©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Conserved Domain and Membrane Targeting of Nef from HIV and SIV Are Required for Association with a Cellular Serine Kinase Activity (*)

Earl T. Sawai (§) , Andreas S. Baur (1)(¶), B. Matija Peterlin (1)(**), Jay A. Levy , Cecilia Cheng-Mayer (§§)

From the (1)Cancer Research Institute, Department of Medicine, and Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0128

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Among the primate lentiviruses (human immunodeficiency virus (HIV) -1, HIV-2, and simian immunodeficiency virus (SIV)), the nef gene is highly conserved and encodes a myristylated protein of 27 kDa (HIV-1) or 34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIV and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1 Nef have the same effect when introduced into SIV Nef.


INTRODUCTION

The nef gene was first identified as an open reading frame that overlaps with the 3`-long terminal repeat of the human immunodeficiency virus type 1 (HIV()-1)(1) . It is conserved among all primate lentiviruses, i.e. HIV-1, HIV-2, and simian immunodeficiency virus (SIV)(2, 3) . The viral gene product is translated from multiply spliced transcripts and is expressed together with the regulatory proteins Tat and Rev early in the viral replicative cycle(4, 5, 6, 7) .

Nef encodes a myristylated, phosphorylated protein of approximately 27 kDa that forms homomeric oligomers and intramolecular disulfide bonds(1, 8, 9, 10, 11, 12, 13, 14) . Myristylation has been reported to be important for its function(13, 14, 15, 16, 17) . In infected cells, Nef primarily localizes to the cytoplasm and intracellular membranes (18) and preferentially associates with the cytoskeleton(15) . However, it has also been reported to be present in the nucleus(19, 20) .

Initially, Nef was reported to have a negative effect on virus replication and transcription(12, 13, 21, 22, 23, 24, 25, 26, 27, 28, 29) . However, later studies demonstrated that Nef expression has no effect on viral replication in T-cell lines but may have a positive effect on virus replication in peripheral blood mononuclear cells (30, 31, 32) or fetal thymic and liver implants in mice having severe combined immunodeficiency(33) . Thus, the role of this protein on virus replication is controversial. This may be due, in part, to the use of various alleles of Nef and different cell culture systems. Nevertheless, studies in SIV-infected macaques have revealed that the preservation of a full-length Nef is necessary for the maintenance of high viral loads and for the progression of disease(34) . Although the basis for this requirement is still not known, it may be related to a role of Nef in T-cell activation(28, 29, 35, 36, 37, 38, 39, 40) .

Indeed, several studies indicate that the expression of Nef affects a signal transduction pathway. Nef has been shown to down-regulate the expression of CD4 on T-lymphocytes in vitro(41, 42, 43, 44) . This effect of Nef is mediated by an endocytotic mechanism (17, 39, 45) that involves the targeting of CD4 for lysosomal degradation(17, 39, 45) . Furthermore, we recently reported that the expression of a CD8-Nef fusion protein in T-cells leads to inhibition or activation of early T cell signaling events depending on its localization within the cell (40). When expressed at the inner surface of the plasma membrane, the activation markers CD69 and CD25 were induced, and the cells died by apoptosis. Cells that survived contained truncated Nefs.

To elucidate the pathway by which Nef functions, attempts have been made to identify cellular proteins that complex with Nef. Harris and Coates (46) reported that baculovirus-expressed glutathione S-transferase-Nef fusion proteins associate with cellular proteins of various sizes which depend on their subcellular location. We demonstrated that Nef expressed either as a CD8-Nef fusion protein or native Nef itself specifically interacted with a serine kinase in human T-lymphocytes(47) . This kinase was found to phosphorylate proteins of 62 and 72 kDa that coimmunoprecipitated with Nef.

In the present study, the regions of Nef that are important for these interactions with the kinase activity were investigated. Using a transient expression assay, we determined that various alleles of Nef are capable of associating with the kinase, and the kinase is present in non-lymphoid cells from several different species. We found that stability of the molecule affects the association of Nef with the cellular kinase activity. Moreover, two different regions of Nef are critical for the association with this activity; the first has been mapped to a domain that overlaps a centrally located, highly conserved portion of the molecule, and the second represents a membrane targeting signal.


MATERIALS AND METHODS

Cells and Antibodies

The Jurkat T-cell lines that constitutively express the CD8/HIV-1 Nef fusion protein (J.CN) and HUT 78 cells chronically infected with HIV-1 (E-line) were cultured as described previously(47) . COS-7 cells were grown in Dulbecco's minimal essential medium containing 10% fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin. The hybridomas, 51.1 and MH-SVM26, which produce monoclonal antibodies directed against the human CD8 molecule or HIV-1 gp41, respectively, were obtained from the American Type Culture Collection and cultured in HY media containing 20% fetal bovine serum. Hybridoma supernatants containing CD8-specific monoclonal antibody (-CD8) or gp41-specific monoclonal antibody (-gp41) were collected and either partially purified by ammonium sulfate precipitation or used directly for immunoprecipitation analyses. The rabbit anti-HIV-1Nef antibody (-Nef) was provided by Chiron Corporation (Emeryville, CA).

Plasmid Construction and Transfection

To express native Nef in COS-7 cells, the complete nef gene of HIV-1 was inserted into the pRc/CMV expression vector (Invitrogen) generating the plasmid pCMV/SF2Nef. In addition, the complete nef gene of HIV-1 and the pathogenic SIV strain were fused to the extracellular and transmembrane domains of the human CD8 molecule thereby generating a hybrid CD8-SF13Nef or CD8-SIVNef fusion that was subsequently cloned into the pRc/CMV plasmid as described previously(40) . These plasmids were designated pCMV/CD8-SF13Nef and pCMV/CD8-SIVNef, respectively.

Site-specific mutations, carboxyl-terminal truncations, and amino-terminal deletions in Nef were introduced into the pCMV/CD8-SF2Nef, pCMV/CD8-SIVNef, or the pCMV/SF2Nef expression vectors using the single-stranded oligonucleotide-directed mutagenesis strategy (Bio-Rad). The amino-terminal deletion mutants of Nef were fused in-frame to the extracellular and transmembrane domains of CD8. All of the mutations were confirmed by DNA sequencing and by the presence of introduced restriction endonuclease cleavage sites.

Chimeric CD8-Nef plasmids that reciprocally exchanged the centrally located conserved region of HIV-1 Nef for the homologous region of SIV Nef were constructed as follows. Unique KpnI and EcoRV sites, which did not alter the amino acid sequence of the Nef protein, were engineered into the SIV plasmid by site-directed mutagenesis. The KpnI-EcoRV fragments from the HIV-1 and SIV Nefs were subsequently exchanged. The domain substitution results in the replacement of amino acids 78-139 of HIV-1 Nef for amino acids 107-167 of SIV Nef and vice versa.

Plasmid DNA (15 µg) was transfected into 5 10 COS-7 cells by calcium-phosphate precipitation. Twenty-four h after transfection, the cultures were split in half and propagated for an additional 24 h before harvesting for protein labeling and kinase analyses.

Metabolic Labeling, Immunoprecipitation, in Vitro Kinase Assay, and Pulse-chase Analysis

The procedures for metabolic labeling, cellular extraction, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and the in vitro kinase assay were performed as described previously(47) . For pulse-chase analyses, transfected COS-7 cells were starved with methionine- and cysteine-free RPMI 1640 media for 30 min and pulse labeled for 15 min with labeling medium containing 500 µCi/ml S-Translabel (ICN) at 37 °C. After washing the monolayers twice with Dulbecco's phosphate-buffered saline, 10 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum was added to the cells, except for those representing the zero time point which was extracted immediately. After chase times of 30 min, 1, 2 and 4 h, the respective cells were washed twice with Dulbecco's phosphate-buffered saline and extracted. Immunoprecipitations were done as described above.


RESULTS

Kinase Association Is a Highly Conserved Property of Nef and Can Be Detected in Several Cell Types

Previously, using a CD8-Nef fusion protein or native Nef, we identified a serine kinase activity that interacts with HIV-1 Nef expressed in human T-cell lines (47). This kinase was responsible for the phosphorylation of two proteins of 62 and 72 kDa that specifically coimmunoprecipitate with native Nef and CD8-Nef fusion proteins. We also reported that HIV-1 Nef truncated at amino acid 95 (CD8-SF2Nef (1-94)) no longer associated with the kinase activity, as indicated by the absence of the 62 or 72 kDa phosphorylated proteins in Nef immunoprecipitates. In order to develop a rapid assay for screening different alleles and mutants of Nef for the associated kinase activity, transient expression of Nef in simian COS-7 cells was conducted (Fig. 1A). By metabolic labeling, substantial expression of CD8-SF2Nef, CD8-antisense Nef, CD8-SF2Nef (1-94), CD8-SIV Nef, and CD8-SF13 Nef could be detected in transfected COS-7 cells (Fig. 1A, lanes 2-6).


Figure 1: The Nef-associated kinase activity is found in simian fibroblast cells expressing different Nef alleles. A, COS-7 cells transfected with CD8-antisense SF2Nef (Anti, lanes 1, 2, 7, and 8), CD8-SF2Nef (SF2, lanes 3 and 9), CD8-SF2(1-94) (SF2(1-94), lanes 4 and 10), CD8-SF13 Nef (SF13, lanes 5 and 11), and CD8-SIV Nef (SIV, lanes 6 and 12) were extracted with (lanes 1-6) or without (lanes 7-12) metabolic labeling. Immunoprecipitations were done by using a control anti-gp41 monoclonal antibody (lanes 1 and 7) or an anti-CD8 monoclonal antibody (lanes 2-6 and 8-12). An in vitro kinase assay was performed on the unlabeled immunoprecipitates (lanes 7-12). B, kinase assays were performed on immunoprecipitates from either COS-7 (lanes 1 and 2) or Jurkat cells (J.CN,lanes 3 and 4) expressing CD8-SF2Nef. A control anti-gp41 monoclonal antibody (-gp41,lanes 1 and 4) or an anti-CD8 monoclonal antibody (-CD8,lanes 2 and 3) were used. Molecular size standards are marked on the right and the positions of the phosphorylated 62- (p62) and 72-kDa proteins are indicated on the left.



In vitro kinase assays performed on immunoprecipitates from unlabeled, transfected cell extracts demonstrated that besides CD8-SF2Nef, CD8-SF13 Nef, and CD8-SIV Nef also interacted with this kinase (Fig. 1A, lanes 9, 11, and 12). The 62-kDa protein was specifically phosphorylated in these immunoprecipitates but not in immunoprecipitates containing CD8-antisense Nef (anti) or a truncated Nef (SF2Nef(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94) ) (Fig. 1A, lanes 8 and 10). Several other phosphorylated bands are observed in lanes 10 and 11. These were not observed reproducibly and, thus, were considered to be nonspecific (for example compare Fig. 2A, lane 10 and Fig. 1A, lane 10). The phosphorylated 62-kDa protein from T-cell lines and COS-7 cells was similar by partial V8-protease mapping (data not shown). Since three different alleles of Nef were capable of interacting with the kinase, it is likely that this property of Nef is conserved. Moreover, because the kinase activity was found in cultures of human T-cell lines (Jurkat, HUT-78)(47), simian fibroblasts (COS-7), and murine fibroblasts (NIH3T3; data not shown) that express Nef, the kinase itself is conserved. However, the phosphorylation of the 72-kDa substrate was not observed in the latter two cell types (COS-7 and NIH3T3) (Fig. 1B). Thus, phosphorylation of the 72-kDa protein may be T-cell-specific.


Figure 2: The central conserved region of Nef is important for association with the cellular kinase activity. A, COS-7 cells transiently expressing CD8-SF2 Nef (SF2,lanes 1, 2, 8, and 9), CD8-SF2(1-94) (1-94,lanes 3 and 10), CD8-SF2(1-127) (1-127,lanes 4 and 11), CD8-SF2(45-210) (45-210,lanes 5 and 12), and CD8-SF2(70-210) (70-210,lanes 6 and 13) were extracted with (lanes 1-6) or without (lanes 8-13) metabolic labeling as in Fig. 1. Immunoprecipitations were performed either with an anti-gp41 monoclonal antibody (lanes 1 and 8) or a CD8-specific monoclonal antibody (lanes 2-6 and 9-13). An in vitro kinase assay was performed on the immunoprecipitates from unlabeled extracts (lanes 8-13). B, truncation of Nef at amino acid 127 results in an unstable protein. Pulse-chase analysis was performed using cells transiently expressing either CD8-SF2 Nef (lanes 5-8) or CD8-SF2(1-127) (lanes 1-4). Metabolically labeled cells were extracted at 0, 0.5, 1.0, and 2 h after the initial 15-min pulse. Immunoprecipitations were performed using a monoclonal antibody to CD8. The increased mobilities of CD8-SF2 Nef and CD8-SF2(1-127) during the chase period are probably due to the glycosylation of the CD8 portion of the molecule. The positions of CD8-SF2 Nef and CD8-SF2(1-127) are indicated on the right.



The Central Domain of Nef Is Critical for Association with the Kinase Activity

To determine the region of Nef important for the observed kinase activity, amino-terminal and carboxyl-terminal truncations of Nef in the CD8-SF2Nef plasmid were constructed, and the deletion mutants were analyzed for their ability to complex with the kinase activity in COS-7 cells (Fig. 2A). Deletion of amino acids 95-210 resulted in the loss of the associated kinase activity, whereas introduction of a stop codon immediately after amino acid 127 resulted in a greatly reduced, but detectable phosphorylation of the 62-kDa protein (Fig. 2A, lanes 10 and 11). This mutant is truncated at the same position as Nef from the HIV-1 strain(48) . Unlike the CD8-SF2Nef 1-94 protein that is detectable by metabolic labeling, the CD8-SF2Nef 1-127 protein is expressed weakly and appears to undergo proteolytic degradation. Since the stability of Nef may affect kinase association, pulse-chase analyses were performed to compare the metabolic half-life of CD8-SF2Nef 1-127 with wild type CD8-SF2 Nef (Fig. 2B). Previously, we found that the metabolic half-life of CD8-SF2Nef was about 8 h(47) . In contrast, the metabolic half-life of the CD8-SF2Nef 1-127 protein was less than 1 h. Other CD8-SF2Nef truncated proteins encoded by amino acids 1-145 and 1-167 were even less stable than the SF2 Nef 1-127, and therefore had none or barely detectable levels of associated kinase activity (summarized in Fig. 6). From these results, we conclude that carboxyl-terminal truncations of Nef beyond amino acid 94 resulted in the expression of unstable fusion proteins.


Figure 6: Summary of in vitro kinase assay results. A schematic representation of HIV-1 Nef is indicated at the top (white bar). The central domain homologous to HIV-2 and SIV Nefs (SIV Homology) is flanked by acidic regions depicted as partially hatched boxes. The amino-terminal basic domain is represented by a black box. Numbers indicate amino acid position. The conserved cysteines Cys, Cys, and Cys of HIV-1 Nef are indicated. Nef mutants used in this study are indicated below, and numbers indicate the amino acids encoded by each mutant. The results of the in vitro kinase assay are shown on the right. Wild-type (++), reduced (+), weak (±), and undetectable (-) levels of kinase activity in COS-7 cells (as determined by phosphorylation of the 62-kDa protein) are indicated on the right. The SC mutation, which utilizes an alternative open reading frame as described in the text, was introduced into SF2 Nef (SF2(SC)) and is depicted as a box filled with wavy lines. HIV-1 and SIV Nefs are shown as boxes filled with diagonal lines. The chimeric Nef proteins are indicated as SF2(SIV) and SIV(SF2).



Amino-terminal deletions of CD8-SF2Nef that resulted in truncated proteins encoded by amino acids 45-210, 70-210, and 89-210 of Nef (Fig. 6) were also analyzed for the ability to associate with the kinase activity. The deletion of amino acids 1-44 affected neither the stability of the protein nor the ability of the protein to associate with the kinase activity (Fig. 2A, lanes 5 and 12). The deletion of amino acids 1-69 was stable yet did not exhibit associated kinase activity (Fig. 2A, lanes 6 and 13). However, the deletion of amino acids 1-88 adversely affected protein stability, and consequently no kinase activity was detected (Fig. 6). From these amino-terminal and carboxyl-terminal deletions, we have provisionally mapped the domain of Nef important for association with kinase activity to amino acids 45-127.

Results from studies with CD8-SF2Nef 1-94 and CD8-SF2Nef 1-127 suggest that the highly conserved central region of Nef (HIV-1 Nef amino acids 74-152, Fig. 3) contains an important domain for associated kinase activity. To further examine this possibility, we constructed a CD8-SF2Nef mutant that resembles the HIV-1 allele(49) . In nef of HIV-1, a single nucleotide deletion at position 9117 results in the utilization of an alternative open reading frame that continues for 34 amino acids until another single nucleotide insertion at position 9219 restores the original nef open reading frame. These mutations were placed into the CD8-SF2nef gene so that the alternate open reading frame was utilized between amino acids 105 and 138. The mutant protein was metabolically stable (data not shown), yet no associated kinase activity was detected (summarized in Fig. 6). These findings further indicate that the central region between amino acids 94 and 127 of Nef is important for the association with the kinase activity.


Figure 3: Alignment of HIV-1, HIV-2, and SIV Nefs in the central, conserved region. Sequence comparisons were done using the protein sequence alignment program, BLAST (50). All the sequences (51) were aligned and compared to the Nefs of HIV-1 and HIV-1. HXB2 Nef is prematurely truncated at amino acid 123 as indicated by a stop codon (asterisk). Dashes indicate positions where the amino acids are identical to the HXB2 and BRU sequences. Non-identical amino acids are indicated in single-letter amino acid code. The coordinate of the last amino acid residue in each of the aligned sequences is indicated on the right. A gap (indicated by a period) was introduced in the HIV-1 sequence to facilitate the alignment. A consensus sequence (Consensus) of amino acid residues that are identical among all of the aligned sequences (with the exception of HIV-1; see text for a description of the SC allele) are shown in capital letters, and their positions (#) are indicated; other positions of non-identity are shown (). Dots are placed above the Nef residues that were mutagenized in the SF2 allele for this study. The position of the stop codon for the SF2(1-94) truncated Nef is indicated (+). The truncation of SF2 Nef at amino acid 127 (CD8-SF2(1-127)) directly corresponds to the site of truncation in HXB2 Nef (amino acid 123). Potential protein kinase C (PKC) phosphorylation sites, and the conserved acidic region (Acidic) are indicated on the bottom by bars.



Specific Amino Acid Substitutions in the Conserved Region of Nef Can Affect Association with the Kinase Activity

Alignment of HIV-1, HIV-2, and SIV Nef sequences indicated that several amino acids in the region between 94-127 were identical among all primate lentiviral Nef proteins (Fig. 3, note consensus sequence between 94 and 127). Of the conserved amino acids, 2 adjacent arginine residues at positions 109 and 110 of SF2 Nef represented a charged region. Because hydrophobic moment analyses (52) performed on the entire SF2 Nef molecule indicated that this region had a high degree of hydrophilic character, both of these amino acids were mutated to leucine residues. The mutant protein (CD8-SF2Nef RR-LL) was stable but lost the ability to associate with the kinase activity (Fig. 4A, lanes 3 and 12). Several other phosphorylated bands are observed in various lanes (lanes 11-18). These were not observed reproducibly and were considered to be nonspecific (for example compare Fig. 4A, lane 12 and Fig. 4B, lane 7). Mutation of the individual arginines resulted in the loss of associated kinase activity only when arginine 110, but not when arginine 109, was changed to leucine (Fig. 4A, lanes 13 and 14).


Figure 4: Specific, highly conserved amino acids in the central homology domain of Nef are important for the association with the cellular kinase activity. A, COS-7 cells transiently expressing CD8-SF2Nef (SF2,lanes 1, 2, 10, and 11), CD8-SF2(RR-LL) (R-R-LL,lanes 3 and 12), CD8-SF2(R-L) (R-L,lanes 4 and 13), CD8-SF2(R-L) (R-L,lanes 5 and 14), CD8-SF2(T-A) (T-A,lanes 6 and 15), CD8-SF2(S-A) (S-A,lanes 7 and 16), CD8-SIV Nef (SIV,lanes 8 and 17), and CD8-SIV(RR-LL) (SIV),lanes 9 and 18) were extracted with (lanes 1-9) or without (lanes 9-18) metabolic labeling. Immunoprecipitations were performed with monoclonal antibodies against gp41 (lanes 1 and 15) or CD8 (lanes 2-9, and 11-18). An in vitro kinase assay (lanes 10-18) was done as described in Fig. 1. The positions of p62 and CD8-Nef are marked on the right. B, a point mutation in CD8-SF2Nef eliminates the ability of Nef to associate with the cellular kinase activity in T-cell lines. Jurkat cells that constitutively express CD8-SF2Nef (lanes 1, 2, 4, and 5) and CD8-SF2Nef(R-R-LL) (lanes 3, 6, and 7) were extracted with (lanes 1-3) or without (lanes 4-7) metabolic labeling. Immunoprecipitations were performed with monoclonal antibodies against gp41 (lanes 1, 4, and 6) or CD8 (lanes 2, 3, 5, and 7). An in vitro kinase assay (lanes 11-20) was performed as described in Fig. 1. A 34 kDa band representing a proteolytic degradation product of CD8-SF2Nef(R-R-LL) is observed in lane 3. The positions of CD8-Nef, p62, and p72 are marked.



To determine whether the CD8-Nef RR-LL fusion protein associates with the cellular kinase activity in T-cell lines, we have stabily expressed this mutant in Jurkat T-cells and have found that the protein is metabolically stable, yet does not associate with the cellular kinase activity (Fig. 4B). These results confirm those obtained in COS cells with this mutant (CD8-Nef RR-LL, Fig. 4A, lane 12).

Since the arginines at positions 109 and 110 of SF2Nef are also present in SIVNef, the arginine to leucine substitutions were introduced into CD8-SIVNef at positions 137 and 138. This SIVNef mutant, CD8-SIVNef RR-LL, like its HIV-1 counterpart CD8-SF2Nef RR-LL, was metabolically stable but defective for the associated kinase activity (Fig. 4A, compare lanes 11 and 12 with lanes 17 and 18). These results indicate that the homologous arginine residues in SIV are also critical for the association with the kinase activity.

Because Nef has been reported to be phosphorylated by protein kinase C (8, 9) and phosphorylation of Nef may be important for function(12) , potential phosphorylation sites were also selected for mutation. Serine 14, threonine 84, and serine 107 of Nef were mutated to alanine residues. In each case, these amino acid substitutions did not affect the associated kinase activity (Fig. 4A, lanes 15 and 16 and Fig. 6). In addition, the conserved cysteine at position 146 also was converted to an alanine. This residue has been implicated as being important for intrachain disulfide bond formation and function(11) . A change in this residue also did not affect the associated kinase activity (Fig. 6). These findings suggest that neither protein kinase C phosphorylation of Nef nor the formation of intrachain disulfide bonds are critical for this association.

Finally, we found that the reciprocal exchange of the centrally located conserved domain between CD8-SF2Nef and CD8-SIVNef gave rise to stable chimeric proteins that did not associate with the kinase activity despite retaining the conserved arginine at residue 110 (SF2) or 138 (SIV) (SF2(SIV) and SIV(SF2), Fig. 5). These results suggest that protein conformation may be important for association of Nef with the kinase activity.


Figure 5: Analysis of HIV/SIV Nef chimeras in the kinase assay. COS-7 cells transiently expressing CD8-SF2Nef (SF2,lanes 1, 5, 9, and 12), CD8-SIV (SIV,lanes 2 and 6), CD8-SIV(SF2KpnI-EcoRV) (SIV(SF2), lanes 3, 4, 7, 8, 11, and 14), and CD8-SF2(SIVKpnI-EcoRV) (SF2(SIV), lanes 10 and 13) were extracted with (lanes 1-4 and 9-11) and without (lanes 5-8 and 12-14) metabolic labeling. Immunoprecipitations were done using a monoclonal antibody to CD8. The positions of p62 and CD8-Nef are indicated on the right.



Myristylation of Native Nef Is Critical for the Associated Kinase Activity

Several reports have indicated that myristylation is important for Nef function(13, 15, 16, 17, 44) . To determine whether myristylation of native Nef was important for the Nef-associated kinase activity, a myristylation defective mutant, that converts glycine at position 2 to alanine, was constructed. The ability of the mutant Nef to associate with the kinase activity in transient assays was tested. Whereas native SF2 Nef was observed to associate with the kinase activity ( Fig. 6and Fig. 7, lanes 6 and 8), the myristylation negative mutant (G-A) and the control antisense Nef (fen) did not associate with the kinase activity (Fig. 7, lanes 5 and 7). These results indicate that the myristylation of Nef is also a critical determinant for the association with the kinase activity. Because both the CD8-Nef fusion protein and the native Nef associated with the kinase activity, and the myristylation-defective Nef did not, we conclude that the targeting of Nef to intracellular membranes is important for association with a cellular kinase activity.


Figure 7: The myristylation of Nef is critical for the association with the cellular serine kinase activity. COS-7 cells transiently expressing HIV-1 antisense Nef (fen, lanes 1 and 5), HIV-1 native Nef (Nef, lanes 2 and 6), a myristylation defective HIV-1 Nef (myr, lanes 3 and 7), together with HUT 78 cells chronically infected with HIV-1 (E-line, lanes 4 and 8) were extracted with (lanes 1-4) or without (lanes 5-8) metabolic labeling. Immunoprecipitations were done with a polyclonal rabbit anti-Nef serum. An in vitro kinase assay was performed as described in Fig. 1 (lanes 5-8). The positions of the 72-kDa phosphorylated protein (p72), p62, and Nef are indicated on the right.




DISCUSSION

We previously reported the association of a cellular serine kinase with HIV Nef in T-cell lines(47) . In the present study, we demonstrate that the association with this kinase is a highly conserved property of Nef and is found in lymphoid and non-lymphoid cells from different species. We have also found that Nef from HIV-1-infected peripheral blood mononuclear cells is capable of associating with the cellular serine kinase activity (data not shown). Since phosphorylation of the 72-kDa protein in COS-7 and NIH3T3 cells was not detectable, the association of this protein with Nef and/or its phosphorylation by the serine kinase may be specific for T-cells.

Using amino-terminal and carboxyl-terminal deletion mutants of Nef, we localized the region of Nef that is important for the association with the kinase activity to amino acids 45-127. This region overlaps the centrally located, highly conserved domain (amino acids 74-152, Fig. 3) of HIV-1 and SIV Nef proteins. While the basic region and the putative amino-terminal phosphorylation site (Thr) of Nef are dispensable for association with the kinase activity (45-210, Fig. 6), further deletion of the region between amino acids 45 and 70, which includes the amino-terminal acidic domain (amino acids 63-69), eliminates the Nef-associated kinase activity (70-210, Fig. 6). These findings indicate that the retention of only the conserved domain of Nef is not sufficient for its ability to associate with the kinase activity and suggest that multiple sites along this protein interact with the kinase or its substrates. Alternatively, the amino-terminal region (amino acids 45-70) might be required to maintain a proper conformation of Nef. The latter possibility is consistent with the prediction from NMR spectrographic studies of proteolytic peptide fragments that amino acids 66-206 of HIV-1 Nef may represent a separable functional domain (53). Furthermore, we observe that carboxyl-terminal truncations of Nef are relatively unstable. This effect may be due to the presence of internal PEST-like sequences (54) in Nef which, as a result of truncations, are now present near the carboxyl-terminus, and signal the targeting of newly synthesized proteins for premature degradation.

Using point mutations in HIV-1 Nef, we have determined that several conserved amino acids that are located in different regions of the central Nef homology domain are important for the association with the kinase activity. While mutation of potential protein kinase C phosphorylation sites did not affect the ability of Nef to complex with the cellular kinase activity, mutation of the double arginines at positions 109 and 110 of HIV-1 Nef and positions 137 and 138 of SIV Nef did. One of these residues, arginine 110 of HIV-1 Nef, was critical since conversion of this residue to leucine abrogates the associated kinase activity. Because reciprocal substitution of the conserved domains of HIV-1 Nef and SIV Nef did not result in kinase association, it appears that the conserved central domain of Nef interacts with its flanking regions in conferring a conformation that is important for association with the kinase activity.

Although we have mapped the region of Nef critical for the association with the kinase activity, we cannot differentiate between the possibilities that this region functions as a binding domain for the serine kinase and/or, one or both of the phosphorylated substrates (62- or 72-kDa proteins). It remains possible that one or both of the phosphorylated proteins may be the kinase(s) in lymphoid cells. The lack of phosphorylation of the 72-kDa protein in non-lymphoid cells indicates that this protein is not the kinase in these cells. Identification and characterization of the 62- and 72-kDa proteins should resolve this question.

Our finding with the myristylation mutant indicates that Nef interacts directly with a membrane-associated kinase activity and supports the idea that membrane targeting is likely to be important for Nef function. In this cellular compartment, Nef may affect an intracellular signaling pathway(s).

In this regard, we observed differential effects of Nef on early signaling events in T-cells depending on its subcellular expression (40). When Nef was expressed on the inner surface of the plasma membrane, an activated, apoptotic phenotype was observed. Only cells expressing truncated forms of Nef survived this event. Because membrane targeted Nef associates with the kinase activity and truncated forms of Nef do not, these results suggest that Nef may influence intracellular activation through its interaction with the cellular serine kinase. Whether the Nef-associated kinase activity is linked to other functions of Nef (e.g. CD4 down-regulation and enhancement of viral infectivity) remains to be determined.

Nef has been shown to be important for the maintenance of high viral loads and the progression of SIV-infected macaques to disease(34) . Our observations that Nef of the pathogenic strain SIV also associates with the kinase activity and that the region of Nef important for this association maps to the central homology domain raise the possibility that this association with the kinase may be important for viral pathogenesis in vivo.

In summary, our results define two regions of Nef that regulate one of its biochemical properties: the central homology region and the myristylation signal sequence. Moreover, this study demonstrates that the Nef-associated kinase activity represents a conserved function of primate lentiviral Nef proteins. It will be important to determine whether this association is necessary for viral replication and/or disease progression in vivo.


FOOTNOTES

*
This study was supported by National Institutes of Health Grant RO1 AI25284 (to C. C. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the University of California University-wide AIDS research program. Present address: Dept. of Medical Pathology, University of California, Davis, Davis, CA 95616.

Supported by the Howard Hughes Institute for Medical Research. Present address: Institut fur Klinische und Molekulare Virologie, Schlossgarten 4, D-91054 Erlangen, Germany.

**
Supported by the Howard Hughes Institute for Medical Research.

§§
Present address: Aaron Diamond AIDS Research Center, 455 1st Ave., 7th Floor, New York, NY 10016.

The abbreviations used are: HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus.


ACKNOWLEDGEMENTS

We thank Greg Harrowe for his expert technical assistance. The full-length nef gene of SIV was constructed and provided by Paul Luciw (University of California, Davis, CA).


REFERENCES
  1. Allan, J. S., Coligan, J. E., Lee, T. H., McLane, M. F., Kanki, P. J., Groopman, J. E., and Essex, M.(1985) Science230, 810-813 [Medline] [Order article via Infotrieve]
  2. Colombini, S., Arya, S. K., Reitz, M. S., Jagodzinski, L., Beaver, B., and Wong-Staal, F.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 4813-4817 [Abstract]
  3. Shibata, R., Miura, T., Hayami, M., Ogawa, K., Sakai, H., Kiyomasu, T., Ishimoto, A., and Adachi, A.(1990) J. Virol.64, 742-747 [Medline] [Order article via Infotrieve]
  4. Feinberg, M. B., Jarrett, R. F., Aldovini, A., Gallo, R. C., and Wong-Staal, F.(1986) Cell46, 807-817 [Medline] [Order article via Infotrieve]
  5. Robert-Guroff, M., Popovic, M., Gartner, S., Markham, P., Gallo, R. C., and Reitz, M. S.(1990) J. Virol.64, 3391-3398 [Medline] [Order article via Infotrieve]
  6. Klotman, M. E., Kim, S., Buchbinder, A., DeRossi, A., Baltimore, D., and Wong-Staal, F.(1991) Proc . Natl. Acad. Sci. U. S. A.88, 5011-5015 [Abstract]
  7. Munis, J. R., Kornbluth, R. S., Guatelli, J. C., and Richman, D. D. (1992) J. Gen. Virol.73, 1899-1901 [Abstract]
  8. Guy, B., Kieny, M. P., Riviere, Y., Le Peuch, C., Dott, K., Girard, M., and Montagnier, L.(1987) Nature330, 266-269 [CrossRef][Medline] [Order article via Infotrieve]
  9. Guy, B., Riviere, Y., Dott, K., Regnault, A., and Kieny, M. P.(1990) Virology176, 413-425 [CrossRef][Medline] [Order article via Infotrieve]
  10. Kienzle, N., Freund, J., Kalbitzer, H. R., and Mueller-Lantzsch, N. (1993) Eur. J. Biochem.214, 451-457 [Abstract]
  11. Zazopoulos, E., and Haseltine, W. A.(1993) J. Virol.67, 1676-1680 [Abstract]
  12. Bandres, J. C., Luria, S., and Ratner, L.(1994) Virology201, 157-161 [CrossRef][Medline] [Order article via Infotrieve]
  13. Yu, G., and Felsted, R. L.(1992) Virology187, 46-55 [Medline] [Order article via Infotrieve]
  14. Zazopoulos, E., and Haseltine, W. A.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 6634-6638 [Abstract]
  15. Niederman, T. M. J., Randall-Hastings, and Ratner, L.(1993) Virology197, 420-425 [CrossRef][Medline] [Order article via Infotrieve]
  16. Chowers, M. Y., Spina, C. A., Kwoh, T. J., Fitch, N. J., Richman, D. D., and Guatelli, J. C.(1994) J. Virol.68, 2906-2914 [Abstract]
  17. Aiken, C., Konner, J., Landau, N. R., Lenburg, M. E., and Trono, D. (1994) Cell76, 853-864 [Medline] [Order article via Infotrieve]
  18. Franchini, G., Robert-Guroff, M., Ghrayeb, J., Chang, N. T., and Wong-Staal, F.(1986) Virology155, 593-599 [Medline] [Order article via Infotrieve]
  19. Kohleisen, B., Neumann, M., Herrmann, R., Brack-Werner, R., Krohn, K. J., Ovod, V., Ranki, A., and Erfle, V.(1992) AIDS6, 1427-1436 [Medline] [Order article via Infotrieve]
  20. Murti, K. G., Brown, P. S., Ratner, L., and Garcia, J. V.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 11895-11899 [Abstract]
  21. Terwilliger, E. F., Sodroski, J. G., Rosen, C. A., and Hazeltine, W. A. (1986) J. Virol.60, 754-760 [Medline] [Order article via Infotrieve]
  22. Luciw, P. A., Cheng-Mayer, C., and Levy, J. A.(1987) Proc. Natl. Acad. Sci. U. S. A.84, 1434-1438 [Abstract]
  23. Cheng-Mayer, C., Iannello, P., Shaw, K., Luciw, P. A., and Levy, J. A. (1989) Science246, 1629-1632 [Medline] [Order article via Infotrieve]
  24. Tsunetsugu-Yokota, Y., Matsuda, S., Maekawa, M., Saito, T., Takemori, T., and Takebe, Y.(1992) Virology191, 960-963 [Medline] [Order article via Infotrieve]
  25. Ahmad, N., and Venkatesan S.(1988) Science241, 1481-1485 [Medline] [Order article via Infotrieve]
  26. Niederman, T. M. J., Thielan, B. J., and Ratner, L.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 1186-1132
  27. Maitra, R. K., Ahmad, N., Holland, S. M., and Venkatesan, S.(1991) Virology182, 522-533 [Medline] [Order article via Infotrieve]
  28. Niederman, T. M. J., Garcia, J. V., Randall-Hastings, W., Luria, S., and Ratner, L.(1992) J. Virol.66, 6213-6219 [Abstract]
  29. Niederman, T. M. J., Randall-Hastings, W., Luria, S., Bandres, J. C., and Ratner, L.(1993) Virology194, 338-344 [CrossRef][Medline] [Order article via Infotrieve]
  30. de Ronde, A., Klaver, B., Keulen, W., Smit, L, and Goudsmit, J.(1992) Virology188, 391-395 [Medline] [Order article via Infotrieve]
  31. Spina, C. A., Kwoh, T. J., Chowers, M. Y., Guatelli, J. C., and Richman, D. D.(1994) J. Exp. Med.179, 115-123 [Abstract]
  32. Miller, M. D., Warmerdam, M. T., Gaston, I., Greene, W. C., and Feinberg, M. B.(1994) J. Exp. Med.179, 101-113 [Abstract]
  33. Jamieson, B. D., Aldrovandi, G. M., Planelles, V., Jowett, J. B., Gao, L., Bloch, L. M., Chen, I. S., and Zack, J. A.(1994) J. Virol.68, 3478-3485 [Abstract]
  34. Kestler, H. W., III, Ringler, D. J., Mori, K., Panicali, D. L., Sehgal, P. K., Daniel, M. D., and Desrosiers, R. C.(1991) Cell65, 651-662 [Medline] [Order article via Infotrieve]
  35. Luria, S., Chambers, I., and Berg, P.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 5326-5330 [Abstract]
  36. Skowronski, J., Parks, D. and Mariani, R.(1993) EMBO J.12, 703-713 [Abstract]
  37. De, S. K., and Marsh, J. W.(1994) J. Biol. Chem.269, 6656-6660 [Abstract/Free Full Text]
  38. Rhee, S. S., and Marsh, J. W.(1994) J. Immunol.152, 5128-5134 [Abstract/Free Full Text]
  39. Rhee, S. S., and Marsh, J. W.(1994) J. Virol.68, 5156-5163 [Abstract]
  40. Baur, A. S., Sawai, E. T., Dazin, P., Fantl, W. J., Cheng-Mayer, C., and Peterlin, B. M.(1994) Immunity1, 373-384 [Medline] [Order article via Infotrieve]
  41. Garcia, J. V., and Miller, A. D.(1991) Nature350, 508-511 [CrossRef][Medline] [Order article via Infotrieve]
  42. Garcia, J. V., Alfano, J., and Miller, A. D.(1993) J. Virol.67, 1511-1516 [Abstract]
  43. Inoue, M., Koga, Y., Djordjijevic, D., Fukuma, T., Reddy, E. P., Yokoyama, M. M., and Sagawa, K.(1993) Int. Immunol.5, 1067-1073 [Abstract]
  44. Mariani, R., and Skowronski, J.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 5549-5553 [Abstract]
  45. Sanfridson, A., Cullen, B. R., and Doyle, C.(1994) J. Biol. Chem.269, 3917-3920 [Abstract/Free Full Text]
  46. Harris, M., and Coates, K.(1993) J. Gen. Virol.74, 1581-1589 [Abstract]
  47. Sawai, E. T., Baur, A., Struble, H., Peterlin, B. M., Levy, J. A., and Cheng-Mayer, C.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 1539-1543 [Abstract]
  48. Ratner, L., Starchich, B., Josephs, S. F., Hahn, B. H., Reddy, E. P., Livak, K. J., Petteway, S. R., Jr., Pearson, M. L., Haseltine, W. A., Arya, S. K., and Wong-Staal, F.(1985) Nucleic Acids Res.13, 8219-8229 [Abstract]
  49. Gurgo, C., Guo, H.-G., Franchini, G., Aldovini, A., Collati, E., Farrell, K., Wong-Staal, F., Gallo, R. C., and Reitz, M. S., Jr.(1988) Virology164, 531-536 [Medline] [Order article via Infotrieve]
  50. Altschul, S., F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol.215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  51. Meyers, G., Berzofsky, J. A., Korber, B., Smith, R. F., and Pavlakis, G. N.(1993) Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Los Alamos National Laboratory, Los Alamos, New Mexico
  52. Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R.(1984) J. Mol. Biol.179, 125-142 [Medline] [Order article via Infotrieve]
  53. Freund, J., Kellner, R., Houthaeve, T., and Kalbitzer, H. R.(1994) Eur. J. Biochem.221, 811-819 [Abstract]
  54. Rogers, S., Wells, R., and Rechesteiner, M.(1986) Science234, 364-368 [Medline] [Order article via Infotrieve]

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