interaction of HIV-1 Nef with Lck in human T-cells and show that this interaction is associated with impaired Lck kinase activity as well as with defects in both proximal and distal signaling events mediated by Lck. Our results provide a molecular basis by which Nef affects T-cell functions and also possibly CD4 cell surface expression and viral growth.

MATERIALS AND METHODS

Antibodies, Recombinant Fusion Proteins, and Chemicals

MATG0020 is a monoclonal antibody provided by Transgene (Strasbourg), which recognizes the 161-175 amino acid residues from HIV-1 nefBru. Anti-Nef sheep polyclonal serum was also developed by immunization with the Nef-GST recombinant protein (see below). The CD4 monoclonal antibody (13B8.2) was developed in the laboratory(72) . Anti-phosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and anti-Lck(45) , anti-CD4, and anti-GST polyclonal antibodies were a kind gift from M. Marsh (Medical Research Council (MRC) Laboratory, London), Q. Sattentau (Centre D'Immunologie de Marseille-Luminy (CIML), Marseille), and J. P. Borg (U119 INSERM, Marseille), respectively.

GST-Lck and -Csk fusion proteins were a kind gift from P. Jullien and C. Bougéret (ICGM, Paris), and p50NF-kappaB was kindly provided by P. Lecine (U119 INSERM, Marseille). The GST-Nef and Nef production was described previously (35, 57) as well as that of the GST-Lck SH2, GST-Lck SH3, GST-Lck SH2+SH3, GST-Fyn SH2, GST-phospholipase C SH2, and phosphatidylinositol 3-OH-kinase SH2(C)(73, 74, 75) .

Nef peptides produced by Neosystem S.A. (Strasbourg, France) and distributed by ANRS were coupled to activated agarose beads (Steragene) at a ratio of 1 mg of peptide/ml of activated beads according to instructions provided by the manufacturer. PMA and Ionomycin were purchased from Sigma. Peptides Glu-Pro-Gln-Tyr(P)-Glu-Glu-Ile-Pro-Ile and Glu-Pro-Gln-Tyr-Glu-Glu-Ile-Pro-Ile were kindly provided by O. Acuto (Institut Pasteur, Paris, France).

Cells and Cell Culture

The Jurkat cell line JH6.2 was described previously (76) and was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. JBru.2 was obtained by stable transfection of JH6.2 with the HIV-1 Bru/Laï-derived nef gene as described previously(77) .^2

Preparation of Cell Extracts

For total protein extraction, cultured cells were lysed for 15 min at 4 °C as indicated, in 1% (v/v) Triton X-100, 25 mM Hepes-NaOH, pH 7.8, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM vanadate, or lysed in 0.5% (w/v) Brij 96, 25 mM Hepes-NaOH, pH 7.8, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM vanadate. After lysis, cell extracts were centrifuged for 10 min at 4 °C, and postnuclear supernatant was further processed for analysis.

Biochemical Assays

Immunoprecipitations, immunoblotting, and kinase assays were performed as described previously(73, 74, 77) . Briefly, cell extracts were precipitated either by use of recombinant fusion proteins immobilized on agarose beads or by use of antibodies subsequently harvested with appropriate Sepharose beads (Protein A or G, Pharmacia Biotech Inc.). Precipitated proteins were then extensively washed in lysis buffer and fractionated by SDS-PAGE as indicated. To analyze the CD4-associated Lck kinase activity, the previously described technique from Biffen et al.(56) was used. Briefly, magnetic beads (Immunotech) were bound to goat anti-mouse IgG (Immunotech) and incubated with CD4 monoclonal antibody (13B8.2) as recommended by the manufacturer. After washing in culture medium (RPMI), the preformed magnetic beads-IgG-CD4 monoclonal antibody conjugates were added to cell suspension (2.10^7/4 ml of RPMI, 20% fetal calf serum) and rotated on a wheel for 15 min at 4 °C. Cells were collected using a magnet, washed, and then lysed in ice-cold lysis buffer.

For immunoblotting, fractionated proteins were transferred to polyvinylidene difluoride membranes (Millipore), and filters were blocked for 2 h at room temperature in 5% bovine serum albumin (Sigma) in phosphate-buffered saline containing 0.01% Tween 20 detergent. Filters were successively incubated for 1 h at room temperature with appropriate primary and secondary antibodies. Each incubation period was followed by three washes in phosphate-buffered saline with 0.01% Tween 20. Proteins were finally detected by enhanced chemiluminescence following instructions of the manufacturer (Amersham Corp.).

For kinase assays, precipitates were further washed in kinase buffer (50 mM Tris-Cl, pH 7.4; 10 mM MnCl(2)) and resuspended in 25 µl of this buffer. For autophosphorylation analysis, the reaction was processed in the presence of 1 µCi of [-P]ATP (5000 Ci/mmol, ICN) for 15 min at room temperature and stopped by the addition of SDS-PAGE reducing sample buffer. To analyze for the phosphorylation of exogenous substrates, experiments were identically performed except that 5 µg of acid-denatured enolase and 3 µM of unlabeled ATP were added. Phosphorylated proteins were then analyzed by SDS-PAGE and autoradiography. Integrated signal intensity of phosphorylated proteins was determined by use of the BioImage system (Millipore Corp.).

For determination of CAT activity, cells were harvested, washed in phosphate-buffered saline, and resuspended in lysis buffer (0, 25 M Tris-Cl, pH 7.8), and proteins were extracted by successive freezing/thawing cycles. After centrifugation of the lysates, supernatants were collected, and the protein concentration was determined by the Bradford method (Bio-Rad). CAT enzyme activity present in the sample was then determined as described previously(78) , using an equivalent amount of total protein. Reaction products were analyzed by thin-layer chromatography followed by autoradiography and determination of integrated signal intensity by use of the BioImage system (Millipore). CAT activity was expressed as the ratio of radioactivity present in the acetylated forms of chloramphenicol to the sum of both acetylated and unacetylated forms.

RESULTS

Specific Precipitation of Nef by both Isolated Recombinant Lck SH2 and SH3 Domains

It was recently reported that a GST-Nef recombinant protein could precipitate the Lck tyrosine kinase from human T-cells(54) . To gain insight into the possible interaction of Nef with Lck, we first determine whether the SH2 and SH3 domains from Lck mediated interaction with cell-derived Nef. Lysates from JBru.2, a cellular clone derived from stably nef-transfected lymphoid Jurkat cells(52) ,^2 were precipitated with a GST-Lck SH2+SH3 fusion protein and immunoblotted with a Nef monoclonal antibody. Expression of Nef in the JBru.2 cells is under the control of the hCMV promotor regions and is very low but can be up-regulated in presence of PMA, which tightly controls the transcriptional activity of this promotor(52, 53) .^2 A Nef-immunoreactive polypeptide was specifically detected in precipitates from PMA-treated JBru.2 cells (Fig. 1A, lane 4) but not from similarly treated untransfected cells (lane 3). As expected, a lower amount of the Nef-immunoreactive band was detected in precipitates from untreated JBru.2 cells (lane 5). In contrast, GST-beads were found to be unable to precipitate Nef (data not shown). Similar results were obtained by use of various representative stably nef-transfected clones and also upon transient transfection of nef (data not shown). To discriminate between SH2- and SH3-mediated binding to Nef, cell lysates were precipitated with either GST-Lck SH2 or Lck SH3 fusion proteins and compared with GST-Lck SH2+SH3 precipitates (Fig. 1B). Both GST-Lck SH2 and GST-Lck SH3 were found to precipitate Nef (0.5 and 2%, respectively, of the Nef protein present in total cell lysates) and to cooperate in a synergistic manner, as SH2 increased by 18-fold the efficiency of GST-Lck SH3 recombinant protein to precipitate Nef. The specificity of this precipitation of Nef by GST-Lck recombinant proteins was determined by Nef immunoblotting of precipitates obtained by use of recombinant proteins that contained SH2 or SH2+SH3 domains from the Src tyrosine kinase Fyn, the non-Src tyrosine kinase Csk, and the unrelated proteins phospholipase C, phosphatidylinositol 3-OH-kinase (Fig. 2B), and Grb2 (not shown). Strikingly, only Lck isolated domains allowed significant precipitation of cell-derived Nef protein.

Figure 1: Specific precipitation of Nef by immobilized GST-Lck-SH2+SH3, GST-Lck-SH3, and GST-Lck-SH2 recombinant proteins. A, Jurkat and JBru.2 T-cell lysates (10^7 cell equivalent in 500 µl) were precipitated with immobilized GST-Lck SH2+SH3 fusion protein (10 µg of recombinant protein recoupled to glutathione-agarose beads). Precipitates were fractionated on a 12% SDS-PAGE, followed by immunoblotting with the Nef monoclonal antibody and enhanced chemiluminescence. Cells were left uninduced or induced by PMA (15 ng/ml) and ionomycin (0.5 µg/ml) to increase nef expression, and whole cell lysates from Jurkat (lane 1) and JBru.2 (lane 2) induced cells were included as control. Lane 3, induced Jurkat cells; Lanes 4 and 5, respectively, induced and uninduced JBru.2 cells. B and C, as A with the exception that lysates from induced cells were precipitated with the indicated GST-Lck (B) or GST-Lck, GST-Fyn, GST-Csk, GST-p85, or GST-phospholipase C (C) recombinant proteins. WCL, whole cell lysate; AU, arbitrary units.


Figure 2: HIV-1 Nef is tyrosine-phosphorylated. A, JBru.2 cell lysates were precipitated by the GST-Lck SH2 recombinant protein as described in Fig. 1, except that 5 µM unphosphorylated (YEEI, lane 3) or tyrosine-phosphorylated (pYEEI, lane 4) EPQYEEIPI peptide was added during the precipitation step. Precipitates were subsequently fractionated by SDS-PAGE and analyzed by Nef immunoblotting. Whole cell lysates from Jurkat and JBru.2 cells were added as control (lanes 1 and 2, respectively). B, lysates from induced Jurkat(-) and JBru.2 (+) cells were precipitated by use of GST-Lck SH2+SH3 recombinant protein, separated by SDS-PAGE and analyzed by Nef immunoblotting (left panel) followed by phosphotyrosine immunoblotting (right panel). Whole cell lysates were added as control where indicated. C, cells were either left unstimulated(-) or stimulated (+) in the presence of PMA (20 ng/ml) for 15 h to increase nef expression. Cell lysates (4 times 10^7 cells equivalent) were immunoprecipitated with a Nef polyclonal sheep antiserum, and analyzed by anti-phosphotyrosine immunoblotting (left panel). After stripping, the same membrane was also probed with a Nef monoclonal antibody (right panel). Lane 1, stimulated Jurkat cells; lanes 2 and 3: unstimulated and stimulated JBru.2 cells, respectively. PTYR, anti-phosphotyrosine; Ig, light chain immunoglobulins.


Nef is Tyrosine-phosphorylated and Contains an SH3-binding Domain

SH2 domains interact specifically with amino acid motifs containing phosphorylated tyrosine residues. To test the hypothesis that tyrosine phosphorylation of Nef could be involved in the interaction with Lck SH2, cell lysates were precipitated with GST-Lck SH2 recombinant protein in the presence of the phosphopeptide (EPQpYEEIPI) containing the sequence predicted to be optimal for binding to the Lck SH2 domain(60) . The precipitation of Nef was specifically inhibited in the presence of 5 µM concentration of this phosphopeptide as compared with its unphosphorylated counterpart (Fig. 2A, lanes 3-4). Similar results were obtained in a dose-dependent manner upon precipitation of Nef by the Lck SH2+SH3 recombinant protein (data not shown). The tyrosine phosphorylation of Nef was further investigated by probing Lck SH2+SH3 precipitates by anti-phosphotyrosine immunoblotting (Fig. 2B). An immunoreactive product specifically precipitated from Nef-transfected cells was evidenced (right panel), which at least partly corresponded to Nef as indicated by reprobing the filter with Nef antibodies (left panel). Finally, the tyrosine phosphorylation of Nef was verified by direct immunoprecipitation with the Nef antibody and anti-phosphotyrosine immunoblotting (Fig. 2C). Indeed, a significant signal was detected in immunoprecipitates from induced nef-expressing cells (Fig. 2C, left panel) that coincided with the Nef-specific band detected after stripping and reprobing of the membrane with a Nef monoclonal antibody (Fig. 2C, right panel).

The binding of the GST-Lck SH3 fusion protein to Nef suggested that Nef might contain a proline-rich domain that interacted with the Lck SH3 domain. Fig. 3A identifies a proline-rich domain that matches the consensus proline-rich motif defined by Yu et al.(71) and present in the nefBru/Laï primary sequence (residues 68-78) but also in various HIV-1, HIV-2, and SIV nef isolates. Interestingly, this motif corresponds to the prototypic class II proline-rich motif with the consensus sequence Pro-X-q-Pro-X-Arg, where X represents any amino acid residue and q represents a hydrophobic residue. To determine whether this motif of Nef mediated interaction with Lck SH3 domain, a peptide encompassing HIV-1 NefBru residues 66-80 was directly coupled to activated agarose beads and used to precipitate GST fusion proteins (Fig. 3B). This proline-rich domain of Nef precipitated the GST-Lck SH2+SH3 fusion protein as efficiently as glutathione-agarose beads (Fig. 3B, compare lanes 2 and 5). Similarly, GST-Lck SH3 fusion protein could be efficiently precipitated (Fig. 3B, lanes 3 and 6), whereas GST-Lck SH2 was not (Fig. 3B, lanes 1 and 4). This peptide accounts for most of Nef binding to GST-Lck SH3 recombinant protein in vitro, since peptides encompassing residues 34-71 or 137-168 of Nef were poorly or less efficient, respectively, as compared with peptide encompassing residues 66-100 (Fig. 3C). Nef interacted specifically with SH3 domains, since the GST recombinant protein was not precipitated (Fig. 3D) and also because a gradual affinity for Src-like derived SH3 domains, in particular for Lck, was observed (Fig. 3D).

Figure 3: Nef contains a functional Lck SH3 binding domain. A, sequence alignment of HIV-1 Bru/Laï(70) , HIV-1 consensus(38) , HIV-2 consensus(38) , and SIV consensus (38) reveals the presence of a consensus (71) proline-rich domain in Nef that corresponds to a class II motif (-Pro-X--Pro-X-Arg). Position numbering is indicated on the left of each sequence. The single letter amino acid code is used, X represents nonconserved residues, represents hydrophobic residues, and P represents residues that are likely to be proline. B, a Nef peptide encompassing amino acid residues 66-80 from HIV-1 Bru/Laï Nef was immobilized on agarose beads (1 mg/ml). 10 µg of peptide equivalent were then incubated for 2 h at 4 °C in 500 µl of lysis buffer with 2 µg of soluble GST-Lck SH2, GST-Lck SH2+SH3, and GST-Lck SH3 fusion proteins. Precipitates were extensively washed and subsequently fractionated by 10% SDS-PAGE. Precipitated proteins were visualized by Coomassie Blue staining. The various GST-recombinant proteins were also precipitated by use of glutathione-agarose beads (GA-beads) as a loading control. C, peptides encompassing the indicated amino acid residues from HIV-1 NefBru were immobilized on agarose beads and used to precipitate soluble GST-Lck SH3 recombinant protein as described in B. Signal intensities were determined by use of the BioImage system (Millipore), and results are presented as percentage of maximal binding determined for the Nef peptide encompassing residues 66-80. D, as B and C except that immobilized Nef peptide (amino acids 66-80) was used to precipitate the indicated soluble GST recombinant proteins. Results, determined as in C, are expressed as arbitrary units of optical density.


Nef Binds and Modulates Lck in Vitro

To examine the possibility of a direct interaction of Nef with full-length Lck, we investigated by in vitro kinase assay whether immobilized recombinant Nef protein could precipitate active recombinant Lck. Indeed, significant kinase activity was detected in Nef precipitates (Fig. 4A, lane 2) that co-migrated with autophosphorylated GST-Lck (Fig. 4A, lane 1). Specificity of GST-Lck binding to Nef was verified by the absence of activity in precipitates obtained with an irrelevant protein (p50NF-kappaB) incubated with Lck-GST (Fig. 4A, lane 3). When precipitates obtained by use of immobilized Nef were probed by immunoblotting, co-precipitated recombinant GST-Lck was detected both by anti-Lck (Fig. 4B, upper panel, lanes 4 and 5) and anti-GST antibodies (lower panel), whereas the GST recombinant protein used as a control was not detected (Fig. 4B, lanes 2 and 3). Conversely, as evidenced by Nef immunoblotting, anti-Lck antibodies allowed immunoprecipitation of Nef recombinant protein using the GST-Lck fusion protein as an intermediate substrate (Fig. 4B, lane 6). Also, recombinant GST-Nef could precipitate cell-derived Lck produced in insect cells infected by recombinant baculovirus(83) .

Figure 4: In vitro physical and functional interaction of HIV-1 Nef with Lck. A, kinase-active recombinant GST-Lck fusion protein (100 ng) was incubated in 1% Brij 96 buffer for 2 h at 4 °C with 1 µg of either Nef or p50NF-kappaB immobilized on Ni-agarose beads. After precipitation and extensive washing, kinase activity that precipitated with immobilized recombinant proteins was determined by in vitro kinase assay in the presence of [-P]ATP followed by SDS-PAGE fractionation and autoradiography. The GST-Lck and GST recombinant proteins were directly tested as controls (Lanes 1 and 4, respectively). Lane 2, Nef precipitates; lane 3, p50NF-kappaB precipitates. B, as A, except that precipitates were analyzed by immunoblotting as indicated. Immobilized Nef beads were used to precipitate different amounts of GST or GST-Lck recombinant proteins (lanes 1-5 ), and precipitates were separated by SDS-PAGE and subsequently analyzed by Lck immunoblotting (upper left panel, lanes 1-5). Filters were then stripped and reprobed by GST immunoblotting (lower left panel, lanes 1-5). Conversely, soluble Nef or p50NF-kappaB recombinant proteins were incubated in Brij 96 buffer with the GST-Lck fusion protein and immunoprecipitated by use of an Lck polyclonal antibody directed against the unique region of Lck (lanes 6-7). SDS-PAGE-fractionated proteins were probed by Nef immunoblotting (upper right panel, lanes 6-7), and after stripping of the filter, by GST immunoblotting (lower right panel, lanes 6-7). GST-Lck recombinant protein was directly loaded as a control (Lane 1). Lanes 2 and 3, 0.2 and 1 µg of GST protein, respectively; lanes 4 and 5, 0.2 and 1 µg GST-Lck fusion protein, respectively; lane 6, p50NF-kappaB recombinant protein; lane 7, Nef recombinant protein. Note that the Nef protein migrates in SDS-PAGE with an apparent molecular mass of 33-34 kDa according to the addition of the six histidine residues to the Nef primary sequences. C, GST-Lck or GST recombinant proteins were incubated with Nef or p50Rel as indicated and in identical conditions as described for A, except that kinase activity present in each sample was directly determined by in vitro kinase assay without precipitation.


The GST-Lck kinase activity precipitated by immobilized-Nef was very weak, suggesting that Lck might be affected by Nef. To test this hypothesis, GST-Lck was incubated with soluble Nef and simultaneously assayed by in vitro kinase assay. A 9-fold reduction of recombinant Lck kinase activity was found in the presence of Nef relative to the GST-Lck activity determined in the presence of p50NF-kappaB (Fig. 4C, compare lanes 3 and 5). This effect was dose-dependent (IC = 585 ng), the range of specificity being comprised between 0 and 1 µg of Nef recombinant protein, and was prevented by the addition of a Nef monoclonal antibody (data not shown). Interestingly, an Lck-independent phosphorylated band corresponding to Nef was also detected (Fig. 4C, lanes 4 and 5). This band may result from the previously reported autophosphorylation activity of Nef(55) , although conflicting results have been described about such activity(79) . Alternatively, this band may be due to phosphorylation of Nef by a cellular protein kinase of SF9 cells, which binds to and copurifies with Nef.

Interaction of Nef with Lck in Intact Cells

When total Lck was immunoprecipitated and analyzed for kinase activity, a similar level of Lck autophosphorylation was observed in nef transfected cells as compared with control cells (data not shown). It has been recently reported that the activity of CD4-associated Lck is regulated by CD45, although total Lck derived from whole cell lysates is not(56) . As Nef associates with cytoplasmic membranes and down-modulates CD4, we hypothesized that by analogy to the different regulation of Lck by CD45, Nef might differently regulate total and CD4-associated Lck kinase activity. Cross-linked CD4 antibodies coupled to magnetic beads were used to purify CD4+ cells, and subsequently, purified cells were lysed. Immunocomplexes formed of the magnetic bead-linked immunoglobulins, the CD4 receptor, and the associated proteins, were harvested, washed, and analyzed both by immunoblotting and in vitro kinase assay (Fig. 5A). Using this technique, similar amounts of CD4-Lck complex were precipitated from nef-transfected and control cells, as evidenced by immunoblotting with the CD4 and Lck antiserum (Fig. 5A, upper and middle panels, respectively), hence indicating that association of Lck with membrane CD4 was not affected by Nef under these experimental conditions. However, determination of the CD4-associated kinase activity clearly revealed that both CD4-associated Lck kinase activity toward an exogenous substrate (Fig. 5A, lower panel) and Lck autophosphorylation activity (Fig. 5B, lower panel) were reduced by 45-60% respectively, in nef-expressing cells. Cytofluorometric determinations demonstrated that CD45 levels were unaffected by nef expression, indicating that altered Lck activity in these cells was not the result of an up- or down-modulation of the phosphatase (data not shown).

Figure 5: In vivo physical and functional interaction of HIV-1 Nef with Lck. A and B, cells (5.10^6 cells/ml) were incubated for 15 min at 4 °C with magnetic beads coated with CD4 receptor-specific monoclonal antibodies. Bound cells were sorted with a magnet and disrupted in Brij 96 lysis buffer. Immunocomplexes were harvested by centrifugation, washed, and analyzed by CD4 and Lck immunoblotting or by in vitro kinase assay, as indicated. Cell surface CD4-associated Lck kinase activity was evaluated by the phosphorylation of the exogenous substrate enolase (A) or Lck auto-phosphorylation (B). Lane 1, JH6.2 total cell lysate; Lanes 2 and 3, JH6.2 and JBru.2 cells, respectively. Integrated intensity of each signals was determined by use of BioImage system (Millipore). C, cells were either left unstimulated or stimulated by PMA and ionomycin to increase nef expression, and lysed in Brij 96 buffer. 5 times 10^7 cells equivalent were immunoprecipitated with the p56 Lck polyclonal antibody, fractionated on a 11% SDS-PAGE, and immunoblotted with the Nef monoclonal antibody. Lane 1, stimulated JH6.2 cells; lanes 2 and 3, unstimulated and stimulated JBru.2 cells, respectively; lane 4, JBru.2 whole cell lysates (10^6 cell equivalent). Ig, immunoglobulins from anti-Lck polyclonal antibody; KA, kinase assay.


The interaction of Nef with Lck was further investigated by immunoprecipitation of cell-derived Lck from JBru.2 cells and Nef immunoblotting (Fig. 5C). A 29-kDa polypeptide co-migrating with Nef from whole cell lysates was detected in immunoprecipitates from induced nef-expressing cells (Fig. 5C, lane 3) and was absent in control cells (lane 1) but also barely undetectable in uninduced JBru.2 cells (lane 2). Densitometric determination indicated that this signal represented at least 2% of Nef protein present in whole cell lysate (lane 4).

Nef Affects Lck-mediated Proximal and Late Signaling Events

Lysates from unstimulated and stimulated cells (PMA + ionomycin) were precipitated with GST-Lck SH2 fusion protein and analyzed by phosphotyrosine immunoblotting (Fig. 6). At least three different anti-phosphotyrosine immunoreactive polypeptides were precipitated from unstimulated cells (Fig. 6, lane 1). Interestingly, the binding or tyrosine phosphorylation of a 55-60-kDa phosphorylated protein (designated p56 in Fig. 6) was specifically reduced by 76% in nef-transfected cells (compare lanes 1 and 2). Upon stimulation of the cells by PMA and ionomycin for 5 h to increase the nef expression level, both increased tyrosine phosphorylation of these proteins and additional induced tyrosine-phosphorylated proteins were evidenced following precipitation by the GST-Lck SH2 fusion protein (Fig. 6, lanes 3 and 4). The binding or tyrosine phosphorylation of 80-, 70-, 56-, 48-, and 38-kDa proteins was specifically altered by a range of 98-20% in nef-transfected cells (Fig. 6, compare lanes 3 and 4). We then investigated whether late T-cell signaling events known to be dependent on Lck activity were affected in nef-expressing cells. The human IL-2 promoter regions containing the minimal -326 base pair promoter fused to the CAT reporter gene (pIL-2-CAT) was co-transfected in Jurkat cells with the PCDNAI Neo vector containing the nef gene either in antisense orientation (CMV-fen) or sense orientation (CMV-nef). The CMV-fen construct was used as a control, as the primary nef sequence overlaps the 3` long terminal repeat U3 region, and thus contains multiple transcription factor binding sites that may titrate out transcription factors implicated in IL-2 promoter induction. In cells transfected by CMV-fen, the pIL-2-driven CAT expression was induced 84-fold, while upon nef expression, this expression only reached a 10-fold increase (Table 1). When both CMV-fen and CMV-nef constructs were co-transfected with the CMV promoter fused to a rat CD2 truncated reporter gene, the truncated CD2 molecule expression was found similarly induced 16-18-fold, independently of nef expression (Table 1), hence indicating the specificity of the nef-mediated block to IL-2 promoter induction. Finally, both IL-2 promoter transcriptional activity and IL-2 secretion were found inversely correlated with nef expression in various nef stably transfected clones (52) .^2

Figure 6: Binding of GST-Lck SH2 fusion protein to phosphotyrosine-containing proteins from nef-expressing T-cells. Whole cell lysates from uninduced and PMA + ionomycin-induced JH6.2 and JBru.2 cells were obtained as described in the legend top Fig. 1and then precipitated with the GST-Lck SH2 recombinant fusion protein. After extensive washing, bound tyrosine-phosphorylated proteins were fractionated by 12% SDS-PAGE and analyzed by anti-phosphotyrosine immunoblotting. Lanes 1 and 2, unstimulated cells; lanes 3 and 4, induced cells.


DISCUSSION

HIV-1 Nef interacts with various cellular proteins(35, 36, 37, 54) , hence indicating the presence, within its primary sequence, of domains that direct protein-protein interactions. Indeed, Nef contains a proline-rich region (38, 39) that corresponds to the minimal consensus motif required for interaction with SH3 domains. The importance of this domain was outlined by the recent finding that mutations of the proline residues within this motif abolish the ability of Nef to enhance viral growth in infected peripheral blood mononuclear cell cultures(39) . Also, the proline-rich motif of Nef was shown to interact with Hck (39) , an Src-like tyrosine kinase whose expression is restricted to monocytes. While cells from the monocytic lineage clearly represent an important reservoir for HIV-1 infection, CD4+ T lymphocytes also constitute major targets for this virus. The interaction of Nef with monocyte-restricted Src-like tyrosine kinases suggested the possible interaction of Nef, in T-cells, with another Src-like tyrosine kinase, and in particular with the T-cell-restricted Lck. During the preparation of this manuscript, Greenway et al.(54) reported that recombinant GST-Nef fusion protein could precipitate Lck and CD4 from Jurkat T-cells. Also, when co-expressed in insect Sf9 cells, Nef and CD4 produced by recombinant baculoviruses could be co-immunoprecipitated(57) . These observations thus raised the following questions. (i) Does Nef bind directly to Lck, and does this interaction occur in vivo? (ii) If so, what are the molecular bases for this interaction? (iii) What are the consequences of the binding of Nef to Lck?

In this report we demonstrate that cell-derived Nef co-precipitates with recombinant GST-Lck fusion proteins. Conversely, an immobilized peptide encompassing the proline-rich motif of Nef precipitates the GST-Lck SH3 fusion protein. We also present evidence for an interaction between full-length recombinant Nef and Lck proteins. Together, these observations argue for the direct binding of Nef to Lck in vitro. The Nef-Lck interaction also occurs in intact cells as demonstrated in nef-transfected cells both by the co-immunoprecipitation of Nef with Lck and the down-regulation of the CD4-associated Lck kinase activity.

Using recombinant GST fusion proteins, we found that the Nef-Lck interaction involves both SH2 and SH3 domains of Lck, in a synergistic manner. The proline-rich motif identified within Nef was initially proposed to bind selectively Hck and Lyn, in a filter binding assay, and the Nef-PXXP peptide bound neither to Lck nor to Fyn SH3 recombinant proteins under these experimental conditions(39) . Using a similar assay, we confirmed these observations (data not shown). However, we observed the precipitation of cell-derived Nef by the GST-Lck SH3 fusion protein, and conversely, a peptide encompassing the proline-rich domain of Nef allowed the precipitation of soluble GST-Lck SH3 recombinant protein. The involvement of SH3 binding to Nef was further supported by the severalfold increased precipitation of Nef by GST Lck SH2+SH3 recombinant protein as compared to Nef precipitation by either SH2 or SH3 isolated domains. Thus, although the proline-rich motif of Nef might display a higher affinity for Hck and Lyn SH3 domains in filter binding assays(39) , it clearly cooperates with additional domains within Nef to allow its binding to the Lck SH2-SH3 recombinant protein. This cooperation presumably resulted from the previously reported coordinated interplay between SH2 and SH3 domains of Src family kinases(58, 59) . Indeed, occupancy of one domain may influence accessibility of the other(58) . Similarly, the binding of Tip, a Herpesvirus saimiri product that associates with Lck (80) was recently shown to contain at least two Lck-binding motifs(81) . One of these motifs is a proline-rich domain similar to that of Nef and is required but not sufficient for the binding of Tip to Lck(81) . SH2 domains are implicated in mediating protein-protein interactions by binding to tyrosine-phosphorylated proteins(60, 61) . Interestingly, we observed that the specific precipitation of Nef by the GST-Lck SH2 recombinant protein, that was prevented by the presence of a tyrosine-phosphorylated specific peptide substrate of Lck SH2. Together with the anti-phosphotyrosine immunoreactivity of Nef immunoprecipitates, these results strongly argue for the in vivo phosphorylation of Nef on tyrosine residue(s) and for its involvement in Lck SH2 binding. Densitometric determination evidenced that the GST-Lck SH2 domain precipitated at least 2% of the Nef protein present in whole cell lysates, hence indicating the proportion of in vivo tyrosine-phosphorylated Nef. Recently, nef alleles products from SIV viruses were similarly found to be phosphorylated on tyrosine residue(s) when co-expressed with Src in COS-1 cells(82) . The tyrosine residue(s) proposed to be phosphorylated in SIV Nef are not present, however, in HIV-1, suggesting that although the nef gene from these viruses might have similar functions, different mechanisms have been selected in order to reach it. Inspection of the HIV-1 NefBru primary sequence identified seven tyrosine residues, among which at least five are conserved features of different HIV-1 isolates, but also of HIV-2 and SIV Nef proteins. The optimal binding sequence of Lck SH2 domains was determined by use of a random library of tyrosine-phosphorylated peptide and defined as pYEEI(60) . Such a consensus Lck-SH2 binding domain is not present in Nef. However, the possibility cannot be excluded that Nef contains another phosphotyrosine-containing motif that acts as such a substrate for Lck, as previously reported for c-Src SH2 binding to platelet-derived growth factor receptor, or Lck binding to ZAP-70 kinase(62) . Absence of significant precipitation of Nef by phosphatidylinositol 3-OH-kinase p85, phospholipase C, and even by Fyn SH2 domains, the latter belonging to the same tyrosine kinase family as Lck, indicate the high specificity of this Nef-phosphotyrosine for Lck. The kinase activity of the Src family kinases is repressed when the carboxyl terminus tyrosine residue (Tyr in Lck) is phosphorylated(40, 41, 42, 59) . This phosphorylation creates a binding site for the SH2 domain and results in intramolecular interactions that are thought to lock the protein in an inactive conformation. Among the various conserved tyrosine residues identified within Nef, residue Tyr (YXPXP) shares an intriguing similarity with the regulatory Tyr motif from Lck (YXPXP). Strikingly, the YXPXP motif is uniquely shared by Lck, while Fyn and Src have a shorter motif (YXP) also found in Nef (Tyr). The respective contribution of these various tyrosine residues in both Nef phosphorylation and binding to Lck is currently investigated.

Triggering of the T-cell receptor results in the tyrosine phosphorylation of many cytoplasmic and membrane effectors that appear to play an important role in the transcriptional activation of IL-2. Tyrosine phosphorylation is an obligatory event for IL-2 production (63) , and various protein tyrosine kinases have been identified in this process, including Lck (for a review, see (64) ). Interestingly, the tyrosine phosphorylation of several proteins upon TcR triggering is selectively affected in cells expressing a CD8-Nef chimeric protein (29) . In particular, the tyrosine phosphorylation of p36, p48, and at least two additional proteins with molecular mass in the range of 70-80 KDa were reported to be specific targets of Nef. Identification of these cellular proteins is critical in the outstanding comprehension of Nef-mediated T-cell defects. Here, we identified several Nef-sensitive cellular tyrosine-phosphorylated proteins as substrates of Lck SH2 domain. The defective tyrosine phosphorylation pattern observed in the present report by use of the recombinant GST-Lck SH2 fusion protein is very similar to that identified in the CD8-Nef expressing cells. Together with the observation that the transcriptional induction of an IL-2 promoter reporter construct is specifically decreased 8-fold in nef transfected cells, these results suggest a role for the Nef-Lck interaction in perturbation of both tyrosine phosphorylation and IL-2 induction. The function of Lck in T-cell signaling implicates both its catalytic activity and binding to tyrosine-phosphorylated substrate through its SH2 domain(65, 66, 67) . In particular, the CD4-associated Lck is thought to play an important role upon the MHC-restricted recognition of antigenic peptide by the TcR(68) . Here, we found that Nef affects the CD4-associated Lck kinase activity purified from nef transfected cells by more than 50% and decreased the kinase activity of recombinant Lck in vitro 9-fold. As both intact SH2 and SH3 domains are required for Lck catalytic activity, we propose a mechanism by which the impaired Lck kinase activity induced by Nef might result from an inappropriate folding of the kinase in the Lck-Nef complex or from an active repression mediated by occupancy of both SH2 and SH3 domains by Nef. As a consequence, the tyrosine phosphorylation of Lck substrates would be affected (see Fig. 7for the proposed model).

Figure 7: Interaction of Nef and Lck. A general model is proposed that involves multiple consequences on cell functions and viral replication.


The multiple role played by Lck in T-cells suggests foreseeable consequences of the Nef-Lck interaction both on T-cell functions and viral replication. The turnover of CD4 membrane expression is highly dependent on Lck interaction(45) . It is tempting to speculate that the interaction of Nef with Lck might influence the rate of CD4 internalization. Indeed, the Lck binding domain within CD4 is required for Nef-mediated CD4 down-regulation(69) . However, while the Nef-Lck interaction might at least participate in CD4 down-modulation in Lck-positive cells, the molecular basis of CD4 down-regulation by Nef in Lck-negative cells remains to be elucidated. The CD4 cytoplasmic domain also appears to play a critical role during the early stages of HIV infection(51) . The CD4-associated Lck has been proposed to provide a transduction signal that might influence viral transcription. Interaction of Nef with this process would in turn influence the viral replication rate. Indeed, by reducing both Lck enzymatic activity and affinity for tyrosine-phosphorylated substrates, Nef might influence the outcome of T-cell activation while promoting cellular factors needed for viral replication. The recent finding that Nef also binds to another Src family kinase in monocytic cells, namely Hck, further indicates the physiological relevance of such interactions in the HIV life cycle and makes the molecular basis of the Nef-Lck binding an important target for therapeutic strategies.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by INSERM and grants from Agence Nationale de Recherches sur le SIDA (ANRS).

§
Supported by ANRS.

Supported by a grant from Ministère de l'Enseignement Supérieur et de la Recherche.

**
To whom correspondence should be addressed. Tel.: 33-91-75-84-15; Fax: 33-91-26-03-64.

(^1)
The abbreviations used are: IL, interleukin; SH, Src homology; HIV and SIV, human and simian immunodeficiency viruses, respectively; GST, glutathione S-transferase; CMV, cytomegalovirus; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase.

(^2)
Collette, Y., Chang, H S., Cerian, C., Chambost, H., Algarte, M., Mawas, C., Imbert, J., Burny, A., and Olive, D.(1996) J. Immunol.156, 360-370.

ACKNOWLEDGEMENTS

We thank M. Marsh, S. Ward, C. Mawas, and S. Fischer for critical reading of the manuscript and helpful discussions, and we thank P. Jullien and C. Bougéret for providing Lck and Csk constructs.

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