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Journal of General Virology(1993), 74, 1581-1589. Printedin GreatBritain 1581 Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro: a role for myristylation Mark Harris* and Karen Coates MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, U.K. The human immunodeficiency virus (HIV) type 1 nef gene product was expressed as an N-terminal fusion protein with glutathione-S-transferase (GST) in the baculovirus system. The resulting nefGST fusion protein was found to be authentically myristylated at the N terminus and could be purified to homogeneity by one-step affinity chromatography on immobilized glutathione. The high affinity of nefGST for glutathione was exploited to develop an assay to identify cellular proteins capable of interacting with nef Several such proteins were identified in extracts from the Jurkat human T cell line. The interaction between neJZbinding proteins and immobilized nefGST could be specifically competed by the addition of soluble nef Cell fractionation showed that neJ:binding proteins were present in both cytosolic and membrane-associated fractions. A non-myristylated derivative failed to bind to the membrane-associated proteins but was able to bind to the cytosolic group, albeit with reduced affinity. In addition, a single protein present in both soluble and membrane-associated fractions exhibited myristylation-independent binding to nef By analogy with other myristylated proteins such as MARCKS (myristylated alanine-rich C kinase substrate) and the Rous sarcoma virus transforming protein, src, the membrane-associated proteins that bind only to myristylated nef may represent a specific membrane target for nef The cytosolic proteins that interact with nef may constitute soluble components of an as yet unidentified signal transduction pathway which is the target of nefaction in the HIV-l-infected cell. Introduction subsequently identified as important for this enhancement, including Cys143, Cysl70 and Leu182 (Zazapoulos & Haseltine, 1992). Luria et al. (1991) examined the effect of nefon the induction of interleukin 2 receptor (1R2R) mRNA: Jurkat T cells constitutively expressing nefwere unable to respond to a wide range of stimuli that normally induced the synthesis of IL2R mRNA. In contrast, cells expressing an alternative nef allele that differed at only three amino acid residues responded normally. The nefgene product is myristylated at the N terminus (Allan et al., 1985) and this acylation is required for the location of nef on the cytoplasmic face of the plasma membrane in infected cells (Yu & Felsted, 1992). Myristylation of nef enhances HI¥-I replication (Zazapoulos & Haseltine, 1992) and, conversely, suppresses HIV-1 LTR transcription (Yu & Felsted, 1992). Myristylation is thus important for nef function but the precise role of acylation remains to be elucidated. Limited sequence similarity between nefand the GTPbinding domains of ras and G proteins has been reported (Samuel et al., 1987; Guy et al., 1987). Furthermore, bacterially expressed nef was shown to bind and hydrolyse GTP (Guy et al., 1987). Other studies with both bacterial and baculovirus-expressed nef gene pro- The nef gene of human immunodeficiency virus (HIV) type 1 encodes a polypeptide of between 200 and 205 amino acids, varying between isolates (Harris et al., 1992a). Despite intensive work, the precise function of the nefgene product remains the subject of controversy. Initial studies identified the protein as a repressor of viral replication (Fisher et al., 1986; Luciw et al., 1987; Terwilliger et al., 1986) that acted by down-regulation of transcription from the HIV-1 long terminal repeat (LTR) (Ahmad & Venkatesan, 1988; Niederman et al., 1989; Maitra et al., 1991), but other studies failed to reproduce these results (Kim et al., 1989; Hammes et al., 1989; Bachelerie et al., 1990). There is a significant degree of amino acid sequence variation between nefisolates both in vivo and in vitro (Harris et al., 1992a) and the contradictions regarding nef function may represent the consequences of this variation. This problem was highlighted by a number of recent studies. Terwilliger et al. (1991) demonstrated that the replication of the IIIB provirus was suppressed by the corresponding IIIB nef allele but that replacement of nefin the IIIB provirus by the ELI nefallele enhanced the growth of the provirus. A number of amino acid residues in the ELI nef gene were 0001-1605 @1993 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 1582 M. Harris and K. Coates ducts, however, failed to reproduce these findings, suggesting that the activity was due to a contaminating bacterial enzyme (Kaminchik et al., 1990; Nebreda et al., 1991 ; Matsuura et al., 1991 ; Harris et al., 1992a). It has been reported that nef is phosphorylated by protein kinase C, although this observation was made following the expression of nef in BHK21 cells infected with a recombinant vaccinia virus (Guy et al., 1987) and has not been demonstrated for nef expressed in HIV-l-infected human cells. Nef also possesses limited sequence similarity with the human thyrotropin receptor (Burch et al., 1991) and scorpion neuroactive peptides (Werner et al., 1991). All these observations, together with the location of nef on the plasma membrane and its potential ability to modulate IL2R activation, suggest that its role in the viral life cycle involves perturbation of cellular signal transduction pathways. In this context, nef has been reported to down-regulate cell surface expression of CD4 (Guy et al., 1987; Garcia & Miller, 1991) although even this putative function is subject to controversy because Cheng-Mayer et al. (1989) failed to observe this phenomenon. Unambiguous definition of the biochemical function of nef in the life cycle of HIV will involve the precise identification of the target(s) of nef action. We previously described the expression of both laboratory and primary isolates of HIV- 1 nef in Escherichia eoli as fusions with glutathione-S-transferase (GST) (Harris et al., 1992a) using the pGEX system (Smith & Johnson, 1988). By exploiting the high affinity of GST for glutathione immobilized on agarose beads to generate nef affinity reagents, these fusion proteins appeared to provide the ideal method for identifying nef-binding proteins. However, although GST-nef fusion proteins linked to glutathione-agarose (GA) beads were able to precipitate specifically a set of cytoplasmic proteins from extracts of human T cell lines (Harriss et al., 1992b) binding was inefficient and not reproducible. One possible explanation for this might be that nef was not expressed in its authentic eukaryotic conformation. In the pGEX system proteins are expressed as C-terminal fusions with GST and it is likely that the N terminus of nef needs to be exposed. Moreover, myristylation does not occur in prokaryotes and, by analogy with other myristylated proteins such as poliovirus P1 capsid precursor (Ansardi et al., 1992) and the Rous sarcoma virus transforming protein src (Resh & Ling, 1990), myristylation of nef is potentially important for its interaction with cellular proteins. Nef was therefore expressed in the baculovirus expression system as the Nterminal portion of a GST fusion molecule. Nefexpressed in this system was myristylated at the N terminus and could be efficientlypurified by one-step affinity chromatography on GA. This new reagent allowed the definition of three classes of nef-binding proteins on the basis of subcellular localization and myristylation dependence. Methods Plasmid construction. The coding sequences for GST were isolated by PCR using the primer pair NNNGTCGACATGTCCCCTATACTAGGT (coding strand) and NNNGGTACCCCCGGGTTATTTTGGAGGATGGTC (non-coding strand) (where N is any nucleotide) and were cloned into pBluescript as a SalI-KpnI fragment. A KpnI EcoRI-HindIII-BamHI oligonucleotide linker adaptor was ligated into the KpnI site to facilitate further cloning steps. This construct was termed pGST. The coding sequence of the HIV-1 nef gene (strain BH10) was isolated by PCR using the primer pair NNGGATCCTATAAG A T G G G T G G C A A G T G G (coding strand) and NNNNATCGATGCAGTTCTTGAAGTACTCCGG (non-coding strand), and ligated onto the ClaI BamHI fragment of pGST in the presence of BamHI. The ligation product was gel-purified and ligated into the BamHI site of the transfer vector pAcCL29 (Livingstone & Jones, 1989). For GST expression, a BamHI linker-adaptor was ligated to the 5' end of the SalI-BamHI fragment of pGST and this was cloned into pAcCL29. The myristylated GST derivative (myrGST) was constructed by cloning oligonucleotides encoding the myristylation sequence of HIV-1 gag p55 (Met-Gly-Ala-Arg-Ala-Ser) into the SaII site of pGST to generate the plasmid pmGST. For the production of non-myristylated neff the coding strand primer N N G G A T C C T A T A A G A T G A G T G G C A A G T G G was used, replacing the glycine residue at position 2 with a serine. Neftagged with six histidine residues at the C terminus (nef-6H) was produced by PCR using the coding primer described above and NNGGATCCTTAGTGATGGTGATGGTGATGTCTGCAGTTCTTGAAGTACTC as the non-coding primer. This PCR product was cloned directly into pAcCL29 as a BamHI fragment. Isolation and propagation of recombinant baculoviruses. Sf9 cells were grown in TC100 medium (Gibco) containing 10% fetal calf serum (FCS) at 27 °C. Cells (1 x 106 per 35 mm dish) were transfected by the calcium phosphate method with 5 to 10 ~tg of the appropriate pAcCL29 recombinants and 1 lag of AcRPIacZ viral DNA linearized with Bsu36I (Kitts et al., 1990). After 2 days the medium was harvested, filtered through a 0.22 gm filter unit and plated onto Sf9 cells. White plaques were picked and underwent three rounds of plaque purification prior to expansion. Purification of recombinant proteins. Sf9 cells were infected with the appropriate recombinant viruses at 10p.f.u./cell and harvested at 72 h post-infection (p.i.). For the production of GST and GST fusion proteins, infected cells were pelleted, washed twice with ice-cold PBS and lysed in PBS containing 1% Triton X-100, 5 mM-EDTA, 10 mMiodoacetamide and the protease inhibitors aprotinin (2 gg/ml), PMSF (1 mM) and leupeptin (1 lag/ml). After incubation at 4 °C for 30 min the lysate was clarified by centrifugation at 10000r.p.m. for 15 min at 4 °C. Clarified supernatant was incubated with GA beads at 4 °C for 2 h and the beads were then washed extensively in 50 mM-Tris-HC1 pH 8.0. Fusion protein was eluted by the addition of 50 mM-Tris-HC1 pH 8.0 containing 10 m~a reduced glutathione. Fractions containing protein were pooled and dialysed overnight against two changes of 50 mMTris HC1 pH 8"0. Then, 1 mM-PMSF and 5 mM-EDTA were added and purified protein was stored in aliquots at - 7 0 °C. For the production of purified nef-6H, clarified lysate was incubated with nickel nitrilotriacetic acid (Ni-NTA)-agarose, washed with PBS and eluted with 100mM-citrate buffer pH 6.0. Protein-containing fractions were pooled, dialysed against 20 mM-sodium phosphate buffer pH 7.2 and stored at - 7 0 °C. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 Nef binds to cellularproteins in vitro Analysis of myristylation. Sf9 cells (5 x 106 per 25 cm 2 flask) were infected with recombinant viruses at 10 p.f.u./cell. At 18 h.p.i, cells were washed with TC100 medium containing 0-25% BSA and incubated with 2.5ml (per flask) TC100/0.25% BSA containing 100 IxCi/ml [ZH]myristic acid (Amersham; 53.4 Ci/mmol) for 24 h. Cells were harvested, washed once with ice-cold PBS and lysed as described above. Labelling with [3SS]methionine was carried out similarly using methionine-free TC100 containing 1% FCS and 100gCi/ml [35S]methionine (Amersham; 1000 Ci/mmol). nefGSr Western blotting. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and membranes were blocked for 30 min in 25 mM-Tris-HC1 pH 7.5, 150 mM-NaCI, 0'05 % Tween 20 and 2 % dried skimmed milk. Blots were then incubated with primary antibodies followed by goat anti-mouse alkaline phosphate conjugate in 25 mMTris-HC1 pH 7"5, 150 mM-NaCI, 0.5 % Tween 20, 4 % dried skimmed milk and 20 % goat serum for 1 h at room temperature. Blots were developed in 100 mM-Tris-HC1 pH 9"5, 100 mM-NaC1 and 5 mM-MgCI~ with 0"33 mg/ml nitroblue-tetrazolium and 0.17mg/ml 5-bromo-4chloro-3-indolylphosphate. bacGST [ GST myrGST Myr~ GST Metabolic labelling and production of cell lysates. Log phase Jurkat cells (clone E6-1) were pelleted, washed with PBS (37°C) and resuspended in methionine-free RPMI 1640 containing 1% FCS at a concentration of 107 to 2 x 107 cells/ml. After incubation at 37 °C for 1 h the cells were pelleted and resuspended in the same medium containing 100 laCi/ml [35S]methionine (Amersham; 1000 Ci/mmol) and incubated for a further 4 to 5 h at 37 °C. Labelled cells were pelleted, washed twice in PBS and lysed in 10 mM-PIPES-NaOH pH 7.2, 120mM-KCI, 30 mM-NaC1, 5 mM-MgC12, 1% Triton X-100, 10% glycerol containing 10 mM-iodoacetamide, 1 mM-PMSF, 2 ~tg/ml aprotinin and 1 I~g/ml leupeptin at a concentration of 5 x 107 cells/ml. Lysates were incubated at 4 °C for 30 min, clarified by centrifugation (12000g for 5 min) and stored at - 7 0 °C. Cellfractionation. Labelled cells were washed twice in PBS, incubated in hypotonic buffer (10 mM-PIPES-NaOH pH 7"2, 0.5 mM-MgCI2) for I0 rain and lysed by 30 strokes of a tight-fitting Dounce homogenizer. After adjustment of the salt concentration to 120 mM-KC1/30 mMNaC1, nuclei were pelleted (500 g/5 min) and then membranes were pelleted from the clarified lysate (150 000 g for 45 min). The membrane pellet was extracted with lysis buffer as above and the cytosol fraction was adjusted to 5 mM-MgC12, 1% Triton X-100 and 10% glycerol. Nef binding reactions. Lysates were precleared by sequential incubations for 30 min at 4 °C firstly with 1/7 volume GA beads and then with GA beads loaded with GST (expressed in E. colO. Then 100 ~tl cleared lysate was incubated for 1 h at 4 °C with 10 lal GA beads loaded with the appropriate protein. All loaded beads contained approximately 1 mg/ml protein. Beads were washed five times with 0-5 ml lysis buffer, boiled in sample buffer (0.8 % SDS, 8 % glycerol, 2 % 2-mercaptoethanol and 25 mM-Tris-HCl pH 6'8) and separated by SDS-PAGE. Bound proteins were visualized by fluorography. Results Expression and purification of nef affinity reagents Myristylated and non-myristylated nef gene products were expressed as N-terminal fusion proteins with GST [neJGST and nef(m-)GST respectively] using the baculovirus vector system. Myristylated nef was also expressed with a C-terminal tail of six histidine residues to permit purification by metal chelate affinity chromato- Myr~ NEF [ GST nef(m-)GST nef~H 1583 I Myr~ NEF I GST NEF ]RHHHHHH Fig. 1. Structure of recombinant baculovirus-expressed proteins for use as affinity reagents. Myr, Myristic acid residue; R, arginine; H, histidine. graphy. GST was expressed alone and with an additional N-terminal myristylation sequence (Met-Gly-Ata-ArgAla-Ser) to provide negative controls. The structure of the recombinant fusion proteins is shown in Fig. 1, their expression and purification in Fig. 2. Sf9 cells were infected with 10p.f.u./cell of the appropriate recombinant viruses and cytoplasmic lysates were prepared 48 h p.i. Fig. 2 (a) demonstrates that all the recombinant proteins were expressed in the cytoplasm as soluble and largely undegraded polypeptides that could be efficiently purified from lysates by one-step affinity chromatography on either GA or Ni-NTA-agarose. Fig. 2 (b), (e) and (d) confirm that the proteins purified from infected lysates were recognized on Western blots by the appropriate monoclonal antibodies (MAbs). NefGST and nef(m-)GST fusion proteins were recognized by MAbs to both the N and C termini of nefand also by a MAb to GST (vpg66). Nef-6H was recognized only by the anti-nef MAbs and bacGST/myrGST were recognized only by vpg66. The ability of nefGST fusion proteins to bind strongly to GA indicates that the GST portion of the fusion protein is in the native state. Interactions between the two parts of the fusion protein which could potentially interfere with the binding of cellular proteins to nefare therefore unlikely, but cannot be ruled out. Interestingly, the N terminus-specific antibody recognizes a dimer of nefGST, nef(m-)GST and nef~H that is not recognized by the C terminus MAb or vpg66. This observation suggests that the epitope bound by the C terminus MAb is involved in dimerization and that the epitope on GST recognized by vpg66 might be obscured in the dimer. The significance of this apparent dimerization of nef is not clear. To confirm the nefGST, nef-6H and myrGST were myristylated, Sf9 cells were infected with the appropriate Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 M. Harris and K. Coates 1584 (a) 1 (b) 2 3 4 5 6 7 8 9 1 10 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 9 10 - - 66K - - - 46K - - - 29K - - - 24K - - (a) (c) 1 2 3 4 5 6 7 8 9 1 10 66K 46K o. • ' 29K - - 24K Fig. 2. Expression and purification of recombinant protein affinity reagents. Infected Sf9 cell lysates (lanes 1, 3, 5, 7 and 9) or purified proteins (lanes 2, 4, 6, 8 and 10) were separated by 12% S D S - P A G E and either stained with Coomassie brilliant blue R-250 (a), or transferred to nitrocellulose and probed with an anti-GST M A b (b), M A b s directed against the N terminus (c) or C terminus (at) of nef. Cells were infected with recombinant viruses expressing the following. Lanes 1 and 2, nefGST; lanes 3 and 4, neflm-)GST; lanes 5 and 6, nef-6H; lanes 7 and 8, m y r G S T ; lanes 9 and 10, bacGST. recombinant viruses and labelled with either [35S]methionine or [3H]myristic acid (Fig. 3). Whereas [35S]methionine was incorporated into all the recombinant proteins (Fig. 3 a), only those containing an N-terminal myristylation sequence incorporated [aH]myristic acid (Fig. 3 b). The presence of myristate at the N terminus renders that protein refractory to Nterminal amino acid sequencing by Edman degradation, so this method was used to determine the proportion of myristylated product. Only 1% of a sample of nefGST was accessible to cleavage by Edman reagent, indicating that the protein was essentially completely myristylated (data not shown). The correlation between the expected and observed patterns of myristylation indicated that correct initiation of translation was occurring at the first methionine codon in nef. Initiation has been reported at the second methionine (residue 20) in some expression systems (Ahmad & Venkatesan, 1988; Kaminchik et al., 1991). The data presented in Fig. 2 and 3 thus confirm the physical integrity of the proteins used in this study. Binding of cellular proteins to nef in vitro Lysates were prepared from either unstimulated or phorbol 12-myristate 13-acetate (PMA)-stimulated Iurkat cells as described in Methods. Aliquots of the lysates were sequentially precleared by incubation with GA beads alone followed by E. col#expressed GST bound to GA beads. Cleared lysates were then incubated with baculovirus-expressed bacGST or nefGST bound to GA beads. Following extensive washing of the beads with lysis buffer, bound proteins were eluted with sample buffer and analysed by SDS-PAGE on 6 to 20 % linear gradient gels followed by fluorography (Fig. 4). A number of proteins in the Jurkat extracts bound strongly either to GST, glutathione or to agarose beads and thus were retained by bacGST-GA beads (lanes 1, 2). In the presence of nefGST (lanes 3, 4), an additional subset of proteins, designated p280, p97, p75, p55/57 and p35 according to their apparent Mrs, were retained on the beads. A number of tess abundant proteins of lower M~, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 Nef binds to cellularproteins in vitro (a) 1 2 3 4 5 6 7 8 9 1 10 11 12 2 3 1585 4 - 69K - 46K 30K - -21K 4 200K (b) 1 2 3 4 5 6 7 8 9 10 11 12 - 69K - 46K - 30K 97K - 4 -21K 69K Fig. 3. Baculovirus-expressed neJ~3STis myristylated at the N terminus. Autoradiograph of lysates (lanes 1, 2, 3, 5, 7, 9 and 11) and purified fusion proteins (lanes 4, 6, 8, 10 and 12), prepared from infected Sf9 cells labelled with either [35S]methionine (a) or [3H]myristic acid (b) and separated by 12 % SDS-PAGE. Cells were infected with recombinant viruses expressing proteins as follows. Lanes 1, Mock-infected; lanes 2, 13-galactosidase; lanes 3 and 4, nefGST; lanes 5 and 6, nef(m-)GST; lanes 7 and 8, nef-6H; lanes 9 and 10, myrGST; lanes 11 and 12, bacGST. "4 46K - 4 30K - 21-5K - p32, p28 and p26, were also observed. Only one difference could be discerned between unstimulated and stimulated extracts, a protein of 45K, that bound to nefGST but not bacGST was present only in extracts of PMAstimulated Jurkat ceils (Fig. 4, lane 4). The subcellular location of these nef-binding proteins was determined by separating a Jurkat cell lysate into cytosolic and membrane-associated protein fractions. These fractions were precleared as described and incubated with either bacGST- (Fig. 5, lanes 1 to 3) or nefGST~GA beads (Fig 5, lanes 4 to 6). The results show that nef-binding proteins could be separated into three classes. The first is proteins that were exclusively membrane-bound, including p280, p35, p32, p28 and p26, and that were only observed in the total cytoplasmic fraction (lane 4) or the membrane-bound fraction (lane 5). The second class is proteins that were present in the total cytoplasmic fraction (lane 4) and the cytosol fraction (lane 6, p57/55). A number of nef-binding proteins, and in particular a doublet of p75 and a previously unobserved protein of 180K, were present in the cytosolic fraction but not in the total cytoplasmic lysate. This may have been due to differences in extraction procedures or it could be that the absence of proteins such as p280 and p35 permitted the binding of other proteins to nef. Thirdly, only one protein, p97, was Fig. 4. NefGST interacts with cytoplasmic proteins from unstimulated or PMA-stimulated Jurkat T cells. Autoradiograph of extracts from unstimulated (lanes l, 3) or PMA-stimulated (50 riM) (lanes 2, 4) Jurkat cells metabolically labelled with [35S]methionine, precleared as described, incubated with either b a c G S T - G A beads (lanes 1,2) or nefGST GA beads (lanes 3, 4) and separated by 6 to 20 % SDS-PAGE. Arrowheads, subset of proteins, p280, p97, p75 p55/57 and p35. Arrow, 45K protein binding only to nefGST in PMA-stimulated Jurkat cell extracts. present in both cytosolic and membrane-associated fractions (lanes 4 to 6). The effect of N-terminal myristylation on the binding of nefto cellular proteins was investigated using the nonmyristylated nef(m-)GST fusion protein. Precleared Jurkat lysate was incubated with myrGST, bacGST, nefGST or nef(m-)GST as described. The results are shown in Fig. 6. A number of proteins, p280, p32 and p28, that bound to myristylated nef(lane 3) failed to bind to non-myristylated nef(lane 4). It is unclear whether this is also the case for p35 and p26 because the higher background obtained in the presence of myristylated nef (lane 3) obscures these bands. Proteins p75 and p57/55 (migrating as a single band in Fig. 6) still bound to nonmyristylated nef but with markedly lower affinity. The binding of p97 was independent of myristylation: p97 bound equally well to nefGST or nef(m-)GST. A Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 1586 M. Harris and K. Coates 1 2 3 4 5 6 1 2 3 4 200K 200K - 97K - 97K .q 69K - 69K 46K 46K - 30K 30K - 21.5K 21.5K - Fig. 5. Nef-binding proteins are b o t h cytosolic and m e m b r a n e associated. J u r k a t cells labelled with [aSS]methionine were fractionated as described and incubated with b a c G S T G A beads (lanes 1 to 3) or n e f G S T - G A beads (lanes 4 to 6). Lanes 1 and 4, total cytoplasmic extract; lanes 2 and 5, m e m b r a n e fraction; lanes 3 and 6, soluble fraction. myristylated GST derivative (myrGST; lane 1), consisting of bacGST with the addition of the N-terminal six amino acids from HIV-1 gag p55 (Met-Gly-Ala-ArgAla-Ser), failed to bind to any of the proteins that bound only to myristylated nef. This confirmed that these proteins were specifically binding to myristylated nef, and were not simply interacting with the myristate moiety at the N terminus. To confirm the specificity of the binding of these cellular proteins to nef, a competition assay was performed (Fig. 7). Increasing amounts of soluble myristylated nef (nef-6H) (lanes 3 to 5) or BSA (lanes 6 to 8) were added to aliquots of precleared Jurkat extract prior to incubation with nefGST-GA beads. Addition of exogenous soluble nefwas able to inhibit the binding of p280, p97, p75, and p35 to nefGST-GA beads [compare lane 2 (no competitor) with lane 5 (40 gg nef-6H)], Fig. 6. Role o f myristylation in the binding of cellular proteins to nef Extract f r o m J u r k a t cells labelled with [3~S]methionine was incubated with m y r G S T G A beads (lane 1), b a c G S T - G A beads (lane 2), nefGST G A beads (lane 3) or neflm-)GST-GA beads (lane 4). presumably by itself binding to these proteins. Lack of definition of low Mr proteins resulted in ambigious visualization of competition for the p26, p28 and p32 proteins. Intriguingly, in the presence of 40 gg nef-6H (lane 5) more p55/57 was bound by nefGST. This result was reproducible and may be explained by the fact that in the presence of high concentrations of soluble nef-6H a small amount was found tightly associated with beads (data not shown). By comparison of the intensity of the bands the relative amounts of p55/57 were clearly higher than those of the other nef-binding proteins and, thus, it would not be as efficiently competed by soluble nef-6H. The presence of nef-6H on the beads, whether by nonspecific association or specific dimerization with nefGST as discussed above, would therefore result in the binding of more p55/57. Addition of a non-specific protein (BSA) to the assay had no effect on the binding of these proteins to nefGST-GA beads. These proteins are thus binding to nef amino acid sequences alone and are not Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 Nef binds to cellular proteins in vitro 1 2 3 4 5 6 7 8 200K- 97K 69K 46K - 30K - Fig. 7. Solublemyristylatednefcompeteswith immobilizednefGST for the bindingof cellularproteins. Solubleprotein was addedto precleared extract from Jurkat cells labelled with [zSS]methionine prior to incubation with bacGST-GA beads (lane 1) or nefGST GA beads 0anes 2 to 8). Lanes 1 and 2, no solubleprotein; lanes 3 to 5, 10, 20 and 40 lag nef-6H; lanes 6 to 8, 10, 20 and 40 tag BSA. Table 1. Classification of nef-binding proteins from Jurkat T cells Localization Cytosol Myristylationdependent Partially dependent on myristylation Myristylationindependent Membrane and cytosol Membrane p280, p35, p32, p28, p26 p55/57, p75 p97 binding to an artefactual combination of nef and GST sequences. Discussion In this study, a baculovirus-expressed nefGST fusion protein was used as an affinity reagent to identify cellular proteins that specifically interact with nef. A number of such proteins, both soluble and membrane-associated, were identified in cytoplasmic extracts of the Jurkat human T cell line. These proteins could be divided into 1587 three classes according to their subcellular location and dependence on N-terminal myristylation of nef for binding. This classification is summarized in Table 1. Prokaryotic GST fusion proteins (expressed in the pGEX system; Smith & Johnson, 1988) have previously been used to identify proteins binding to the retinoblastoma gene product (Kaelin et at., 1991) and the SH2 domain of the actin-binding protein, tensin (Davis et at., 1991). However, as discussed above, GSTnef expressed from pGEX proved to be an unsuitable reagent for the identification of nef-binding proteins. One reason for this is that N-terminal myristylation ofnefis required for the interaction with three of the proteins identified in this study. These proteins (p280, p32 and p28), which failed to bind to non-myristylated nef, are all associated with the cytoplasmic membrane, raising the possibility that one or more of them represents a membrane target for nef. Recent data for two other myristylated proteins, the myristylated alanine-rich C kinase substrate M A R C K S (Li & Aderem, 1992) and src (Resh & Ling 1990), show that association of myristylated proteins with the membrane is not merely the result of insertion of the fatty acid moiety into the lipid bilayer. Instead, a combination of this fatty acid moiety and amino acid sequences at the N terminus interact with specific receptor molecules on the inner surface of the membrane. In the case of src (Resh & Ling, 1990), a myristylated peptide representing the N-terminal 11 amino acids, but not the corresponding non-myristylated peptide, could be cross-linked to a 32K plasma membrane protein. It is unlikely that this 32K protein is identical to p32 described here because the N-terminal amino acid sequences of nef and src are distinct, and myristylpeptides other than src did not cross-link to the 32K protein (Resh & Ling, 1990). Both nefGST and n e f ( m - ) G S T bound to p97 with equal affinity (Fig. 6). p97 was detected in both cytosolic and membrane-bound fractions (Fig. 5). p97 may be only loosely associated with the membrane and readily dislodged by the extraction procedure, or it may be present in both soluble and membrane-bound forms. Interestingly, p97 is the only protein reproducibly bound by pGEX-expressed GSTnef (Harris et al., 1992b; data not shown). These observations suggest that the interaction between nef and p97 is independent of the membrane-bound class of nef-binding proteins and does not involve the N terminus of nef. A third group of proteins including p55/p57 and possibly p75 bind to non-myristylated nef, but the presence of a myristyl residue increases the affinity of binding. These proteins appear to be exclusively cytosolic although they could be very loosely associated with the membrane and completely dislodged during extraction. The fact that these proteins bind to non-myristylated nef Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 1588 M. Harris and K. Coates in the absence of the membrane-associated proteins again suggests that, as with p97, their interaction with nef is independent of this membrane-associated class. It is not clear whether or not they are associated with p97. The interactions of nef with both cytosolic and membrane-bound proteins supports the hypothesis that the mode of nefaction is in some way to perturb specific signal transduction pathway(s) in infected cells. By binding independently to both soluble and membranebound components of a particular signal transduction pathway nefcould effectively prevent the protein-protein interactions required for passage of the signal. Alternatively, nefmight alter the signal in some way, perhaps by diverting it down an alternative pathway so as to elicit an inappropriate response from the cell. Both of these actions could contribute to the cytopathogenesis of viral infection. Further speculation must await the identification of these nef-binding proteins, a task that is in progress. Thanks are due to Dr Bob Possee for the transfer vector pAcCL29, to Dr George Reid for plasmid pGST, to Celia Cannon for producing the anti-nef MAbs and to Tom Dunsford for the anti-GST MAb. Thanks also to Professor Jim Nell and Dr George Reid for critical reading of the manuscript. This work was supported by the MRC AIDS Directed Programme (ADP). Jurkat E6-1 cells were obtained from the ADP Reagent Repository. References AHMAD, N. & VENKATESSAN,S. (1988). Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241, 1481-1485. ALLAN, J. S., COLIGAN, J.E., LEE, T.-H., MCLANE, M.F., KANKI, P. J., GROOPMAN,J. E. & ESSEX,M. (1985). A new HTLV-III/LAVencoded antigen detected by antibodies from AIDS patients. Science 230, 810-813. ANSARDI, D.C., PORTER, D.C. & MORROW, C.D. (1992). Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles. Journal of Virology 66, 4556-4563. BACHELERm, F., ALCAMI, J., HAZAN, U., ISRAEL, N., GOUD, B., ARENZANA-SEISDEDOSF. & VIRELIZIER,J.-L. (1990). Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. Journal of Virology 64, 3059-3062. BURCH, H. B., NAGY, E. V., LUKES,Y. G., CAI,W. Y., WARTOFSKY,L. & BtmMAN, K.D. (1991). Nucleotide and amino acid homology between the human thyrotropin receptor and the HIV-1 nefprotein: identification and functional analysis. Biochemical and Biophysical Research Communications 181, 498-505. CHENG-MAX'ER,C., IANNELLO,P., SHAW,K., Luclw, P. & LEVY, J. A. (1989). Differential effects of nefon HIV replication: implications for viral pathogenesis in the host. Science 246, 1629-1632. DAVIS, S., Lu, M. L., LO, S. H., LIN, S., BUTLER,J. A., DRUKER,B. J., ROBERTS,T. M., AN, Q. & CHEN, L. B. (1991). Presence of an SH2 domain in the actin-binding protein tensin. Science 252, 712-715. FISHER, A. G., RATNER,L., MITSUYA,H., MARSELLE,L. M., HARPER, M.E., BRODER, S., GALLO, R.C. & WONG-STAAL, F. (1986). Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects. Science 233, 655-659. GARCIA, J.V. & MILLER, A.D. (1991). Serine phosphorylationindependent downregulation of cell-surface CD4 by nef Nature, London 350, 508-511. Guy, B., KIENY, M. P., RiVtERE,Y., LE PEUCH, C. Do'r'r, K., GIRARD, M., MONTAGNIER,L. & LECOCQJ.-P. (1987). HIV F/3'orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature, London 330 266-269. HAMMES, S., DIXON, E. P., MALIM, M. H., CULLEN, B. R. & GREENE, W. C. (1989). Nefprotein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proceedingsof the National Academy of Sciences, U.S.A. 86, 9549-9553. HARRIS,M., HISLOP, S., PATSILINACOS,P. & NEIL, J. C. (1992a). In vivo derived HIV-1 nef gene products are heterogeneous and lack detectable nucleotide binding activity. AIDS Research and Human Retroviruses 8, 53%543. HARRIS, M., TALBOT, S., PATSILINACOS,P. & NEIL, J. C. (1992b) The HIV-I nef gene product. Biochemical Society Transactions 20, 517-520. KAELIN, W.G., PALLAS, D.C., DECAPRIO, J.A., KAYE, F.J. & LIVINGSTON,D. M. (1991). Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64, 521-532. KAMINCHIK, J., BASHAN, N., PINCHAS1, D., AMIT, B., SARVER, N., JOEINSTON,M. I., FISCHER,M., YAVIN,Z., GOI~CKI, M. & PANET,A. (1990). Expression and biochemical characterization of human immunodeficiency virus type 1 nefgene product. Journal of Virology 64, 3447-3454. KAMINCHIK, J., BASHAN,N., ITACH, A., SARVER,N., GORECKI,M. & PANET, A. (1991). Genetic characterization of human immunodeficiency virus type l nef gene products translated in vitro and expressed in mammalian cells. Journal of Virology 65, 583-588. KIM, S., IKEUCHI,K., BYRN,R., GROOPMAN,J. & BALTIMORE,D. (1989). Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences, U.S.A. 86, 9544-9548. KITTS, P. A., AYFmS,M. D. & POSSEE, R.D. (1990). Linearization of baculovirus DNA enhances recovery of recombinant virus expression vectors. Nucleic Acids Research 18, 5667-5672. LI, J. & ADEREM,A. (1992). MacMARCKS, a novel member of the MARCKS family of protein kinase C substrates. Cell 70, 791-801. LIVINGSTONE, C. & JONES, I. (1989). Baculovirus expression vectors with single strand capability. Nucleic Acids Research 17, 2366. LUClW, P.A., CHENG-MAYER,C. & LEvY, J. A. (1987). Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proceedings of the National Academy of Sciences, U.S.A. 84, 1434-1438. LURIA, S., CHAMBERS,I. & BERG, P. (1991). Expression of the type 1 human immunodeficiency virus nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. Proceedings of the National Academy of Sciences, U.S.A. 88, 5326-5330. MAITRA,R. K., AHMAD,N., HOLLAND,S. M. & VENKAaXSAN,S. (1991). Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines. Virology 182, 522-533. MATSUURA,Y., MAEKAWA,M., HATTORI,S., IKEGAMI,N., HAYASHI, A., YAMAZAKI,S., MOR1TA,C. &;TAKEBE,Y. (1991). Purification and characterisation of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus. Virology 184, 580-586. NEBREDA,A. R., BRYAN,T., SEGADE,F., WINGFIELD,P., VENKATESAN, S. & SANTOS,E. (1991). Biochemical and biological comparison of HIV-1 NEF and ras gene products. Virology 183, 151-159. NIEDERMA~, T. M. J., TrimLAN, B.J. & RATNER, L. (1989). Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proceedings of the National Academy of Sciences, U.S.A. 86, 1128-1132. RESH, M. D. & LING, H.-P. (1990). Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60 ..... Nature, London 346, 84-86. SAMUEL,K. P., SETH,A., KONOPKA,A., LUTENBERGER,J. A. & PAPAS, T. S. (1987). The 3'-orf protein of human immunodeficiency virus shows structural homology with the phosphorylation domain of human interleukin-2 receptor and the ATP-binding site of the protein kinase family. FEBS Letters 218, 81-86. SMITH, D.B. & JOHNSON, K.S. (1988). Single-step purification of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29 N e f binds to cellular proteins in v i t r o polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 31-40. TERWILLIGER,E., SODROSKI,J. G., ROSEN, C. A. & HASELTINE,W. A. (1986). Effects of mutations within the 3'orf open reading frame region of human T-lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. Journal of Virology 60, 754-760. TERVClLLIGER,E. F., LANGHOFF,E., GABUZDA,D., ZAZAPOULOS,E. & HASELTINE,W. A. (1991). Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences, U.S.A. 88, 10971-10975. WERNER,T., FERRONI,S., SAARMARK,T., BRACK-WERNER,R., BANATI, R. B., MAGER, R., STErNAA,L., KREUTZBERG,G. W. & ERFLE, V. 1589 (1991). HIV- 1 nefprotein exhibits structural and functional similarity to scorpion peptides interacting with K ÷ channels. AIDS 5, 1301-1308. Yu, G. & FELSTED, R. L. (1992). Effect of myristoylation on p27nef subcellular distribution and suppression of HIV-LTR transcription. Virology 187, 46-55. ZAZAPOULOS,E. & HASELTINE,W. A. (1992). Mutational analysis of the human immunodeficiency virus type 1 Eli nef function. Proceedings of the National Academy of Sciences, U.S.A. 89, 6634~6638. (Received 2 February 1993; Accepted 19 March 1993) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 04 Aug 2017 00:59:29