Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

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.

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) synthe-sized 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 SIV mac239 model for nef gene function in vivo, Nef has been shown to be necessary for maintaining high viral loads and for induction of an AIDSlike disease in rhesus monkeys infected with recombinant SIV mac239 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 ␣-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 p56 Lck 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 p56 Lck -binding site (23,24). The molecular interactions between Nef and p56 Lck 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 ␤-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][34][35][36][37][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-1 LAI 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 3 Gly) or 175 (Asp-175 3 Gly) and by combined Asp-174 3 Gly and Asp-175 3 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 Nefinduced T-cell signaling defects involving the c-Raf1-MAP kinase signaling pathway.
c-Raf1 Expression Plasmids-The pGEX-Raf-(50 -133) expression plasmid was created by polymerase chain reaction cloning in which the primer pairs, 5Ј-GTAGGATCCGATCCTTCTAAGACAAGC-3Ј and 5Ј-CTAGAATTCATCCAGGAAATCTACTTG-3Ј containing a 5Ј-BamHI and 3Ј-EcoRI restriction site, were used to release a 245-bp EcoRI-BamHI fragment from the pKSϩcRaf-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 fulllength 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.

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 glutathioneagarose 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.

Coprecipitation Assay
Soluble CEM whole cell lysates containing 5 ϫ 10 7 CEM cell eq/ml were incubated with 50 l (1 g 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 g of thrombin-cleaved soluble recombinant Nef proteins were incubated with 50 l (1 g 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 2ϫ 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 antihuman 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-1 LAI (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 ϫ 10 8 CEM cell eq/ml were prepared from permanent CEM T-cell lines transduced with the HIV-1 LAI (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 l 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.

Recombinant Nef Fusion Proteins-Recombinant
GST-⌬Nef fusion proteins containing overlapping amino acid deletions between residues 136 and 206 at the carboxyl-terminal region of the HIV-1 LAI 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 g 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-⌬12Nef (lane 3), GST-⌬31Nef (lane 5), and GST-⌬41Nef (lane 6), respectively, show anomalous mobilities on the SDS gel. In addition, samples of the glutathione-agarose beads containing 1 g 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.
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 se-quence 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][34][35][36][37][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-1 LAI , positions 204 and 205 of HIV-2 ISY , and positions 203 and 204 in SIV MM239 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.
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 ␤-sheet structure formed between two anti-parallel ␤-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 ␤-strand structures within which the conserved acidic sequence motif in the HIV-1 Nef (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-1 LAI 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 However, the affinity beads containing amino acids 136 -165 of HIV-1 Nef (fusion protein GST-⌬41Nef) also did not precipitate the c-Raf1 protein from the CEM extract ( Fig. 2A, lane 6). The missing sequence from GST-⌬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.
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-⌬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 wildtype and mutant Nef were expressed in E. coli as GST-Nef fusion proteins and were purified by coupling to glutathioneagarose 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 wildtype 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.
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   Table I. 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).
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-1 LAI (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).
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
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)(14)(15)(16)(17)(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-Raf1binding 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-⌬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 carboxylterminal 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 3 Gly or Asp-175 3 Gly changes are sufficient to completely abrogate c-Raf1 binding in vitro. Significantly, we demonstrated that the Asp-174 3 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 3 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 3 Gly and Asp-175 3 Gly and triple Asp-174 3 Gly, Asp-175 3 Gly, and Glu-179 3 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 mitogenactivated 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 3 Lys or pairwise Asp-174 3 Lys and Asp-175 3 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 3 Gly or Asp-175 3 Gly substitution and a double Asp-174 3 Gly and Asp-175 3 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 3 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 Nefc-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)(14)(15)(16)(17)(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 Nef-c-Raf1 Interactions 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 chargecharge 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.