Binding of c-Raf1 Kinase to a Conserved Acidic Sequence within the Carboxyl-terminal Region of the HIV-1 Nef Protein*

David R. HodgeDagger , K. Joyce Dunn§, Gou Kui Pei, Mrinal K. Chakrabartyparallel , Gisela HeideckerDagger parallel , James A. Lautenberger, and Kenneth P. Samuel**Dagger Dagger

From the Dagger  Laboratory of Leukocyte Biology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, § Laboratory for Genetic Disease Research, National Human Genome Research Laboratory, Bethesda, Maryland 20892,  Laboratory of Genomic Diversity, NCI-Frederick Cancer Research and Development Center, parallel  SAIC, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and ** Department of Biology, Morgan State University, Baltimore, Maryland 21239

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The Nef protein of the human and simian immunodeficiency viruses (HIV-1,1 HIV-2, and SIV) is predominantly a membrane-associated cytoplasmic phosphoprotein (1-3) synthesized from multiply spliced transcripts (4) early during the viral infection process and is also packaged into mature virions (5, 6). By using in vitro cell culture assay systems, Nef has been shown to be dispensable for virus replication (7-9) but is necessary for enhancing virus production and infection of recipient cells in vitro (5, 6, 9-11). In the SIVmac239 model for nef gene function in vivo, Nef has been shown to be necessary for maintaining high viral loads and for induction of an AIDS-like disease in rhesus monkeys infected with recombinant SIVmac239 with an intact nef gene (12). In the transgenic mouse model for Nef function in vivo, it has also been found that the HIV-1 Nef alters normal T-cell activation responses in thymocytes (13), suggesting that the function of Nef in vivo is tied to its ability to perturb T-cell signaling pathways. In addition, evidence from several in vitro studies have also supported a link between Nef expression and defective T-cell signaling and activation. Notably, Nef expression has been tied to its ability to interfere with signaling and cellular activation in T-lymphocytes (14-18), phosphatidylinositol 3-kinase signaling in NIH/3T3 cells (19), and tumor necrosis factor alpha -activated sphingomyelin signaling in human glial cells (20).

One of the best characterized signaling defects induced by Nef expression is the down-regulation of the cell-surface CD4 viral receptor molecule (2, 13, 21) by a mechanism that involves physical interaction with p56Lck kinase (22) and requires a common di-leucine containing sequence element at the cytoplasmic tail of the CD4 receptor molecule that also overlaps with the p56Lck-binding site (23, 24). The molecular interactions between Nef and p56Lck kinase (22) and between Nef and other members of the Src family of protein tyrosine kinases (25) occur through the highly conserved proline (PXXP)3 repeat motif in Nef and the conserved Src homology 2 (SH2) and/or SH3 domains of the protein kinases (22, 25). Other classes of cellular proteins have been reported to associate with the Nef protein in vitro or in vivo, including the p21-activated serine/threonine protein kinase (26-28), the MAP/ERK-1 protein kinase (17), p53 (17), protein kinase C (29, 30), a 46-kDa cellular phosphoprotein (31, 32), numerous T-cell-derived cytosolic and membrane-associated proteins (3, 17, 32, 33), and beta -cop protein (34), an important component of non-clathrin-coated pit vesicles involved in cellular membrane trafficking.

The ability of Nef to alter T-cell signaling and activation pathways in vitro and in vivo suggests a mechanism that may involve specific molecular interactions between Nef and a limited number of cellular signaling proteins. In order to define this mechanism, we set out to identify and map the T-cell signaling proteins and their specific protein-protein interaction domains that are targets of the HIV-1 Nef protein in vitro and in vivo. In the present report, we describe the identification of a relatively well conserved acidic sequence motif, consensus Asp/Glu-Asp-X-X-X-Glu (where X represents any other amino acid), at the carboxyl-terminal region of the primate lentiviral Nef proteins (35), that is structurally quite similar to the corresponding acidic sequence, Asp-Pro-Thr-Ile-Glu-Asp (amino acids 33-38), at the c-Raf1-binding site within the conserved core effector loop of the Ras oncoprotein (36). Furthermore, we describe that the c-Raf1 kinase physically interacts with the HIV-1LAI Nef protein at the acidic sequence motif which can be effectively blocked by single point mutations that substituted a nonpolar glycine residue for either of the conserved aspartic acid residues at positions 174 (Asp-174 right-arrow Gly) or 175 (Asp-175 right-arrow Gly) and by combined Asp-174 right-arrow Gly and Asp-175 right-arrow Gly mutations at both positions. Our results define the direct binding of c-Raf1 kinase to a conserved acidic sequence in the carboxyl-terminal region of the HIV-1 Nef protein in vitro and suggest a possible molecular model for investigating the Nef-induced T-cell signaling defects involving the c-Raf1-MAP kinase signaling pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutagenesis and Construction of nef Expression Plasmids

Site-specific Mutagenesis-- Oligonucleotide-directed mutagenesis (37) was performed on plasmid pSG5-Delta 9Nef which contains the full-length HIV-1Lai (HTLV-111B) nef gene (GenBankTM M15654, nucleotides 8152-8773) (38), utilizing the Muta-Gene kit (Bio-Rad). The mutagenic oligonucleotide, 5'-CTGCATGGAATGGGTGACCCGGAGAGA-3' (mutated nucleotides are in boldface and codon changes are underlined), which substituted a glycine for the aspartic acid at position 174 (D174G change), spans nucleotide positions 8659-8685 of the HIV-1Lai nef gene (38); the oligonucleotide, 5'-CTGCATGGAATGGATGGCCCGGAGAGAGA-3' (nucleotide positions 8659-8685) substituted a glycine for the aspartic acid at position 175 (D175G change); oligonucleotide 5'-CTGCATGGAATGGGTGGCCCGGAGAGAGAA-3' spans nucleotides 8659-8688 and introduced double aspartic acid to glycine substitutions at positions 174 and 175, respectively (D174G/D175G change); and oligonucleotide 5'-CTGCATGGAATGGGTGGCCCGGAGAGAGGAGTGTTAGAGTGG-3', spans nucleotides 8659-8700 and introduced triple point mutations in which aspartic acids 174 and 175 and glutamic acid 179, respectively, were changed to glycine (D174G/D175G/E179G change). The resulting wild-type and mutated full-length nef genes were cloned into the BamHI and EcoRI sites of the pGEX-2T plasmid (Amersham Pharmacia Biotech) (39), generating the pGEX-wtNef and pGEX-Nef174D-, pGEX-Nef175D-, pGEX-Nef174:175DD-, and pGEX-Nef174:175:179DDE-nef expression plasmids, respectively. The correct clones were verified by DNA sequence analysis.

Deletion Mutagenesis-- A 360-bp EcoRV-Ecl136II nef gene fragment spanning nucleotides 8555-8929 and encoding amino acid residues 136-206 at the carboxyl-terminal region of the HIV-1LAI(HTLV-111B) Nef protein was subcloned into the SmaI site of pBluescript plasmid (Stratagene, CA), then released with BamHI and EcoRI enzymes, and cloned into the BamHI and EcoRI sites of the pGEX-2T plasmid (39) to generate the pGEX-Nef1C expression plasmid. The 360-bp BamHI-EcoRI fragment was used to generate a panel of carboxyl-terminal nef deletion mutants by digestion with unique restriction enzymes, followed by repair of sticky ends with Klenow enzyme. The blunt-ended fragments were then cloned into the pGEX-2T or pGEX-3X plasmids (39) to generate the following expression plasmids: pGEX-Delta 12nef, which contains a 180-bp BamHI-HaeI11 nef gene fragment (codons 136-194); pGEX-Delta 31nef containing codons 136-177 encoded by a 123-bp BamHI-Nci1 fragment; pGEX-Delta 41nef containing an 88-bp BamHI-Mae111 fragment encoding codons 136-165; and pGEX-Delta 29nef containing a 290-bp MaeIII-Ecl136II fragment encoding codons 165-206.

c-Raf1 Expression Plasmids-- The pGEX-Raf-(50-133) expression plasmid was created by polymerase chain reaction cloning in which the primer pairs, 5'-GTAGGATCCGATCCTTCTAAGACAAGC-3' and 5'-CTAGAATTCATCCAGGAAATCTACTTG-3' containing a 5'-BamHI and 3'-EcoRI restriction site, were used to release a 245-bp EcoRI-BamHI fragment from the pKS+cRaf-1 plasmid (a gift from Dr. Debra Morrison, FCRDC, Frederick, MD) which encodes amino acids 50-133 of the human c-Raf1 protein. The fragment was then cloned into the BamHI-EcoRI sites of pGEX-2T vector. The correct clone was authenticated by DNA sequence analysis. The expression vectors were then used to transform competent Escherichia coli XL-1 or JM109 cells (Stratagene, La Jolla, CA) for expression of fusion proteins. The full-length human c-Raf1 protein (residues 1-648) was expressed as a GST-Raf-(1-648) fusion protein in the Sf9 insect (Spodeptera frugiperda) cell line with the pVL1393 baculovirus expression vector.

Protein Expression and Purification

Fresh overnight cultures of E. coli XL-1 or JM109 cells (Stratagene, La Jolla, CA) harboring the various pGEX expression plasmids were diluted 1:10 in Luria-Bertani (LB) broth supplemented with 100 µg/ml ampicillin, and the cultures were grown and induced with 1 mM isopropyl-beta -D-thiogalactoside (Sigma) as described previously (39). Cell pellets were collected by centrifugation and washed in ice-cold PBS (pH 7.4, Mg2+/Ca2+-free) supplemented with 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone, 100 µg/ml L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone, 10 µg/ml pepstatin A, 20 µg/ml aprotinin, and 10 µg/ml leupeptin, and Triton X-100-soluble extracts containing the recombinant proteins were prepared essentially as described before (40). The detergent-soluble protein extracts were dialyzed against PBS, pH 7.4, containing 0.5% Triton X-100 and protease inhibitors (Boehringer Mannheim), and the GST and recombinant GST fusion proteins were purified by coupling to glutathione-agarose beads (Sigma) by gentle mixing at 4 °C for 1 h as described (39, 40). The coupled affinity beads were sequentially washed with excess PBS, pH 7.4, containing 0.5% Triton X-100, PBS containing 0.5% Triton X-100 and 300 mM NaCl, and finally with PBS. The protein-bound affinity beads were analyzed and quantitated by Coomassie Blue R-250 staining following SDS-PAGE analysis (41). The coupled affinity beads were then adjusted to approximately 1 µg of protein per 50-µl beads and stored as a 33% (v/v) slurry at 4 °C in PBS supplemented with protease inhibitors.

Preparation of Soluble Recombinant Nef Proteins

Full-length wild-type and mutant recombinant GST-Nef fusion proteins bound to glutathione-agarose beads were digested with thrombin protease enzyme according to the recommendations of the manufacturer (Boehringer Mannheim) to remove the 26-kDa GST fusion tag, and the thrombin activity was inactivated with the protease inhibitor mixture (Boehringer Mannheim). The resulting protease-cleaved soluble Nef proteins were recovered by centrifugation of the glutathione-agarose beads to which the GST tag was still bound, followed by buffer exchange through a small Sephadex G-25 column (Amersham Pharmacia Biotech). The recovered recombinant Nef proteins were analyzed by SDS-PAGE and quantitated by staining with Coomassie Brilliant Blue R-250. The samples were adjusted to contain the same amount of protein.

Preparation of Soluble CEM Cell Lysate

The CEM T-lymphoblastoid cell line was maintained at 37 °C in a humidified 5% CO2 incubator in RPMI 1640 media supplemented with 4 mM glutamine, 15% heat-inactivated fetal bovine serum, and 100 units each of streptomycin and penicillin (Atlanta Biologicals, Norcross, GA). Cells in exponential growth phase were collected at 500 × g for 5 min at 4 °C and washed once with ice-cold PBS. Whole cell lysates were prepared in cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100) supplemented with 0.1 mM sodium orthovanadate, 5 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone, 100 µg/ml L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone, and 10 µg/ml each of leupeptin and pepstatin A (Boehringer Mannheim) at 4 °C, then cleared by centrifugation at 100,000 × g for 30 min at 4 °C. Cleared cell lysates containing 5 × 107 CEM cell eq/ml were used immediately or frozen at -70 °C until use.

Coprecipitation Assay

Soluble CEM whole cell lysates containing 5 × 107 CEM cell eq/ml were incubated with 50 µl (1 µg of protein) of GST beads or GST-Nef affinity beads with gentle mixing for 2 h at 4 °C, and the protein-bound beads were extensively washed with an excess of ice-cold cell lysis buffer containing protease inhibitors and processed for SDS-PAGE and Western blot analysis as described above.

Direct Protein Binding Assay

For the direct protein binding assay, aliquots containing 1 µg of thrombin-cleaved soluble recombinant Nef proteins were incubated with 50 µl (1 µg of protein) of glutathione-agarose affinity beads containing either the bound recombinant GST-Raf-(50-133) or GST-Raf-(1-648) fusion proteins for 2 h at 4 °C as before. The protein-bound beads were then extensively washed with cell lysis buffer and processed for SDS-PAGE and Western blot analysis as before.

SDS-PAGE and Immunoblot Analysis

Cellular proteins bound to the GST beads and GST fusion protein affinity beads were solubilized by boiling with 2× SDS-PAGE sample buffer and resolved by fractionation on reducing SDS-12% polyacrylamide gels (41). The fractionated proteins were then electrophoretically transferred (42) to Hybond-polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Burkinghamshire, UK), and the membranes were blocked for 2 h at 23 °C in TBS (10 mM Tris, pH 7.5, 100 mM NaCl) buffer containing 0.1% Tween 20 and 5% BSA (fraction V) (Sigma). The membranes were then incubated with a 1:1000 dilution of mouse anti-human c-Raf1 monoclonal antibody (mAb) that was raised against amino acids 162-378 of the human c-Raf1 kinase (Transduction Laboratories) or with a 1:500 dilution of the 55S mAb against the HIV-1LAI(HTLV-111B) Nef protein (43), in TBS-T (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 1% BSA for 12 h at 4 °C. Following extensive washes for 10 min each in TBS-T, the membranes were incubated with a 1:5000 dilution of a sheep anti-mouse IgG:horseradish peroxidase conjugate in TBS-T with 1% BSA at 23 °C for 60 min. The membranes were then extensively washed as before, and the complexes were visualized with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Burkinghamshire, UK).

Coimmunoprecipitation Assay

Whole cell lysates containing 5 × 108 CEM cell eq/ml were prepared from permanent CEM T-cell lines transduced with the HIV-1LAI(HTLV-111B) wild-type nef allele in the forward (NEF) and reverse (FEN) directions with the pLXSN retroviral expression vector (CLONTECH, Inc., La Jolla, CA) and were incubated either with the 55S anti-Nef (43) or the anti-c-Raf1 mAb for 12 h at 4 °C in cell lysis buffer containing protease and phosphatase inhibitors, followed by incubation with 100 µl of 33% (v/v) protein G-Sepharose beads at 4 °C for an additional 2 h. The immunoprecipitates were washed extensively with cell lysis buffer, then solubilized by boiling in SDS-PAGE sample buffer, and resolved by fractionation on SDS-PAGE as described before. The fractionated proteins were transferred to Hybond-polyvinylidene difluoride membrane as before; the membrane was then incubated either with the anti-Raf1 or anti-Nef mAbs, and specifically bound antigen-antibody complexes were visualized with the enhanced chemiluminescence system as before.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Recombinant Nef Fusion Proteins-- Recombinant GST-Delta Nef fusion proteins containing overlapping amino acid deletions between residues 136 and 206 at the carboxyl-terminal region of the HIV-1LAI Nef protein were expressed in an insoluble form in E. coli bacteria and then solubilized by extraction with the Sarkosyl-Triton X-100 procedure (40). The detergent-soluble fusion proteins were finally purified by directly coupling to glutathione-agarose beads as described previously (39, 40). Aliquots of affinity beads containing 1-3 µg of the purified GST fusion proteins were resolved by fractionation on SDS-PAGE and quantitated by staining the gel with Coomassie Brilliant Blue R-250 (Fig. 1, lanes 1-6). Three of the resulting Nef fusion proteins carrying carboxyl- or amino-terminal deletions, GST-Delta 12Nef (lane 3), GST-Delta 31Nef (lane 5), and GST-Delta 41Nef (lane 6), respectively, show anomalous mobilities on the SDS gel. In addition, samples of the glutathione-agarose beads containing 1 µg of the GST-bound wild-type full-length Nef (Fig. 3B, lane 2) and mutant full-length Nef proteins containing individual or combined point mutations within the conserved acidic sequence motif (Fig. 3B, lanes 3-6) were also analyzed by SDS-PAGE and quantitated by Coomassie Blue staining.


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Fig. 1.   Purification of recombinant Nef deletion proteins. Bacterially expressed GST-Delta Nef deletion mutants were affinity purified by coupling to glutathione-agarose beads, and samples containing 1-3 µg of protein were separated by electrophoresis on an SDS-12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250 as described under "Experimental Procedures." The fusion protein sample beads are GST (lane 1), GST-Nef1C (lane 2), GST-Delta 12Nef (lane 3), GST-Delta 29Nef (lane 4), GST-Delta 31Nef (lane 5), and GST-Delta 41Nef (lane 6), respectively. Molecular mass markers are shown by numbers in the margin.

Similarity between the Nef Acidic Sequence and a Corresponding c-Raf1-binding Site Sequence in Ras-- During structure-function analysis on the predicted Nef protein sequences of HIV-1, HIV-2, and SIV, we observed a well conserved acidic hexapeptide sequence with the general consensus, Asp/Glu-Asp-X-X-X-Glu, located at the carboxyl-terminal region of the primate lentiviral proteins (35), which suggests that this sequence may have an important role in the function of Nef. Interestingly, this sequence has a number of characteristic features in common with a corresponding acidic sequence, Asp-Pro-Thr-Ile-Glu-Asp (amino acids 33-38), at the c-Raf1-binding site sequence within the highly conserved core effector region of the mammalian Ras oncoprotein (36). In particular, both the Nef and Ras acidic sequence motifs share a similar pair of conserved acidic amino acids located at one end of the sequence and a single glutamic or aspartic acid residue positioned at the opposite end of the sequence (see Table I). Similarly, the relative spacing of three residues between the conserved terminal acidic amino acids is also well conserved within the Nef and Ras sequence motifs. However, these oppositely positioned single and double acidic amino acids are oriented differently in the two proteins, such that the two aspartic acid residues at positions 174 and 175 in the HIV-1LAI, positions 204 and 205 of HIV-2ISY, and positions 203 and 204 in SIVMM239 Nef proteins, respectively, are equivalent to the aspartic acid at position 38 and the glutamic acid at position 37 of Ras. Similarly, the highly invariant glutamic acid at positions 179, 209, and 208, respectively, in the HIV-1, HIV-2, and SIV Nef protein sequence, corresponds to the aspartic acid at position 33 within the conserved Ras effector sequence (36). Furthermore, whereas the lone proline residue (position 34) within the Ras sequence is tightly conserved in the subfamily of GTP-binding proteins, the analogous proline residue is conserved only in the predicted Nef protein sequence of the various SIV isolates. In the HIV-1 and HIV-2 Nef protein sequence, this proline residue exhibits a much greater degree of genetic drift.

                              
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Table I
Alignment of the different conserved carboxyl-terminal acidic Nef sequence with a corresponding acidic sequence within the c-Ha-Ras c-Raf1 binding domain (36)
Similar or identical amino acids are in boldface. A gap was introduced between the di-aspartic acid residues to maximize the alignment. The alignment was done by eye. A general consensus sequence motif is also indicated. The single letter amino acid code is used.

It is of interest to also note that residues 32-38 in the effector core regions of Ras and the Ras homologue, Rap1, are important in mediating specific interaction with c-Raf1 (44). The mechanism of the protein-protein interaction is through a conserved inter-protein beta -sheet structure formed between two anti-parallel beta -strands (44). This secondary structural feature is analogous to a recently described disordered tertiary loop structure located between residues 149 and 178 in the predicted HIV-1 Nef protein sequence, which is also connected by two conserved beta -strand structures within which the conserved acidic sequence motif in the HIV-1Nef (residues 174-179) resides (45). This sequence encompases the putative c-Raf1 interaction site in Nef.

Interaction between c-Raf1 and the Carboxyl-terminal Region of Nef-- To determine whether the conserved carboxyl-terminal acidic sequence, Asp-Asp-Pro-Glu-Arg-Glu, spanning amino acids 174-179 within the HIV-1LAI Nef protein is important for mediating specific interaction with c-Raf1 in vitro, we utilized the GST-Nef affinity beads containing different carboxyl-terminal deletions within amino acids 136-206 (see Fig. 1) in precipitation assays to pull down the c-Raf1 protein from detergent lysates of the CEM T-lymphoblastoid cell line. The results, shown in Fig. 2A, demonstrate that the soluble c-Raf1 protein was specifically precipitated from CEM cell lysates with the affinity beads containing amino acid residues 136-206 of Nef (lane 3, GST-Nef1C), residues 136-194 (lane 4, GST-Delta 12Nef), residues 136-177 (lane 5, GST-Delta 31Nef), and residues 165-206 (lane 7, GST-Delta 29Nef), respectively. As expected, the empty GST-agarose beads did not precipitate c-Raf1 (lane 2). However, the affinity beads containing amino acids 136-165 of HIV-1 Nef (fusion protein GST-Delta 41Nef) also did not precipitate the c-Raf1 protein from the CEM extract (Fig. 2A, lane 6). The missing sequence from GST-Delta 41Nef encompasses amino acids 165-177 and includes the conserved acidic sequence, Asp-Asp-Pro-Glu (positions 174-177) within the predicted c-Raf1-binding site (Table I). The results of the experiments presented in Fig. 2A and summarized in Fig. 2B indicate that binding of the CEM cell-derived c-Raf1 protein to the carboxyl terminus of Nef requires a minimal sequence located between amino acids 165 and 177. The results also implicate the importance of the acidic sequence motif at positions 174-179 in the binding.


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Fig. 2.   Mapping of the c-Raf1-binding site by deletion mutagenesis. A, immunoblot analysis of CEM T-cell-derived c-Raf1 protein with a c-Raf1 mAb following precipitation (pull down) assay with glutathione-agarose beads coupled to GST alone (lane 2), GST-Nef1C-(136-206) (lane 3), GST-Delta 12Nef-(136-194) (lane 4), GST-Delta 31Nef-(136-197) (lane 5), GST-Delta 41Nef-(136-165) (lane 6), and GST-Delta 29Nef-(165-206) (lane 7), respectively. Lane 1 contains extract from 5 × 105 CEM cell equivalents. Position of the c-Raf1 band is shown by the arrow. B, diagrammatic map of the carboxyl-terminal Nef sequence between amino acid positions 136 and 206, the deletion mutants and their amino acid boundaries, and their c-Raf1 binding activity in in vitro precipitation assays (+, binding; -, no binding). Dark area represents the proposed c-Raf1-binding domain shown in Table I.

A Di-aspartic Acid Motif Is Critical for C-Raf1 Binding to Nef-- Since the carboxyl-terminal end of the Nef sequence deleted from the GST-Delta 41Nef fusion protein above, Leu-His-Pro-Val-Ser-Leu-His-Gly-Met-Asp-Asp-Pro-Glu (amino acids 165-177), also contains the highly conserved pair of aspartic acid residues (positions 174 and 175), we investigated the contributions of this pair of acidic residues to binding of the c-Raf1 protein in vitro. We introduced single, double, and triple point amino acid substitutions (37) within the conserved acidic Nef sequence such that the conserved aspartic acid residues at positions 174 and 175, and the glutamic acid residue at position 179 within the full-length HIV-1 Nef genetic background (38), were mutated to a glycine residue. Both the full-length wild-type and mutant Nef were expressed in E. coli as GST-Nef fusion proteins and were purified by coupling to glutathione-agarose beads and used in the precipitation assays to pull down the c-Raf1 protein.

The results of this analysis show that introducing the single aspartic acid to glycine change either at position 174 (D174G substitution) (Fig. 3A, lane 3) or at position 175 (lane 4, D175G substitution) was sufficient to totally abrogate binding of the CEM cell-derived c-Raf1 protein to the full-length GST-Nef mutant. Similarly, the Nef affinity beads containing GST-Nef fusion proteins that harbor the double D174G/D175G (Fig. 3A, lane 5) and triple D174G/D175G/E179G (Fig. 3A, lane 6) substitution mutations, respectively, also failed to precipitate the cell-derived c-Raf1 protein. As expected, the empty GST-agarose beads did not pull down any detectable c-Raf1 protein from the CEM cell lysate (Fig. 3A, lane 1), whereas only the wild-type full-length GST-Nef affinity beads precipitated the c-Raf1 protein (Fig. 3A, lane 2). A positive immunoblot reaction of total CEM cell lysate with the anti-c-Raf1 mAb is shown in lane 7.


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Fig. 3.   Site-specific mutagenesis of the putative c-Raf1-binding site in HIV-1 Nef. A, CEM cell lysates containing 5 × 107 cells/ml were precipitated with affinity beads containing 1 µg each of GST (lane 1), full-length wild type (WT) GST-Nef (lane 2), GST-NefD174- with the Asp-174 right-arrow Gly (lane 3), GST-NefD175- with the Asp-175 right-arrow Gly (lane 4), GST-NefDD- with both the Asp-174 right-arrow Gly and Asp-175 right-arrow Gly (lane 5), and GST-NefDDE- with triple Asp-174 right-arrow Gly, Asp-175 right-arrow Gly, and Glu-179 right-arrow Gly (lane 6) substitution mutations, respectively, and separated by electrophoresis on an SDS-12% polyacrylamide gel and immunoblotted with the anti-Raf1 mAb as described under "Experimental Procedures." B, Coomassie Blue-stained SDS gel containing 1 µg of the respective fusion proteins in A. C, predicted amino acid sequence encompassing the conserved acidic motif of the various full-length Nef proteins deduced from the DNA sequence of the different nef alleles. Molecular mass markers are given in the numbers in the margin, A and B.

The stability and relative amount of the different full-length GST-Nef fusion proteins used in the pull-down assay in Fig. 3A above was evaluated by SDS-PAGE analysis and Coomassie Blue staining (Fig. 3B, lanes 2-6). Furthermore, each substitution mutation introduced within the acidic Nef sequence and the presumptive Nef c-Raf1-binding site was verified by DNA sequence analysis (Fig. 3C). Thus, the results of this study demonstrate that CEM cell-derived c-Raf1 protein binds to full-length Nef protein within a conserved acidic sequence motif containing the sequence, Asp-Asp-Pro-Glu-Arg-Glu (positions 174-175). Furthermore, the results also demonstrate the importance of the aspartic acid residues 174 and 175 for binding of c-Raf1 to Nef in vitro.

HIV-1 Nef Protein Directly Interacts with c-Raf1 in Vitro-- The possibility that interaction between matrix-bound GST-Nef fusion proteins and soluble c-Raf1 protein in the CEM T-cell extracts required an additional cellular factor(s) has not been ruled out in the above experiments. Thus, to demonstrate that binding of c-Raf1 to Nef in vitro involves a direct mechanism, which is direct physical interaction between the two proteins, we utilized a direct binding assay in which soluble recombinant Nef proteins free of the GST tag were reacted with matrix-bound full-length GST-Raf1-(1-648) and a truncated GST-Raf1-(50-133) fusion protein (36, 46-49) containing the Ras-binding domain (RBD) within a minimal peptide (36, 46, 48).

As shown in the results of Fig. 4A, affinity beads containing both the truncated GST-Raf-(50-133) with the known RBD (lane 2) and full-length GST-Raf-(1-648) fusion protein (lane 7) effectively pulled down the soluble recombinant wild-type Nef protein but did not precipitate the soluble recombinant Nef proteins containing the single D174G (lanes 3 and 8), double D174G/D175G (lanes 4 and 9), and triple D174G/D175G/E179G (lanes 5 and 10) substitution mutations, respectively, within the conserved acidic Nef sequence motif. As expected, soluble matrix-free bacterial GST protein was not precipitated with the GST-Raf-(50-133) (Fig. 4A, lane 1) nor the GST-Raf-(1-648) (lane 6) affinity beads. The soluble wild-type Nef protein in lane 12 was used as a positive control in the Western blot assay. The integrity of the purified soluble recombinant Nef proteins used in the above direct binding assay was assessed by SDS-PAGE analysis and Coomassie Blue staining (Fig. 4B, lanes 1-5). The results of the direct binding assay demonstrate that Nef and c-Raf1 are sufficient and necessary for direct physical interaction in vitro. In addition, the results confirm the previous findings (Fig. 3A) showing that aspartic acid 174 within the acidic sequence motif is critical for physical interaction with c-Raf1, as the single D174G substitution completely abrogated the binding (Fig. 4A, lanes 3 and 8). Although the single D175G at position 175 was not analyzed in the direct binding assay, we predict that the results will be similar to that with the D174G mutant. Taken together, the results of Fig. 4A demonstrate that the minimal sequence for binding of c-Raf1 to the HIV-1 Nef encompasses the conserved acidic sequence motif, Asp-Asp-X-X-X-Glu (positions 174-175) within the carboxyl-terminal region of Nef, and also indicate that the primary Nef-binding site in the c-Raf1 protein maps within a minimal peptide (residues 50-133) previously shown to be critical for binding to Ras (36, 48-50).


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Fig. 4.   Direct physical interaction between soluble Nef and GST-Raf. Affinity beads containing 1 µg of the truncated GST-Raf-(50-133) (lanes 1-5) and 1 µg of the full-length GST-Raf-(1-648) (lanes 6-10) fusion proteins were used to precipitate approximately 1 µg of full-length GST-free soluble wild type and mutant recombinant Nef proteins as described under "Experimental Procedures." The precipitated Nef proteins were resolved by SDS-PAGE and detected by immunoblot analysis with the anti-Nef mAb. Soluble GST protein (lanes 1 and 6); wild type Nef protein (lanes 2 and 7); mutant D174- protein with Asp-174 right-arrow Gly (lanes 3 and 8); mutant DD- protein with double Asp-174 right-arrow Gly and Asp-175 right-arrow Gly (lanes 4 and 9); and mutant DDE- protein with triple Asp-174 right-arrow Gly, Asp-175 right-arrow Gly, and Glu-179 right-arrow Gly (lanes 5 and 10) substitutions, respectively. Lanes 11 and 13 are blank wells (sample buffer), and lane 12 contains about 1 µg of the soluble wild type WT Nef protein as a positive immunoblot control. B, Coomassie Blue-stained SDS-12% polyacrylamide gel containing GST treated with thrombin (no product released) (lane 1) and about 1 µg of the soluble wild type (lane 2), D- (lane 3), DD- (lane 4), and DDE- (lane 5) Nef proteins. Sizes of the molecular mass markers are shown in kDa units in the margin.

Coimmunoprecipitation of a Nef-c-Raf1 Complex from a Stable Nef Expressing Cell Line-- We next determined whether the interaction we have observed in vitro between the recombinant Nef and c-Raf1 proteins also occurs in vivo. We reasoned that if a stable Nef-c-Raf1 complex exists in vivo, then it can be pulled down with an antibody specific for either the HIV-1 Nef or c-Raf1 protein by coimmunoprecipitation assay on soluble lysates of permanent T-cell lines constitutively expressing the native Nef protein. Thus, using lysates of CEM T-cell lines permanently infected with pLXSN-nef expression vectors carrying the HIV-1LAI (HTLV-111B) wild-type nef allele in either the forward (NEF) or reverse (FEN) orientations, both the 72-kDa c-Raf1 and 27-kDa Nef protein were coimmunoprecipitated as a complex with the 55 S anti-Nef mAb from the NEF cell line (Fig. 5, lane 1) but not from the FEN cell line which does not express the Nef protein (Fig. 5, lane 2).


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Fig. 5.   Coimmunoprecipitation of a stable Nef-c-Raf1 complex from Nef-expressing T-cell lines. Immunoblot analysis of Nef and c-Raf1 proteins coimmunoprecipitated from lysates of permanent NEF and FEN CEM T-cell lines following fractionation by SDS-PAGE. The blot was probed both with the anti-Nef mAb and anti-Raf1 mAb. The NEF (lane 1) and FEN (lane 2) cell lysates were immunoprecipitated with the anti-Nef mAb. The NEF (lane 3) and FEN (lane 4) cell lysates were immunoprecipitated with the anti-Raf mAb. The NEF (lane 5) and FEN (lane 6) cell lysates were immunoprecipitated with a nonspecific anti-rabbit mAb used as negative control. The NEF (lane 7) and FEN (lane 8) cell lysates each containing 1 × 105 CEM cells were run as positive controls for total Nef and c-Raf1 proteins, respectively. Arrows identify the 27-kDa Nef and 72-kDa c-Raf1 proteins. Molecular mass markers are shown in kDa units in the margin.

In the reciprocal experiment, the anti-Raf1 mAb also coimmunoprecipitated both the 27-kDa HIV-1 Nef protein and the native 72-kDa c-Raf1 protein complex from the NEF cell line (Fig. 5, lane 3) but not from the FEN cell line (Fig. 5, lane 4). As expected, the anti-Raf1 mAb also immunoprecipitated the c-Raf1 protein from the FEN cell line (lane 4). The 27-kDa Nef band migrates slightly behind the 25-26-kDa light chain IgG band of the antibody species (Fig. 5, lanes 2 and 4). In addition, a nonspecific monoclonal antibody was also used in immunoprecipitation assays with the same CEM T-cell lysates but did not bring down either Nef or c-Raf1 protein from both the NEF (lane 5) and the FEN (lane 6) cell lines. Immunoreactivities of the anti-Nef and c-Raf1 mAbs with total lysates from the NEF (lane 7) and FEN (lane 8) cell lines are also shown. Therefore, results of the coimmunoprecipitation experiment demonstrate that interaction between the native HIV-1 Nef protein and c-Raf1 protein occurs in vivo, suggesting that the stable Nef-c-Raf1 complex may be important for Nef action in vivo. Studies are currently underway to characterize further the role of the Nef-c-Raf1 interaction in modulating signal transduction in T-cells.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Several studies suggest that the Nef proteins of HIV-1, HIV-2, and SIV have a critical role in modulating transduction of T-cell activation signals as well as in T-cell activation (2, 7, 13-18, 50). It is possible that Nef exerts its effects on T-cell signaling and activation by its ability to associate with specific factors involved in signaling pathways in vivo (17, 22, 23, 25-28, 30, 33). Thus, we investigated interaction between HIV-1 Nef and the c-Raf1 protein in vitro and identified a well conserved acidic sequence motif with consensus, Asp/Glu-Asp-X-X-X-Glu (where X represents any other amino acids), found at the carboxyl-terminal regions of the HIV-1, HIV-2, and SIV Nef proteins, that is critical for interaction with c-Raf1 in vitro. We have shown that the Nef acidic sequence is structurally quite similar to the corresponding sequence, Asp-Pro-Thr-Ile-Glu-Asp, that spans amino acids 33-38 within the c-Raf1-binding site in the conserved core effector loop of the Ras GTP-binding protein (36, 46). However, this sequence is oriented in the reverse position relative to the Nef sequence (Table I). Analysis of GST-Delta Nef truncations has allowed us to define the minimal region for binding c-Raf1 in vitro in precipitation pull-down assays. This region was mapped to a sequence spanning amino acids 165-177 within the carboxyl-terminal region of the HIV-1 Nef protein, which is critical for the Nef-c-Raf1 interaction. Additionally, we showed that c-Raf1 residues spanning the region between amino acids 50 and 133, which contains a minimal domain for binding Ras (46, 48, 50), are also sufficient for binding the full-length recombinant HIV-1 Nef, since single substitution mutations involving either Asp-174 right-arrow Gly or Asp-175 right-arrow Gly changes are sufficient to completely abrogate c-Raf1 binding in vitro. Significantly, we demonstrated that the Asp-174 right-arrow Gly mutation was sufficient to block direct binding of the soluble Nef mutant to both the full-length GST-Raf-(1-648) protein and a truncated GST-Raf-(50-133) containing the RBD. Although the Asp-175 right-arrow Gly point mutation was not analyzed in the direct binding assay, we expect that it will also effectively block direct binding with c-Raf1 in vitro. In addition, we also showed that a pairwise Asp-174 right-arrow Gly and Asp-175 right-arrow Gly and triple Asp-174 right-arrow Gly, Asp-175 right-arrow Gly, and Glu-179 right-arrow Gly substitution mutation within the conserved acidic Nef sequence completely blocked direct interaction with c-Raf1 in vitro.

We have also demonstrated the in vivo association between the native HIV-1 Nef protein and c-Raf1 proteins in a stable complex that was coimmunoprecipitated from lysates of the NEF cell line expressing both the Nef and c-Raf1 proteins but not from the FEN cell line that expressed the c-Raf1 protein but did not express Nef.

We occasionally were able to precipitate a tripartite Nef-c-Raf1-MEK-1 complex in our in vitro precipitation assay, but we did not observe a similar triple complex in lysates of the NEF expressing CEM T-cell (data not shown). Since a highly stable c-Raf1-MEK-1 complex is known to exist in vivo and is important for signaling through the c-Raf1-MAP kinase pathway (51), we cannot totally rule out the possibility that Nef may interact with such a complex in vivo. It has previously been shown that the HIV-1 Nef exists in a complex with mitogen-activated protein kinase in vitro (21), but no evidence was presented to show whether Nef and MEK-1 associate with each other. Presumably, MEK-1 forms a specific complex with c-Raf1 (51) without interference by the complex formed between Nef and c-Raf1 in vitro. Although we do not yet know the reason for the discrepancy between our in vitro and in vivo results with regard to the Nef-c-Raf1-MEK-1 tripartite complex, it may be that such a triple complex is not sufficiently stable in vivo to allow detection with the coimmunoprecipitation assay used in the present study.

While the present work was in progress, we were excited to learn of the findings of Aiken et al. (52) and LaFrate et al. (53) showing that introduction of a single Asp-174 right-arrow Lys or pairwise Asp-174 right-arrow Lys and Asp-175 right-arrow Lys substitutions of aspartic acids 174 and 175 for lysine effectively blocked the ability of both the HIV-1 and SIV Nef proteins to down-regulate the CD4 receptor (52, 53). These results parallel our present findings showing that a single Asp-174 right-arrow Gly or Asp-175 right-arrow Gly substitution and a double Asp-174 right-arrow Gly and Asp-175 right-arrow Gly substitution introduced within the HIV-1 Nef acidic sequence, Asp-Asp-Pro-Glu-Arg-Glu (residues 174-179), can effectively abrogate interaction between Nef and c-Raf1 proteins in vitro. The ability of the same pairwise mutations at the highly conserved pair of acidic amino acids at positions 174 and 175 to simultaneously block the Nef-c-Raf1 binding in vitro and the Nef-induced CD4 down-regulation function in vivo strongly suggests that c-Raf1 may have a role in Nef-induced CD4 modulation.

In studies with long term survivors with non-progressive HIV-1 infection, it has been shown that these individuals are infected with an HIV-1 strain that has a high frequency of defective nef alleles (54). It is of interest to also note that a significant number of these defective nef alleles also harbor the aspartic acid to lysine (Asp-174 right-arrow Lys) substitution at position 174 in the Nef protein that are also defective in the ability to down-regulate the CD4 receptor (55). Whether or not the Nef-c-Raf1 interaction we described in the present study is also linked to other Nef functions such as CD4 down-regulation (2, 13, 21) and modulation of T-cell signaling pathways (2, 13-18) remains to be determined. However, the ability of Nef to interfere with transduction of signals suggests that the viral protein may directly interact with specific proteins of T-cell signaling pathways.

Activated Ras, a target of the c-Raf1 protein, has been shown to recruit c-Raf1 to the plasma membrane where it becomes activated by tyrosine phosphorylation (56, 57). Whether Nef functions by disrupting interaction between Ras and c-Raf1 and therefore interfering with the Ras-Raf-MAP kinase signaling cascade remains to be determined. However, our present findings provide the first demonstration of the direct interaction between Nef and the c-Raf1 kinase in vitro. Although we have no data to suggest that Nef and Ras compete for binding c-Raf1, it is of interest to note the close similarity in amino acid sequence conservation between the c-Raf1-binding sites in Nef (residues 174-179) and Ras (residues 33-38) (36). Both sequences are acidic with similar orientation and spacing of critical acidic amino acids, and they are similarly located within conserved flexible or disordered secondary loop structures (44, 45).

Abrogation of the Nef-c-Raf1 interaction in vitro (this study) and the Nef-induced CD4 down-regulation in vivo (52, 53) by single or double mutations that substituted an uncharged nonpolar (aliphatic) or positively charged (basic) amino acid for the negatively charged (acidic) amino acid residues at positions 174 and 175 in the Nef sequence suggests that strong charge-charge interactions may be involved in both functions. A mechanism was previously suggested for involvement of conserved charge-charge interactions between the acidic side chain groups of Asp-33, Glu-37, and Asp-38 of Ras with the corresponding basic side chain groups of Lys-84, Lys-87, and Arg-89, respectively, within the RBD of c-Raf1 (36, 49). Therefore, it is tempting to speculate that Nef may function by presenting itself as a "false" G-protein (2, 58), and it may also participate as a downstream mediator or interceptor of signals passing through the Ras-Raf-MAP kinase signaling pathway. Like Ras (56, 57), Nef is associated with the inner face of the plasma membrane (1-3) and may physically interact with c-Raf1 upon recruitment to the membrane by Ras (56, 57). Therefore, direct physical interaction between Nef and the c-Raf1 kinase within the described acidic sequence motif could have important significance for the mechanism(s) of the Nef-induced T-cell signaling defects and CD4 down-regulation functions in vivo.

    ACKNOWLEDGEMENTS

We thank Dr. Frank Ruscetti and Dr. David Derse for their critical reading of this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 2P20RR011606-02.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Biology, Spencer Hall, Rm. 110, Morgan State University, 1700 Cold Spring Ln., Baltimore, MD 21251. Tel.: 410-319-4005; Fax: 410-426-4732; E-mail: ksamuel{at}moac.morgan.edu.

1 The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; GST, glutathione S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; bp, base pair; MAP, mitogen-activated protein; RBD, Ras-binding domain.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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