Human Immunodeficiency Virus Type 1 Nef Binds ... - Journal of Virology

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ALISON GREENWAY,1* AHMED AZAD,2 JOHN MILLS,3. AND DALE MCPHEE1. AIDS Cellular ...... Macreadie, and A. Azad. 1994. Nef 27, but not the Nef 25 ...

JOURNAL OF VIROLOGY, Oct. 1996, p. 6701–6708 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 70, No. 10

Human Immunodeficiency Virus Type 1 Nef Binds Directly to Lck and Mitogen-Activated Protein Kinase, Inhibiting Kinase Activity ALISON GREENWAY,1* AHMED AZAD,2 JOHN MILLS,3

AND

DALE MCPHEE1

AIDS Cellular Biology Unit,1 Macfarlane Burnet Centre for Medical Research,2 National Centre in HIV Virology Research,3 Fairfield, Victoria, Australia 3078, and Biomolecular Research Institute, Parkville, Victoria, Australia 30522 Received 21 March 1996/Accepted 14 June 1996

It is now well established that human immunodeficiency virus type 1 (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis. Nef on viral replication are most consistently observed in vitro, using quiescent peripheral blood mononuclear cells and not mitogen-stimulated peripheral blood mononuclear cells or Tcell lines (32, 53). There are multiple pathways for T-cell activation which use at least some common signal transduction elements (37). Nef has been shown to affect cellular receptors and bind to intermediary elements in a number of these pathways. Nef causes the down-regulation in expression of cell surface CD4 and of the alpha chain of the interleukin-2 (IL-2) receptor complex (IL-2Ra) on activated peripheral blood mononuclear cells and CD41 T-cell lines and prevents the induction of IL-2 mRNA in response to stimuli which normally result in increased transcription of this growth factor (16, 17, 19, 31). It has also been reported that stimulation of cells through the T-cell receptor– CD3–CD4 complex is inhibited by Nef as a result of downregulation of the transcription factors NF-kB and AP-1 (3, 34). Recently, we reported that Nef interacts with multiple cellular proteins, including the src family kinase Lck, CD4, tumor suppressor protein p53, and the serine/threonine mitogen-activated protein kinase (MAPK), all of which are intricately linked in signal transduction. Interaction of Nef with these proteins may result in its observed activities (18). The molecular basis of how Nef interacts with cellular proteins such as Lck, CD4, MAPK, and p53 is unknown; however, identification of the domains of Nef responsible for targeting it to specific cellular proteins will provide further insight into its function and possible targets for antiviral therapy. One of the four most highly conserved regions of Nef is a proline-rich (PxxP)4 repeat sequence occurring between amino acid residues 69 and 78 which displays striking similarity to a consensus sequence in-

The nef gene from human immunodeficiency virus type 1 (HIV-1) encodes a 25- to 30-kDa myristoylated protein produced early during infection by translation from several singly and multiply spliced mRNA species (25, 39, 42, 48). In infected cells, Nef localizes predominantly at the plasma membrane and preferentially associates with the cytoskeleton (15, 24, 35). Controversy surrounded the initial investigations of Nef effect on virus replication and long terminal repeat-driven transcription (1, 7, 20, 30, 36, 54). More recent studies using primary T cells and monocytes indicate that Nef acts as a positive factor in virus replication (32, 53). The importance of nef in development of AIDS and maintenance of high virus load is clearly evident during infection of rhesus monkeys with the closely related simian immunodeficiency virus (23). Detection of partial deletions in nef in HIV-1 isolates from one patient with long-term nonprogressive infection (26) and identification of HIV-1 sequences which contain deletions in the nef gene and in the nef/long terminal repeat U3 overlap region from an HIV-1-infected blood donor and a cohort of six recipients who remain free of HIV-1-related disease 10 to 14 years after infection (12) confirm the importance of this gene in determining pathogenicity of HIV-1. Although the mechanism by which Nef contributes to viral replication is not clear, it appears that Nef may act by disturbing T-cell activation (18, 19, 31, 34, 52). Indeed, the positive effects of * Corresponding author. Mailing address: AIDS Cellular Biology Unit, National Centre in HIV Virology Research, Macfarlane Burnet Centre for Medical Research, P.O. Box 254, Fairfield, Victoria, Australia 3078. Phone: 61-3 9282 2112. Fax: 61-3 9282 2100. Electronic mail address: [email protected] 6701

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volved in binding to the Src homology 3 (SH3) domain of protein kinases involved in signal transduction and hence is a candidate sequence for binding to Lck, especially given the fact that Nef has been shown to interact with the SH3 domain of the src family kinases Hck and Lyn (8, 29, 40, 41, 44, 51, 56). Thus, to investigate how Nef interacts with its targets, purified protein and synthetic peptides corresponding to selected regions were used in conjunction with Lck and glutathione Stransferase (GST)–Lck fusion constructs in binding studies to identify regions of Nef and Lck responsible for interaction. Similar studies using purified MAPK and Nef were also performed to determine how these two proteins interact. We report that the interaction between Nef and Lck is direct and can occur through the proline-rich amino acid site between amino acid residues 69 to 83 of Nef and the SH3 domain of the src family kinase Lck; Nef also binds directly to MAPK via the same proline-rich domain. We also show that Nef inhibits the tyrosine kinase and serine/threonine kinase activities of Lck and MAPK, respectively, presumably as a consequence of direct binding. MATERIALS AND METHODS Peptide synthesis and protein expression. Short synthetic peptides corresponding to amino acid residues 1 to 19 (MGGKWSKSSVIGWPAVRERM), 44 to 65 (TSSNTAANNAACAWLEAQEEEE), 69 to 83 (PVTPQVPLRPMTY KA), 72 to 83 (PQVPLRPMTYKA), 75 to 83 (PLRPMTYKA), 87 to 101 (LSH FLKEKGGLEGLI), 108 to 114 (DILDLWI), 164 to 186 (LLHPVSLHGMDD PEREVLEWRFD), and 187 to 206 (SRLAFHHVARELHPEYFKNC) of Nef derived from HIV-1NL4-3 were synthesized as described previously (14). The large-scale expression in Escherichia coli and purification of Nef 27 HIV-1NL4-3 either alone or as a GST-Nef fusion protein have been described elsewhere (2). Coprecipitation of Lck with GST-Nef. Purified Lck (0.7 mmol per sample; Upstate Biotechnology, Lake Placid, N.Y.), derived from bovine thymus and maintained in 25 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES; pH 7.0)–10% glycerol–0.1% Nonidet P-40 (NP-40), was diluted in phosphate-buffered saline (PBS) containing 0.05% NP-40 and incubated with GST-Nef 27 (0.7 mmol per sample) or, as a control, GST alone (0.7 mmol per sample) for 2 h at 48C. Following this incubation period, 50 ml of a 50% slurry of glutathione-Sepharose beads was added to each tube, and the tubes were incubated with continuous agitation for 30 min at room temperature. Each suspension was then cooled on ice for 15 min before the Sepharose beads were washed three times with 1.5 ml of ice-cold PBS containing 0.05% (vol/vol) NP-40. Bound GST-Nef and Lck were then eluted from the beads by incubation of the beads with 20 ml of 10 mM glutathione for 10 min at room temperature. After centrifugation, the supernatant was removed and stored, and the elution step was repeated twice. Aliquots (20 ml) of the eluted material were then electrophoresed on a 13% polyacrylamide gel, and the separated material was transferred to Hybond-C (Amersham International, Amersham, England). After transfer, the membranes were blocked with 5% (wt/vol) BLOTTO for 2 h at 378C and then incubated with anti-Lck (rabbit polyclonal antibodies with epitopes corresponding to amino acid residues 476 to 505; diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, Calif.) overnight at 48C. The next day, the membranes were washed extensively in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-Tween 20) and then incubated with donkey anti-rabbit immunoglobulin (Ig)-conjugated to biotin (diluted 1:1,000; Amersham) for 1 h at room temperature. After being washed in TBS-Tween 20, the membranes were incubated with streptavidin-conjugated horseradish peroxidase (strep-HRP; diluted 1:1,000; Dako, Carpinteria, Calif.) for 30 min at room temperature. After further washing, the membranes were developed by using 1,4-dichloronaphthol as the substrate. The specificity of the GST-Nef-Lck interaction was verified by competitive inhibition studies with purified recombinant Nef 27. For the competition studies, recombinant Nef 27 (at 0.35, 0.7, 1.4, or 2.8 mmol per sample) was incubated with Lck at the same time that GST-Nef 27 or, as a control, GST was added to the solution containing purified Lck. Detection of Nef-Lck and Nef-MAPK interaction by direct ELISA. Two different enzyme-linked immunosorbent assay (ELISA) formats were developed for detection of Lck-Nef interaction. The first format involved interaction of Lck with immobilized Nef, while the second format involved interaction of Nef with immobilized Lck. In the first case, 96-well polystyrene microtiter plates (Nuncimmunoplate I; A/S Nunc, Kamstrup, Denmark) were coated for 2 h at 378C with 50 ml of purified recombinant Nef 27 (75 nmol) or, as a control, bovine serum albumin (BSA; 75 nmol) diluted in PBS. The wells were then washed three times with PBS containing 0.05% Tween 20 (PBS-Tween). To block the remaining sites of the wells, 150 ml of 1% (wt/vol) gelatin dissolved in PBS was added to each well, and the plates were incubated at 378C for 1 h. After further washing with

J. VIROL. PBS-Tween, 50 ml of purified Lck (0 to 150 nmol) or PBS alone was added to the wells, and the plates were incubated for 2 h at 378C. Following washing with PBS-Tween, rabbit anti-Lck (Santa Cruz) or unrelated rabbit antibodies, all diluted in PBS to the same IgG concentration (1 mg/ml), were added to each well (50 ml per well), and the plates were incubated at 378C for 1.5 h. Wells were again washed as described above and then incubated with donkey anti-rabbit Ig conjugated to biotin (50 ml per well; Amersham), diluted 1:1,000 in PBS, for 1 h at 378C. Following further washing steps, the wells were incubated with strep-HRP (diluted 1:1,000; Dako) for 30 min at 378C, after which binding was detected by using o-phenylenediamine (Sigma) as the substrate. Absorbance was measured by using a plate reader at dual wavelengths of 450 and 630 nm. In the case of the second format, polystyrene microtiter plates (Nunc) were coated for 2 h at 378C with 50 ml of purified recombinant Lck (70 nmol) diluted in PBS. After this incubation period, the wells were washed three times with PBS-Tween. To block the remaining sites of the wells, 150 ml of 1% (wt/vol) gelatin dissolved in PBS was added to each well, and the plates were incubated at 378C for 1 h. After further washing with PBS-Tween, 50 ml of purified Nef (0 to 150 nmol) or PBS alone was added to the wells, and the plates were incubated for 2 h at 378C. Following washing with PBS-Tween, anti-Nef monoclonal antibody (MAb) AE6 (AIDS Reference Reagent Program) or an isotype-matched control MAb diluted in PBS to the same Ig concentration was added to each well (50 ml per well), and the plates were incubated at 378C for 1.5 h. Wells were again washed as described above and then incubated with sheep anti-mouse Ig conjugated to biotin (50 ml per well; Amersham), diluted 1:1,000 in PBS, for 1 h at 378C. Following further washing steps, the wells were incubated with strep-HRP (diluted 1:1,000; Dako) for 30 min at 378C, after which binding was detected as described above. For detection of MAPK interaction with Nef, wells were coated with purified MAPK (50 nmol; Santa Cruz) or BSA (50 nmol) for 2 h at 378C, after which time remaining sites were blocked with 1% gelatin as described above. After washing, Nef (0 to 75 nmol) was added to the wells, and the plates were incubated for 2 h at 378C. Anti-Nef (MAb AE6), diluted in PBS to 1:1,000, or an isotype-matched control diluted to the same Ig concentration, was then added. Binding of anti-Nef to the Nef-MAPK complex was then detected by using anti-mouse Ig conjugated to biotin (diluted 1:1,000; Amersham) followed by strep-HRP (diluted 1:1,000; Dako) and substrate as described above. Identification of amino acid residues of Nef which interact with Lck and MAPK. Short synthetic peptides corresponding to amino acid residues 1 to 19, 20 to 36, 44 to 65, 69 to 83, 72 to 83, 75 to 83, 87 to 101, 108 to 114, 164 to 186, and 187 to 206 of HIV-1NL4-3 Nef were coated (5 mmol; diluted in PBS) onto the wells of 96-well plates for 2 h at 378C. Following washing with PBS-Tween and blocking of the remaining sites with 1% (wt/vol) gelatin for 1 h at 378C, purified Lck (0 to 150 nmol) or MAPK (0 to 75 nmol) was added to the wells in 50-ml aliquots, and the plates were incubated for 2 h at 378C. The next day, the wells were washed with PBS-Tween, and then anti-Lck (Santa Cruz), anti-MAPK (Santa Cruz), or unrelated rabbit antibodies, all diluted in PBS to 1 mg/ml of Ig, were added to the wells (50 ml per well) for 1.5 h at 378C. Binding of anti-Lck to Nef peptide-associated Lck or anti-MAPK to Nef peptide-associated MAPK was detected as described above. Controls for the peptide binding assay included incubation of the immobilized peptides with TBS alone in place of Lck or MAPK and incubation of the peptide-Lck or peptide-MAPK complex with unrelated rabbit antibodies to verify the specificity of anti-Lck and anti-MAPK. Binding of Lck or MAPK to full-length Nef was competed for with Nef peptides corresponding to amino acid residues 1 to 19 or 69 to 83. For these experiments, Nef (75 nmol) was coated onto wells of polystyrene microtiter plates as described above. After blocking of remaining sites with 1% gelatin, Lck (100 nmol) or MAPK (50 nmol) which had been preincubated with peptide 69 to 83 or peptide 1 to 19 (75 to 750 nmol of each peptide) for 1 h at 48C was then added to the immobilized Nef as described above. Binding of Lck or MAPK to Nef was then detected as described above. Coprecipitation of Nef with GST-Lck constructs. Several GST-Lck fusion proteins which contain only the SH2 (GST-LckSH2), SH3 (GST-LckSH3), or SH2 and SH3 (GST-LckSH2SH3) domains of human Lck were used with highly purified Nef in coprecipitation studies to identify whether these regions of Lck can interact with Nef. GST-LckSH2 (1 mmol), GST-LckSH3 (1 mmol), GSTLckSH2SH3 (1 mmol), or, as a control, GST alone was incubated with Nef (1 mmol) or an unrelated protein (BSA; 1 mmol) in PBS for 2 h at 48C. After this incubation period, GST-Lck constructs with associated Nef protein were precipitated and eluted from glutathione-Sepharose beads as described above. Aliquots (20 ml) of the eluted material were then electrophoresed on a 13% polyacrylamide gel, and the separated proteins were detected by silver staining. Lck tyrosine kinase assay. The phosphorylation of p34cdc2[Lys 19](6-20)NH2 by Lck in the presence of Nef or short Nef peptides was assessed by in vitro kinase assays. Lck (100 nM) diluted in 80 mM HEPES (pH 7.0)–4% (vol/vol) glycerol–0.05% (vol/vol) NP-40 was preincubated with purified Nef (0 to 300 nmol), a Nef peptide (0 to 10 mmol) corresponding to amino acid residues 69 to 83 or 1 to 20, or, as a control, GST (0 to 300 nM), BSA (0 to 300 nM), or TBS alone for 1 h at 48C. Following this incubation period, the enzyme assays were carried out at 308C for up to 30 min in a 25-ml volume containing the assay buffer (50 mM Tris-HCl [pH 7.0], 25 mM MgCl2, 0.05 mM Na3VO4, 100 mM [g-32P] ATP) and 300 mmol of peptide substrate [p34cdc2(6-20)NH2]. As a control, p34cdc2[Lys 19, Phe 15](6-20)NH2 or p34cdc2[Lys 19, Ser 14, Val 12](6-20)NH2

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matography paper (Whatman). After four washes with excess phosphoric acid (0.75%, vol/vol) and one wash with excess acetone, the discs were counted in a scintillation counter.

RESULTS

FIG. 1. Direct interaction between recombinant Nef and purified Lck demonstrated by coprecipitation. Purified Lck was reacted with GST (track 3) or GST-Nef (track 5), purified, electrophoresed, and immunoblotted with polyclonal antibodies specific for Lck (see Materials and Methods). For competition studies, Lck was preincubated with purified Nef protein at 0.5-fold (track 6), 1-fold (track 7), 2-fold (track 8), or 4-fold (track 9) molar excess before reaction with GST-Nef and processing as described above. Tracks 1, 2, and 4 represent purified GST, GST-Nef, and Lck alone, respectively, reacted with anti-Lck.

peptide was substituted as the substrate. Tubes containing the reaction mixture but no peptide substrate were included to measure Lck autophosphorylation activity and the effect of Nef on this activity. Background levels of Lck autophosphorylation kinase activity were measured by incorporation of 32P into Lck when Lck alone was incubated at 08C for the length of the assay. The reaction was stopped by the addition of 10 ml of 50% (vol/vol) acetic acid, and then 25 ml of the reaction mixture was spotted onto P81 chromatography paper (Whatman). After four washes with excess phosphoric acid (0.75%, vol/vol) and one wash with excess acetone, the discs were counted in a scintillation counter. MAPK assay. The phosphorylation of myelin basic protein (MBP) by MAPK in the presence or absence of Nef or short Nef peptides was assessed. MAPK (25 nmol) diluted in assay buffer (25 mM Tris-HCl [pH 7.0], 0.1 mM EGTA, 0.1 mM NaVO3, 10 mM magnesium acetate) was preincubated with purified Nef (0 to 75 nmol), a peptide (0 to 2.5 mnmol) corresponding to Nef amino acid residues 69 to 83 or 1 to 20, or, as a control, GST (0 to 75 nmol), BSA (0 to 75 nmol), or TBS alone for 1 h at 48C. Following this incubation period, the enzyme assay was carried out at 308C for 30 min in a 50-ml volume of assay buffer containing 100 mM [g-32P]ATP and 300 mM peptide substrate (MBP). For the blank, the reaction mixture contained no peptide substrate. As controls, an unrelated peptide corresponding to amino acid residues 89 to 97 of Nef was substituted as the substrate. The reaction was stopped by the addition of 10 ml of 50% (vol/vol) acetic acid, and then 25 ml of the reaction mixture was spotted onto P81 chro-

Coprecipitation of Lck with GST-Nef. A direct and specific interaction between recombinant Nef and purified Lck was shown by coprecipitation and immunoblotting (Fig. 1). There were no nonspecific interactions detected. Proteins such as BSA did not bind to GST-Nef, while the binding of Lck to GST-Nef could be inhibited by purified recombinant Nef in a concentration-dependent manner (Fig. 1). To confirm the interaction between Lck and Nef, we developed a microtiter plate binding assay using immobilized Nef or Lck. Lck bound to immobilized Nef in a concentration-dependent manner, plateauing above 100 nmol, with a molar ratio of 1:1 (Fig. 2A). No detectable binding of Lck to BSA or other unrelated proteins was observed, confirming the specific nature of the Nef-Lck interaction (data not shown). Nef also bound to immobilized Lck in a concentration-dependent fashion (Fig. 2B), and saturation again occurred at a molar ratio of approximately 1:1. The molar ratios were calculated according to the concentration of Nef or Lck proteins initially used to coat the plate. Binding of Nef to the SH3 domain of Lck. To investigate if the Src homology domains of Lck are involved in the Nef-Lck interaction, C-terminal GST-Lck fusion proteins containing the SH2, SH3, or both SH2 and SH3 domains of Lck were used in coprecipitation studies. Nef bound to a GST-LckSH2SH3 fusion protein (amino acid residues 54 to 226 of human Lck) and to a GST-LckSH3 protein (amino acid residues 54 to 120) (Fig. 3, tracks 10 and 7, respectively) but did not bind to GST alone (track 3) or to the GST-LckSH2 construct (amino acid

FIG. 2. Direct binding of recombinant Nef to purified Lck as determined by ELISA. (A) Recombinant Nef protein was coated onto the wells of a 96-well polystyrene microtiter plate and incubated with increasing amounts of Lck (0 to 300 nmol). Binding of Lck was detected with an anti-Lck antibody (■) followed by incubation with a biotin-conjugated anti-rabbit Ig, strep-HRP, and o-phenylenediamine substrate solution, and optical density was measured at 450 and 630 nm. The control (F) was an unrelated rabbit IgG used at the same concentration as the specific antibody. In contrast to the binding observed with anti-Lck, only low background binding was seen when the control Ig was used in place of anti-Lck, indicating the specificity of anti-Lck binding. (B) Purified Lck protein was coated onto the wells of a 96-well polystyrene microtiter plate and incubated with increasing amounts of Nef (0 to 200 nmol). Binding of Nef was detected with an anti-Nef antibody (F), and the control was unrelated rabbit IgG (■) used at the same concentration as the specific antibody. Other steps were as for panel A. In contrast to the binding observed with anti-Nef, only low background binding was seen when the isotype control antibody was used in place of anti-Nef.

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FIG. 3. Nef binds to the SH3 domain of Lck. GST-Lck fusion protein constructs which contained only the SH2, the SH3, and the SH2 and SH3 domains of Lck were incubated with Nef (tracks 4, 7, and 10, respectively), BSA (tracks 5, 8, and 11, respectively), or TBS alone (tracks 6, 9, and 12, respectively), affinity purified by using glutathione-Sepharose beads, and electrophoresed, and the gel was silver stained (see Materials and Methods). Purified Nef and BSA alone are in tracks 1 and 2, respectively. Nef was also incubated with GST alone and affinity purified by using glutathione-Sepharose beads to confirm that Nef does not bind to GST alone (track 3). The interaction of Lck-SH3 with Nef is thought to be specific, since control protein (BSA) did not bind to either the SH2 and SH3 domains, SH2 domain, or SH3 domain of Lck.

residues 120 to 226) (track 4). The interaction of Nef with GST-LckSH2SH3 may therefore be via the SH3 domain of Lck. The interaction of Nef with the GST-LckSH3 and GSTLckSH2SH3 constructs was specific for Nef, since unrelated proteins such as BSA did not coprecipitate with either construct (tracks 8 and 11, respectively). Quantitation of input Nef versus Nef bound to the GST-Lck constructs showed that approximately 70% of input Nef bound to the constructs. Further assessment of Nef interaction with the GST-Lck constructs by using a 96-well binding format showed no significant difference between GST-LckSH2SH3 and GST-LckSH2 constructs in terms of ability to bind Nef (data not shown). Region of Nef binding to the SH3 domain of Lck. A prolinerich (Pxx)4 repeat sequence between amino acid residues 69 to 78 is one of the most highly conserved regions in the HIV-1 Nef protein (51). This sequence strongly resembles sequences in proteins which are involved in binding to the SH3 motifs of a large number of cytoskeletal and signalling molecules (8, 40, 41, 56). To determine the role of this proline-rich domain of Nef in binding to Lck, synthetic peptides corresponding to selected regions of Nef were used as capture antigens in the binding assay. Lck bound to the Nef peptide corresponding to amino acid residues 69 to 83 (Fig. 4). Lck did not bind to other domains of Nef or BSA (see Fig. 4 for data concerning Nef peptide amino acid residues 1 to 19 and Table 1 for data on other regions of Nef). These results indicate that binding of Lck to Nef peptide 69 to 83 represents a specific interaction of Lck with these amino acid residues of Nef. To further delineate the minimum region between amino acid residues 69 to 83 of Nef essential for binding to Lck, derivatives of this peptide corresponding to amino acid residues 72 to 83 (PQVPLRPMT YKA ) and 75 to 83 (PLRPMTYKA ) were used in the binding assay. Peptide 72 to 83 lacks the first proline residue, while peptide 75 to 83 contains only the last two proline residues. Lck bound to peptide 72 to 83 slightly less well than to the longer peptide 69 to 83, which contains all four proline repeat sequences (Table 1). However, when peptide 75 to 83 was used as the capture antigen, binding of Lck was markedly reduced, being approximately 30% of that observed with peptide 69 to 83, suggesting that the first two proline repeat sequences in the Nef peptide PVTPQVPLRPMTYKA are essential for maximal binding of Nef to Lck (Table 1). The Nef proline-rich peptide (amino acid residues 69 to 83) inhibited binding of Lck to full-length Nef in a concentrationdependent manner; 50% inhibition occurred with a 10-fold

FIG. 4. Lck binds to the proline-rich repeat sequence between residues 69 to 83 of Nef. A synthetic peptide corresponding to amino acid residues 69 to 83 (■ and h) or amino acid residues 1 to 19 (F and E) was coated onto wells of a 96-well polystyrene microtiter plate and incubated with purified Lck. Binding of Lck to the peptides was detected with anti-Lck (■ and F) followed by speciesspecific Ig-biotin and strep-HRP and assayed as described for Fig. 2. The control was an unrelated polyclonal rabbit antibody used at the same IgG concentration as the specific antibody (h and E). In contrast to anti-Lck, only low background level binding was obtained when the control Ig was reacted with the Nef peptideLck complex, verifying the specificity of anti-Lck.

molar excess of peptide relative to full-length Nef (Table 2). Preincubation of Lck with a peptide corresponding to amino acid residues 1 to 19 of Nef did not block binding of Nef to Lck (Table 2). The 10-fold molar excess of peptide 69 to 83 required to achieve 50% inhibition of Lck to full-length Nef suggests that other determinants present in Nef are required for maximal binding to Lck or that the peptide is in a different conformation, while the 50% inhibition of MAPK serine/

TABLE 1. Identification of the precise domains of Nef which are involved in Lck and MAPK bindinga Substrate

% Binding of Lck to Nef peptides relative to Nef peptide 69 to 83

Nef peptide sequences (amino acid residues) 1–19.......................................................................................... 3 44–65........................................................................................ 2 69–83........................................................................................ 100 72–83........................................................................................ 96 75–83........................................................................................ 32 87–101...................................................................................... 5 108–114.................................................................................... 7 164–186.................................................................................... 3 187–206.................................................................................... 4 BSA.............................................................................................. 3 a Synthetic peptides corresponding to amino acid residues 69 to 83, 72 to 83, or 75 to 83 of Nef were coated onto wells of a 96-well polystyrene microtiter plate and incubated with either purified Lck or MAPK. Binding of Lck and MAPK to the peptides was detected with anti-Lck and anti-MAPK, respectively, or, as a control, an unrelated polyclonal rabbit antibody, followed by species-specific Ig-biotin and strep-HRP, and assayed as described for Fig. 2.

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TABLE 2. Determination that Nef peptide 69 to 83 competes with full-length Nef for binding to Lck and MAPKa Nef peptide competitor (amino acid residues)

% Binding of MAPK to full-length Nef

% Binding of Lck to full-length Nef

69–83 1-fold molar excess 3-fold molar excess 10-fold molar excess 30-fold molar excess 1–19 (30-fold molar excess)

74 53 15 8 98

88 65 54 11 100

a Recombinant Nef protein was coated onto wells of microtiter plates, and purified Lck or MAPK which had been preincubated with increasing concentrations of a Nef peptide corresponding to the proline repeat motif (amino acid residues 69 to 83) or, as a control, amino acid residues 1 to 19 was added to the wells.

threonine kinase activity by only a 3-fold molar excess of peptide 69 to 83 suggests that this may be the only domain of Nef required for binding to MAPK. Nef inhibits Lck kinase activity. To investigate the effect of Nef on Lck tyrosine kinase activity, we assessed the ability of Lck to phosphorylate a substrate p34cdc2[Lys](6-20)NH2 peptide in the presence of increasing amounts of full-length Nef or fragments of Nef shown to block Lck-Nef interactions. It has been reported previously that p34cdc2[Lys 19](6-20)NH2 is a highly efficient substrate of the src family kinase Lck (5, 6). Phosphorylation of the p34cdc2[Lys 19](6-20)NH2 peptide by Lck is markedly attenuated by replacement of Y-15 by Phe (5, 6). Lck rapidly phosphorylated peptide p34cdc2[Lys 19](620)NH2 (Fig. 5), while the alternate substrate peptide, negative control peptide p34cdc2[Lys 19, Phe15](6-20)NH2, was a markedly less efficient substrate for Lck (Fig. 5). Preincubating Lck with increasing amounts of Nef inhibited Lck kinase activity in a concentration-dependent manner (Fig. 5), with 50% inhibition occurring when Lck was pretreated with between 10 and 30 nmol of Nef (Fig. 5). Incubation of Lck with Nef in increasing concentrations from 10 to 300 nmol resulted approximately in a 25 to 100% decrease in incorporation of [32P]ATP into the substrate peptide (Fig. 5). Treatment of Lck with GST or unrelated proteins such as BSA had no inhibitory effect on Lck kinase activity. The significant inhibitory effect of Nef when incubated with Lck at a ratio of 1:10 may be indicative of the percentages of active and inactive forms of Lck present in the

FIG. 5. Nef inhibits Lck tyrosine kinase activity. The tyrosine kinase activity of Lck was measured by using p34cdc2 peptide as the substrate. Lck preincubated with either TBS, BSA, GST, or increasing amounts of purified Nef was then added to the peptide p34cdc2[Lys 19](6-20)NH2 or p34cdc2[Lys 19, Phe 15](620)NH2 (control peptide), and the enzyme reaction was allowed to proceed at 308C for up to 30 min. Incorporation of [32P]ATP was measured after 30 min by scintillation counting. Results are representative of three experiments.

FIG. 6. Nef peptide 69 to 83 inhibits Lck tyrosine kinase activity. The in vitro tyrosine kinase activity of Lck was measured in the presence of Nef peptide 69 to 83 or 1 to 19. The tyrosine kinase activity of Lck was measured by using a peptide corresponding to amino acid residues 6 to 20 of p34cdc2 as the substrate (see Materials and Methods). Lck preincubated with either TBS or increasing amounts of a Nef peptide corresponding to amino acid residues 69 to 83 or 1 to 19 was then added to the peptide p34cdc2[Lys 19](6-20)NH2, and the enzyme reaction was allowed to proceed at 308C for up to 30 min. The alternate peptide p34cdc2[Lys 19, Phe 15](6-20)NH2 20)NH2 (control peptide) was included as a negative control. Peptide incorporation of [32P]ATP was measured after 30 min of incubation by scintillation counting and expressed as counts per minute. Results are representative of three similar experiments.

Lck preparation. The autophosphorylation activity of Lck was also dramatically inhibited by treatment with Nef. The autophosphorylation activity of Lck was reduced to background levels (background Lck kinase activity is represented by incorporation of [32P]ATP into Lck when Lck alone is incubated at 08C for the duration of the kinase assay) by prior treatment with 10 to 30 nmol of Nef (data not shown). Treatment of Lck with either BSA or GST had no effect on its autophosphorylation activity (data not shown). The Nef proline repeat peptide (residues 69 to 83) previously shown to bind to Lck and to compete with Nef for Lck binding also inhibited Lck activity in a concentration-dependent manner; 50% inhibition occurred with 0.3 to 1.0 mmol of the peptide (Fig. 6). However, preincubation of Lck with Nef peptide corresponding to amino acid residues 1 to 19 had no effect on Lck activity, suggesting that the inhibitory effect of Nef on Lck tyrosine kinase activity was due largely to the SH3 binding motif present between amino acid residues 69 and 83 in Nef. The relative inefficiency of Nef peptide 69 to 83 in inhibiting Lck tyrosine kinase activity compared with full-length Nef protein suggests that other determinants present in Nef may contribute to binding to Lck. Nef binds directly to MAPK via its proline repeat motif. Nef bound to immobilized MAPK in a concentration-dependent fashion (Fig. 7). Background binding was observed when an anti-Nef MAb was substituted with an isotype control (Fig. 7). No cross-reactivity of anti-Nef with MAPK alone was seen, confirming the specificity of anti-Nef for the MAPK-Nef complex (Fig. 7). The binding of Nef to MAPK was considered specific since Nef did not bind to immobilized BSA (data not shown). Maximal binding of Nef to MAPK was observed when approximately 35 nmol of Nef was added to immobilized MAPK (50 nmol). In assays using the previous series of synthetic peptides corresponding to selected regions of Nef, MAPK, like Lck was shown to interact with the proline repeat sequence present between amino acid residues 69 and 83 of Nef (Fig. 8). MAPK did not bind to peptide fragments of Nef corresponding to the N- or C-terminal region of Nef, indicating the specificity of the interaction of MAPK for the proline repeat sequence (data not shown). MAPK did not bind to the shorter Nef peptides containing a portion of the proline repeat,

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FIG. 7. Direct binding between MAPK and Nef as determined by ELISA. Purified MAPK protein was coated onto the wells of a 96-well polystyrene microtiter plate and incubated with increasing amounts of Nef. Interaction of MAPK with Nef was detected with an antibody specific to Nef (■) or as a control, an isotype-matched control (F) followed by species-specific Ig-biotin and strepHRP. The substrate used was o-phenylenediamine. Background binding was observed when anti-Nef was substituted with an isotype control. No cross-reactivity of anti-Nef with MAPK alone was seen, confirming the specificity of anti-Nef for the MAPK-Nef complex.

corresponding to amino acid residues 72 to 83 and 75 to 83 (Fig. 8). Competition experiments similar to those performed with Nef and Lck showed that Nef peptide corresponding to amino acid residues 69 to 83 competed with full-length Nef for binding to MAPK (Table 2). Binding of MAPK to full-length Nef was inhibited with peptide 69 to 83 in a concentration-dependent manner, with 50% inhibition occurring when a 3-fold molar excess of peptide relative to full-length Nef was preincubated with MAPK. Preincubation of MAPK with a peptide corresponding to amino acid residues 1 to 19 of Nef did not compete with full-length Nef for binding to MAPK (Table 2). Inhibition of MAPK activity by Nef. To investigate the effect of Nef on MAPK serine/threonine kinase activity, we measured the ability of MAPK to phosphorylate a substrate peptide (MBP peptide) in the presence of increasing amounts of Nef or Nef peptide. Preincubation of MAPK with Nef significantly inhibited MAPK activity in a concentration-dependent manner, with 50% inhibition occurring at approximately 2.5 nmol (Fig. 8). No effect on MAPK activity was observed when MAPK was preincubated with unrelated proteins such as BSA or GST (Fig. 8). Pretreatment of MAPK with increasing concentrations of the Nef proline repeat peptide (amino acids 69 to 83) also resulted in decreased MAPK phosphorylation activity; however, Nef peptide corresponding to amino acid residues 1 to 19 had no effect on MAPK activity (Fig. 8). DISCUSSION These data show that the HIV-1NL43 Nef protein associates directly with the critical cellular kinases Lck and MAPK and inhibits their in vitro catalytic activities. In the cases of Lck and Nef, we show that the specific regions within both proteins which facilitate this interaction to be the SH3 domain in Lck

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and the proline-rich motif present between amino acid residues 69 and 83 in Nef. MAPK, despite the absence of an identifiable SH3 motif, also interacts with Nef via the same proline repeat motif. These data show a direct effect of HIV-1 Nef on the cellular kinases Lck and MAPK and extend our previous observation that Nef can coprecipitate several cellular proteins, including Lck, MAPK, CD4, and p53 (18). The region of Nef involved in binding to Lck and MAPK includes at least a proline-rich repeat sequence which occurs between amino acid residues 69 to 83 and represents one of four highly conserved regions of Nef among different HIV-1 isolates (51). The tetrapoline repeat sequence bears some resemblance to a consensus sequence xPxxPPPWxP (P 5 proline, x 5 nonconserved amino acid) which has been implicated as an adapter motif for protein-protein docking with SH3 domain-containing proteins involved in signal transduction and cytoskeletal organization (8, 9, 27, 40, 41, 57). Our binding studies using peptides of progressively smaller size from the N terminus of amino acid residues 69 to 83 showed that the first proline repeat sequence is not essential for binding to Lck but deletion of the first and second repeat sequences eliminated binding altogether. The loss of binding function could be due to an altered conformation of Nef or to a true requirement for both initial proline repeat sequences. An extended peptide corresponding to amino acid residues 65 to 82 of Nef has also been shown to associate with other members of the src family kinases, Hck and Lyn (44). However, in these studies another distantly located proline-rich region which occurs between amino acid residues 147 and 150 of Nef also contributed to binding (44). Our studies do not rule out determinants other than the tetrapoline motif of Nef that may further facilitate binding to Lck. Indeed, full-length Nef protein was more efficient in blocking Lck activity than the Nef peptide corresponding to amino acid residues 69 to 83, which, among other interpretations, could mean that other regions of Nef participate in binding to Lck and inhibiting Lck activity. In the case of MAPK, the first Px(S/T)P sequence between amino acid residues 69 to 71 is essential for binding of MAPK to Nef. Although MAPK does not possess an identifiable SH3 domain, phosphorylation of serine/threonine residues by MAPK is pro-

FIG. 8. Nef inhibits MAPK activity. The in vitro tyrosine kinase activity of MAPK was measured in the presence of Nef or Nef peptide 69 to 83 or 1 to 19. The kinase activity of MAPK was measured by using a peptide corresponding to residues of MBP as the substrate. MAPK preincubated with either TBS, BSA, GST, or increasing amounts of a purified Nef or Nef peptide corresponding to amino acid residues 69 to 83 or 1 to 19 was then added to MBP peptide or an unrelated control peptide, and the enzyme reaction was allowed to proceed at 308C for 30 min. Peptide incorporation of [32P]ATP was measured after 30 min of incubation by scintillation counting and expressed as counts per minute. Results are representative of three experiments.

VOL. 70, 1996

line directed. The threonine residue at position 71 is a potential target for phosphorylation by MAPK. Our data show that the only region of Lck required for binding to Nef is the SH3 domain. This is distinct from the regions of this kinase which are involved in binding to the coreceptors CD4, CD8, and IL-2Rb (21, 49, 50, 55, 56). The unique N-terminal 71 amino acid residues of Lck govern interaction with both CD4 and CD8, while the C-terminal kinase domain of Lck, a domain which is highly conserved among the src family protein tyrosine kinases, associates with IL-2Rb (21, 49, 50, 55). Interaction of Nef with Lck via the SH3 domain of Lck and the subsequent inhibition of tyrosine kinase activity (both autophosphorylation activity and the ability to phosphorylate alternate substrates) highlights the important role of this domain in the regulation of Lck activity. Proposed models for the regulation of activity of the closely related src kinase suggest that the SH3 domain of Src is involved in cooperative intramolecular interactions with the SH2 and C-terminal regions of this protein and maintains the enzyme in an inactive state (9, 11, 27). Deletion or mutation of the SH3 domain can result in the activation of the transforming potential of nonreceptor tyrosine kinases, implying that the SH3 domain exerts a negative effect on the catalytic activity of these kinases (13, 38, 47). A similar model may apply to Lck. Binding of Nef to Lck via the SH3 domain of this kinase may stabilize these intramolecular interactions such that a repressed state is maintained. Hence, in this context, Nef may prove to be a useful tool in studying the involvement of the SH3 motif in the regulation of activity of the src family kinases. Apart from our current description of Nef interaction with Lck, we have further evidence to suggest that Nef also interacts with the SH3 domain of another src family kinase, Fyn (unpublished data). Nef has also been shown to interact with the SH3 domains of Hck and Lyn (44), yet in that study, an interaction between Nef and the SH3 domain of Lck was not observed. However, the integrity of the GST-LckSH3 domain construct used by Saksela et al. (44) must be established by using control proteins known to interact with the SH3 domain of Lck. Confirmation of the Nef-Lck interaction involving the SH3 domain of Lck was reported during the preparation of this report (10). It is possible that Nef binds to the SH3 domain of Hck more strongly than to Lck. Despite this possibility, comparison of the Nef-Lck and Nef-Hck interaction is not particularly relevant to the action of Nef in T lymphocytes, since significant expression of Hck is restricted to monocytes and B lymphocytes (for a review, see reference 4). We are currently investigating the relative binding capacities of src SH3 domains with Nef. Interaction of Nef with several members of the src family kinases pinpoints a principal role for this protein in perturbing cell activation pathways in a number of cell types susceptible to HIV infection and may in part be related to the effect of Nef on modulation of cell surface receptor expression (CD4 and IL-2R) and its effect on viral replication. Both Lck and Fyn, expressed in T lymphocytes, are intricately involved in mediating signals derived from the coreceptors CD4 and CD8, IL-2R (Lck), and the T-cell receptor (Lck and Fyn) (4, 22, 28, 33, 43, 45, 55, 56). The interaction of Nef with Lck in particular may in part be responsible for Nef-induced down-modulation of CD4 and IL-2R expression and relevant to Nef-induced T-cell signalling defects. Perturbation of IL-2Rb signalling by interaction of Nef with Lck and Fyn, described as essential signalling components of the IL-2R complex, may lead to an indirect inhibition in expression of IL-2Ra. The relationship between the SH3 binding motif present in Nef and downregulation of cell surface receptor expression is now being

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assessed. Interaction of Nef with the SH3 domain of Hck has been reported to be required for maximal virus replication but is not necessary for modulation of CD4 surface expression (44). However, this may reflect the differences in physiological function between Lck and Hck, since Lck is known to interact with CD4 but no such interaction has been described for Hck in monocytes (4, 43, 55, 56). The present study extends our previous findings that Nef inhibits normal T-cell function and signalling as a consequence of its interaction with specific intracellular targets such as Lck and MAPK (18). Data obtained by Sawai et al. showing that coprecipitation of Nef with serine phosphoproteins of 62 and 72 kDa suggest that Nef may associate with an unidentified serine kinase or indeed may possess some kinase activity itself and confirm involvement of Nef in manipulating signal transduction events possibly in numerous pathways (46). Nef has also been shown to be an essential factor in the maintenance of high virus load and progression to disease in macaques infected with simian immunodeficiency virus (23). Our data suggest that by directly inhibiting Lck and MAPK activities, and potentially the activities of other members of the signal transduction cascade family involved in cellular activation, Nef may act as a positive factor for the HIV-1 life cycle by protecting the cell against virus-induced apoptosis, postulated as one mechanism of host cell death and other cell activation processes (18). ACKNOWLEDGMENTS We thank Anna Lucantoni for the production of E. coli-derived Nef27 and H.-C. Cheng for the generous gift of src kinase substrate peptides. This work was supported by the Commonwealth AIDS Research Grant Committee through a Commonwealth AIDS Research Grant Post-Doctoral Fellowship, the National Centre in HIV Virology Research, and a Commonwealth AIDS Research Project Grant. It was also supported in part by the Research Fund of the Macfarlane Burnet Centre for Medical Research. REFERENCES 1. Ahmad, N., and S. Venkatesen. 1988. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481–1485. 2. Azad, A., P. Failla, A. Lucantoni, J. Bentley, C. Mardon, A. Wolfe, K. Fuller, D. Hewish, S. Sengupta, S. Sankovich, E. Grgacic, D. A. McPhee, and I. Macreadie. 1994. Large scale production and characterisation of recombinant human immunodeficiency virus type-1 Nef. J. Gen. Virol. 75:651–655. 3. Bandres, J. C., and L. Ratner. 1994. Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NFkB and AP-1 in human T cells in vitro after T-cell receptor stimulation. J. Virol. 68:3243–3249. 4. Bolen, J. B., R. Rowley, C. Spana, and A. Y. Tsygankov. 1992. The src family of tyrosine protein kinases in hemopoietic signal transduction. FASEB J. 6:3403–3409. 5. Cheng, H.-C., C. M. E. Litwin, D. M. Hwang, and J. H. Wang. 1991. Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase. J. Biol. Chem. 266: 17919–17925. 6. Cheng, H.-C., H. Nishio, O. Hatase, S. Ralph, and J. H. Wang. 1992. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases. J. Biol. Chem. 267:9248–9256. 7. Cheng-Mayer, C., P. Iannello, K. Shaw, P. A. Luciw, and J. A. Levy. 1989. Differential effects of nef on HIV replication. Implications for viral pathogenesis in the host. Science 246:1629–1632. 8. Cicchetti, P., B. J. Mayer, G. Thiel, and D. Baltimore. 1992. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science 257:803–806. 9. Cohen, G. B., R. Ren, and D. Baltimore. 1995. Modular binding domains in signal transduction proteins. Cell 80:237–248. 10. Collette, Y., H. Dutartre, A. Benxiane, F. Ramos-Morales, R. Benarous, M. Harris, and D. Olive. 1996. Physical and functional interaction of Nef with Lck. J. Biol. Chem. 271:6333–6341. 11. Cooper, J. A., and B. Howell. 1993. The when and how of src regulation. Cell 73:1051–1054. 12. Deacon, N. J., A. Tsykin, A. Solomon, K. Smith, M. Ludford-Menting, D. J. Hooker, D. A. McPhee, A. L. Greenway, A. Ellett, C. Chatfield, V. A. Lawson,

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