INTRODUCTION

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The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 25- to 27-kDa myristoylated protein unique to primate lentiviruses that is produced early in the infectious cycle and is estimated to comprise up to 80% of early transcripts (51). Its potential role in HIV-1 pathogenesis was established by the seminal study of Kestler et al., in which Nef was demonstrated to be essential for the maintenance of high viral loads and progression to simian AIDS in adult rhesus macaques (26). The existence of individuals infected with forms of HIV-1 from which nef was deleted who remained asymptomatic for over 15 years further supports the idea of Nef playing a role in disease progression (15, 27, 32). The identification and elucidation of the molecular interactions between Nef and host cell factors will therefore be crucial to understanding how Nef manifests its in vivo phenotype and in the rational design of intervention strategies.

Multiple in vitro functions of Nef have been identified and include the downmodulation of the CD4 (18, 22) and major histocompatibility complex class I (58) molecules. A mechanistic basis for these Nef functions has recently been provided in studies showing a connection between Nef's ability to down-regulate specific surface molecules and its ability to bind to components of the cellular protein sorting machinery (7, 19, 34, 37, 49, 50). Nef has also been shown to enhance viral infectivity as measured by single-cycle (11, 44) and peripheral blood mononuclear cell (PBMC) infectivity assays (16, 44, 60). The molecular basis for this phenotype is as yet unclear, but some clues have been obtained from genetic studies showing that the interaction of Nef with cellular kinases contributes to the enhanced replication of Nef⁺ viruses (52, 56, 64). The findings that 10 to 100 molecules of Nef are packaged into the virion (48, 63) and that Nef must be present in the producer cell in order to enhance viral infectivity (1, 45) led to the suggestion that Nef-mediated recruitment of specific cellular factors, such as kinases, results in the modification of the virion so that processes involved in uncoating and/or reverse transcription proceed more efficiently (1, 10, 57).

An interesting property of Nef is its ability to alter T-cell signal transduction pathways (4, 23, 25). This activity of Nef is likely to be mediated through interactions with cellular kinases and signaling proteins. In vitro, Nef has been reported to bind the Src family tyrosine kinases Hck (8, 21, 33, 46), Lyn (52), Lck (3, 12, 20), and Src (31). Interactions with protein kinase C theta and delta (3, 59), the zeta chain of the T-cell receptor (5, 24, 65), mitogen-activated protein kinase (20), and lastly a serine/threonine kinase (36, 47, 54) have also been reported. Of these interactions, only that involving the serine/threonine kinase has been shown to be present in virally infected cells. In vitro kinase assays (IVKAs) performed on Nef immunoprecipitates (IPs) from infected and transfected transformed T-cell lines led to the detection of two serine phosphorylated proteins, p62 and p72 (54). It was subsequently shown that the membrane-targeting domain of Nef, critical proline residues in the SH3 domain, and an arginine residue flanking the SH3 domain were required for Nef binding and autophosphorylation of p62 (39, 53, 64). Elements in the C-terminal end of Nef also appear to be important for Nef association with p62 (38).

Several recent studies have provided evidence that the p62 Nef-associated kinase (NAK) is or is closely related to a member of the (p21-activated kinase PAK) family (36, 47, 55). The PAK family of kinases serve as effectors for the small GTP-binding proteins Rac1 and Cdc42 to activate transcriptional events and induce cytoskeletal rearrangements (35, 41). Like that of PAK, NAK activity can be potentiated by constitutively activated forms of the guanine nucleotide binding proteins Cdc42 and Rac1 (36, 47) and blocked by dominant-negative forms of PAK (36). In addition, a membrane-targeted SH3 domain from the adaptor protein Nck was shown to significantly enhance the ability of NL4-3 R71 Nef to activate PAK (39). Nck, which possesses a classical SH3 binding domain as well as a domain capable of binding to a unique proline-rich interaction motif of PAK, was proposed to serve as a connector between Nef and PAK (39).

Although the Nef-NAK interaction can be readily detected in transfected or virally infected immortalized T-cell lines, it has not been examined in the context of more physiologically relevant cell types such as primary peripheral blood lymphocytes and macrophages. In addition to being targets of HIV-1 infection, macrophages may represent an important viral reservoir in infected patients (29). Yet much less is known about the effects of Nef function or its interaction with cellular factors in these cells. Since macrophages play essential roles in the innate immune responses to viral infection and as antigen-presenting cells, it can be envisioned that any effect of Nef on downmodulation of the major histocompatibility complex class I molecules or on signaling pathways may alter their effector functions. In this regard, the observation that Nef can bind and activate Hck catalytic activity in coexpression systems (8, 46) is of interest, as this Src kinase is primarily expressed in macrophages.

In this study, we assess the function of Nef in primary macrophages and look for a physiological interaction of Nef with the host cell kinases, Hck and NAK. Using an HIV-1-infected human macrophage culture system, we were unable to demonstrate an interaction of Nef with Hck. However, we could readily detect Nef binding and activation of p62 NAK in infected macrophages as well as PBMCs. More importantly, we show that the second phosphoprotein, p72, detected in IVKAs performed on Nef IPs from infected T lymphocytes and macrophages represents the recently identified PAK-interacting guanine nucleotide exchange protein (PIX) and its associated p95 protein (42). This finding provides additional evidence that NAK is PAK. The activation of PAK and the phosphorylation of its downstream effectors by Nef in both primary target cells of HIV-1 suggest that this signal transduction pathway is important for Nef-mediated functions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of nef mutant viruses. The SF162 nef and R25 nef viruses were generated by creating a frameshift mutation at the unique XhoI site in nef of SF162 (9) and R25 (30) by fill-in and blunting with Klenow enzyme, followed by religation. R25 is an SF2 env recombinant virus that contains the V1 to V3 regions of SF162 gp120 and is capable of replicating in macrophages (30). Viruses were propagated by cotransfection of linearized plasmids carrying the 3' and 5' halves of the genome into 293T cells and coculture with PBMCs as previously described (9). Two clones of each virus were constructed, sequenced, and characterized to minimize the possibility that the observed effects were due to secondary site mutations.

PBMC isolation and replication assays. PBMCs were isolated from the buffy coats of healthy human donors by Ficoll gradient centrifugation. For the resting cell replication assay, cells were resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Biowhittaker), 1% glutamine, penicillin, and streptomycin (complete medium). Cells (5 × 10⁶) were infected with 10 ng of virus p24 for 4 h. The cells were washed with Hanks' buffered saline solution (HBSS) and resuspended in 7 ml of complete medium in T-25 Falcon flasks. At day 4 postinfection (p.i.) the infected cells were stimulated with 3 µg of phytohemagglutinin (PHA; Sigma) and 5 U of recombinant interleukin 2 (IL-2) per ml for 3 days. The infected and stimulated cells were washed with Hanks' buffered saline solution and resuspended in complete medium containing 5 U of IL-2 per ml. Every 3 to 4 days, 4 ml of medium was removed and saved for p24 quantitation by antigen capture method (Abbott) and 4 ml of fresh medium was added to the cultures. For the stimulated cell assay, cells were resuspended in complete medium containing 5 U of IL-2 and 3 µg of PHA per ml immediately after isolation and stimulated for 3 days. The PBMCs were then washed and resuspended in complete medium with 5 U of IL-2 per ml. Stimulated PBMCs were infected and supernatant was harvested as described above.

Macrophage isolation and virus infection. Enrichment of PBMCs for the monocyte population was obtained by centrifuging the cells through a 46% Percoll gradient (Pharmacia) (13). The monocytes were resuspended in RPMI 1640 complete medium supplemented with 20% heat-inactivated fetal bovine serum and 5% heat-inactivated human AB serum (Biowhittaker). In some experiments, as specified in the text, monocytes were cultured in macrophage serum-free medium (Gibco BRL). No exogenous cytokines were added to serum plus cultured monocytes or serum-free cultured monocytes. Monocytes were allowed to adhere for 3 days to tissue culture flasks that had been coated with polylysine (Sigma) before washing with HBSS to remove any nonadherent cells. In most experiments, monocyte-derived macrophages (MDM) were infected 10 to 14 days postisolation with equal amounts of p24 of the SF162 and R25 wild-type or nef mutant viruses. Infected macrophages were harvested 7 to 10 days p.i. by incubation of monolayers in phosphate-buffered saline (PBS; Biowhittaker) containing 10 mM EDTA for 3 to 5 min, and then cells were gently scraped and collected in PBS. Cells were washed in PBS, counted, and resuspended in the appropriate lysis buffer. CEMx174 cells (2 × 10⁶; obtained from James Hoxie, University of Pennsylvania) chronically infected with SF2 wild-type, SF2 nef P73A, and SF2 nef were subjected to IVKAs as described below.

IVKA, reimmunoprecipitation, and Western blotting. Nef was immunoprecipitated from infected or control MDM with rabbit anti-HIV Nef antisera (1:200) (64). PAK was immunoprecipitated with rabbit anti-PAK (N-20; 1:200); Hck was immunoprecipitated with rabbit anti-Hck (N30; 1:200) and Vav was immunoprecipitated with rabbit anti-Vav (H-211; 1:200) antibodies (Santa Cruz Biotechnology). Affinity-purified anti-PIX SH3 antibodies were the generous gift of Edward Manser and Louis Lim (Glaxo-ICMB Group, Crescent, Singapore). Nef and PAK IPs were subjected to IVKAs as previously described (54). Cells subjected to Hck immunoprecipitation were lysed in a solution containing 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 10 mM MgCl₂, and 1% Triton X-100 (8). Hck IPs were then subjected to IVKAs as described previously (8). For reimmunoprecipitation analyses, proteins were eluted from protein G-Sepharose by incubation in 0.5% sodium dodecyl sulfate (SDS) and heating at 70°C for 10 min. Ten volumes of cold KEB (54) were added, and the supernatant was collected. Before the second antibody was added, the eluted sample was precleared of immunoglobulins by incubation with protein G-Sepharose. Labeled immunoprecipitation analyses were run on SDS-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) gels, dried onto Whatman 3MM filter paper, and subjected to phosphorimager analysis. For detection of Nef or Hck protein by Western blot, immunoprecipitation analyses were run on SDS-10% PAGE and blotted onto Hybond C-extra membranes (Amersham). Membranes were blocked with 5% milk-0.2% Tween 20 (Sigma) in PBS for 1 h at room temperature. Blocked membranes were incubated with anti-Nef (1:500) or anti-Hck (1:200) antibody for 1 to 2 h at room temperature. Membranes were washed with 0.2% Tween 20 in PBS. Bands were visualized with secondary antibodies coupled to horseradish peroxidase in conjunction with the ECL Western blotting kit (Amersham). For reprobing, blots were stripped by incubation in a solution containing 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 7.0, at 50°C for 30 min. They were then washed thoroughly in PBS-0.2% Tween 20 before blocking.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

HIV-1 Nef fails to bind and activate Hck in infected human macrophages. To assess the function of Nef in human macrophages, we generated nef versions of the macrophage-tropic SF162 (9) and R25 (30) viruses. R25 is a recombinant virus that contains regions of the SF162 envelope on the genomic background of the T-cell line-tropic isolate SF2. This construct permitted the study of the well-characterized T-cell line-tropic SF2 nef allele in the context of a macrophage intracellular milieu. Since allelic differences in the dependence on Nef for replication in primary cells have been reported (62), the replication kinetics of the wild-type and nef viruses generated were first examined in PBMCs that were infected immediately after isolation and then stimulated at 4 days p.i. (resting) or in cells that were infected after mitogenic stimulation (stimulated). The two SF162 nef clones replicated with kinetics that were, compared to those of the wild type, slightly delayed in stimulated PBMCs but significantly delayed in resting cells (Fig. 1A). In both cell culture systems, the SF162 nef viruses attained lower viral titers. The kinetics of replication of the R25 wild-type and nef mutant viruses in stimulated PBMCs were comparable to those of the corresponding SF162 viruses. In the resting cell assay, however, wild-type R25 replication was more attenuated than that of wild-type SF162 (Fig. 1A). Only at 7 to 8 days poststimulation was an appreciable amount of p24 detected with R25 wild-type virus. As expected, the delay in replication of the R25 nef viruses was even more pronounced. In MDM, both the SF162 nef and R25 nef viruses showed a highly attenuated replication phenotype (Fig. 1B). These findings are in agreement with previous reports (44, 60) and demonstrate, for these viruses, that Nef is required for efficient growth in the two major target cells of HIV-1.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   (A) Replication kinetics of SF162 and R25 in stimulated and resting PBMCs. PBMCs were stimulated with IL-2 and PHA before infection (stimulated) or infected and then stimulated 4 days postinfection (resting). The amount of p24 capsid antigen released into the culture supernatant was quantitated every 3 to 4 days over periods of 14 and 25 days, respectively, for infected macrophages and PBMCs. , SF162 wild type; , SF162 nef clone 9; , SF162 nef clone 10; , R25 wild type; , R25 nef clone 3; , R25 nef clone 14. (B) Replication kinetics of SF162 and R25 in MDM. MDM were infected on day 10 postisolation with 40 ng of virus p24. , SF162 wild type; , SF162 nef clone 9; , SF162 nef clone 10; , R25 wild type; , R25 nef clone 3; , R25 nef clone 14.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   (A) MDM were grown and infected in medium with serum and harvested as described in Materials and Methods. Approximately 2 × 105 to 5 × 105 cells were used for each immunoprecipitation. Western analysis was performed with an anti-Hck antibody (Anti-Hck) (top row), and the blot was probed with anti-phosphotyrosine antibody (Anti-pTyr) (bottom row). Hck immunoprecipitated from mock-infected cells (lane 1) served as a control for the level of Hck protein present. The two forms of Hck protein are due to alternative translation initiation start sites giving rise to proteins of 59 and 61 kDa. The top edge of a band visible at 49.4 kDa is the immunoglobulin heavy chain. The amount of Nef protein immunoprecipitated was also determined and is shown in Fig. 3, lanes 5 and 6. Anti-Nef IPs from mock-infected cells (lane 2), SF162 wild type-infected cells (lane 3), and R25 wild type-infected cells (lane 4) are shown. (B) IVKAs were performed at 3, 5, and 7 days p.i. on Hck IPs from mock-infected (lanes 1, 3, and 5, respectively) and SF162 wild type-infected (lanes 2, 4, and 6, respectively) macrophages. The reactions were run on SDS-10% PAGE gels and dried on Whatman 3 MM filter paper, and nucleotide incorporation was detected by phosphorimager analysis. The levels of Nef expression were determined by immunoprecipitation and Western analyses.

HIV-1 Nef activates NAK in infected human macrophages and PBMCs. Binding and activation of the PAK-related kinase NAK by Nef was also shown to correlate with the enhancement of viral infectivity (56, 64). Since we could not detect an interaction of Nef with Hck, we looked for the ability of Nef to activate NAK in infected macrophages. The CEMx174 cell line chronically infected with SF2 served as a positive control. We performed IVKAs on Nef IPs from infected CEMx174 cells and from a portion of the same infected macrophages that were used for the Hck experiments whose results are shown in Fig. 2. In contrast to the apparent lack of a Nef-Hck interaction, Nef-mediated activation of NAK was readily detectable in infected human macrophages cultured in the absence or presence of serum. Two phosphorylated proteins were detected, one of 62 kDa and another of ~85 kDa in cells infected with wild-type virus (Fig. 3A, lanes 2 and 3 and 5 through 7) but not in cells infected with virus lacking Nef (lane 9) or encoding the P73A (lane 8) mutation. This mutation was previously shown to abrogate Nef-mediated activation of NAK and was used as a control for nonspecific interactions (53, 64). As in the Hck experiments, we used the R25 recombinant virus to introduce the T-cell line-tropic SF2 wild-type nef allele into the macrophage intracellular environment. Interestingly, although both p62 and p85 species were present in SF2 Nef IPs from infected macrophages and the immortalized T-cell line CEMx174, the pattern of phosphorylation differed (Fig. 3A, compare lanes 6 and 7). NAK was hyperphosphorylated in the CEMx174 cells, while p85 was more highly phosphorylated in macrophages (Fig. 3A). Furthermore, as shown by the experiment results shown in Fig. 3, p85 appeared predominantly as a single band in the donor macrophages while in CEMx174 cells p85 appeared as a doublet. The single p85 band in infected macrophages corresponds to the slower-migrating band in infected CEMx174 cells. In some macrophage experiments, however, a fainter and faster-migrating band was also visible, giving rise to the doublet pattern seen in CEMx174 cells (data not shown).

View larger version (72K): [in this window] [in a new window]   FIG. 3.   (A) Nef IPs from macrophages (lanes 1 to 6) infected and cultured in the presence (+) or absence () of serum for 10 days were subjected to an IVKA. IVKAs of Nef IPs from the chronically infected T-cell line CEMx174 (lanes 7 to 9) were run side by side with the IPs from macrophages for comparison. Nef IVKAs were performed on infected PBMCs at day 12 p.i. (lanes 10 to 13). Results for mock-infected cells (lanes 1, 4, and 10) and cells infected with SF162 wild type (lanes 2, 5, and 12), SF162 nef (lane 11), SF162 Nef P71A (lane 13), R25 wild type (lanes 3 and 6), SF2 wild type (lane 7), SF2 Nef P73A (lane 8), or SF2 nef (lane 9) are shown. Underneath, the levels of Nef protein present in each IP as detected by Western analysis are shown. (B) PAK IPs from macrophages infected and cultured in the presence (+) or absence () of serum were subjected to IVKAs. Results for mock-infected cells (lanes 1 and 4), SF162 wild type-infected cells (lanes 2 and 5) and R25 wild type-infected cells (lanes 3 and 6) are shown. Results of IVKAs of PAK IPs from mock-infected (lane 7) and chronically SF2-infected (lane 8) CEMx174 cells are shown for comparison. The band at 49.4 is the immunoglobulin heavy chain.

The p85 phosphoprotein is a substrate of PAK. We next performed IVKAs on PAK IPs obtained from infected or mock-infected macrophages to determine whether p85 is the substrate for PAK. In macrophages grown in the presence or absence of serum, a phosphorylated protein of 62 kDa representing PAK was observed in both mock-infected and infected cells. However, phosphorylation of an ~85-kDa protein was detected only in infected cells (Fig. 3B, lanes 2, 3, 5, and 6). Phosphorylation of p85 was also observed in IVKAs performed on PAK IPs from CEMx174 cells chronically infected with SF2 wild type as compared to mock-infected cells (Fig. 3B, lanes 7 and 8). Again, the p85 band appears as a doublet in CEMx174 cells. These results demonstrated that p85 is a PAK-associated protein that is phosphorylated in the presence of Nef. In previous studies, the molecular mass of the PAK substrate in SF2- and simian immunodeficiency virus strain mac239-infected CEMx174 cells run on SDS-12% PAGE gels has been assigned a molecular mass of 72 kDa (54, 56). In this study we have assigned a molecular mass of 85 kDa to the PAK substrate. The apparent discrepancy might be related to the fact that different-percentage SDS-PAGE gels were used in these studies. In fact, as shown in Fig. 3, the PAK substrates from T cells and macrophages migrate to the same position on SDS-10% PAGE gels.

PIX complex is the NAK substrate in both HIV-1-infected T lymphocytes and macrophages. Mounting evidence suggests that NAK is related to the PAK family of kinases (36, 39, 47). The recent description of the PAK-interacting guanine nucleotide exchange factor (GEF), PIX (43), as an 85-kDa interacting partner of PAK prompted us to investigate whether the p85 NAK-associated phosphoprotein is related to PIX. As the 95-kDa proto-oncogene and GEF Vav has also been reported to interact with Nef (17), its presence within the Nef immune complex was also investigated. An IVKA was performed on Nef IPs from CEMx174 cells chronically infected with SF2 wild type, SF2 Nef, or SF2 Nef P73A. The phosphorylated protein complexes were eluted from protein G-Sepharose, precleared of released immunoglobulin and then subjected to immunoprecipitation with anti-PAK, anti-PIX, or anti-Vav antibodies. Reimmunoprecipitation with anti-Hck provided a negative control. As expected, no phosphorylated proteins were detected in Nef IPs or from SF2 Nef- or SF2 Nef P73A-infected cells (Fig. 4A, lanes 1 and 3, 4 through 6, and 11 through 13). In agreement with previous reports (47, 56), reimmunoprecipitation of SF2 Nef IPs with anti-PAK antibody brought down the expected phosphorylated species of 62 kDa (Fig. 4A, lane 7). The low level of phosphorylated PAK seen in IP after reimmunoprecipitation (lane 7) compared to the level observed in the IVKA (lane 2) is most likely due to the lower reactivity of the anti-rat PAK1 antibody with human PAK1 (47). Reimmunoprecipitation with the anti-Vav antibody did not result in the specific immunoprecipitation of any phosphorylated species as compared to the anti-Hck control (Fig. 4A, lanes 9 and 10). Significantly, the anti-PIX antibody precipitated two phosphorylated species at 85 kDa representing the PIX complex (Fig. 4A, lane 8). Interestingly, Western blot analysis with anti-PIX antibody on uninfected CEMx174 cell lysates run side by side with the IVKA gels detected only a single protein that comigrated with the lower band in the p85 complex (Fig. 4A, lane 13). Furthermore, Vav expression was barely detectable in this cell line (Fig. 4A, lane 16).

View larger version (44K): [in this window] [in a new window]   FIG. 4.   (A) Reimmunoprecipitation of PAK and PIX from Nef IPs of infected CEMx174 cells. Cell lysates from ~7 × 106 cells were split into two portions (2.5 × 106 and 4.5 × 106 cells) and subjected to Nef immunoprecipitation and IVKA. One portion was reserved as the IVKA control for SF2 nef (Nef) (lane 1), SF2 wild type (SF2) (lane 2) and SF2 Nef P73A (P73) (lane 3). The larger portion was subjected to reimmunoprecipitation with anti-PAK (P) (lanes 4, 7, and 11), anti-PIX (X) (lanes 5, 8, and 12), anti-Vav (V) (lanes 6, 9, and 13), or anti-Hck (H) (lane 10) antibodies as described in Materials and Methods. Western analysis with either anti-PAK (P) (lane 14), anti-PIX (X) (lane 15), or anti-Vav (V) (lane 16) antibody was performed on CEMx174 cell lysates. The band at 49.4 kDa is the immunoglobulin heavy chain. (B) Reimmunoprecipitation of PAK and PIX from infected primary macrophages was performed as described above except that ~1 × 106 cells were used. Results of IVKAs performed as controls for SF162 nef-infected (Nef), SF162 wild-type (SF162), and R25 wild-type (R25) cells are shown in lanes 1, 2, and 3, respectively. Reimmunoprecipitation was performed with anti-PAK (lanes 4, 7, and 10), anti-PIX (lanes 5, 8, and 11), or anti-Vav (lanes 6, 9, and 12) antibodies. Western analysis with anti-PIX, anti-PAK, or anti-Vav antibody was performed on macrophage cell lysates, and results are shown in lanes 13, 14, and 15, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Using a prototypic macrophage-tropic HIV-1 virus and recombinant virus that expressed the nef allele of a T-cell line-tropic isolate, these studies confirmed the importance of Nef function for efficient viral growth in both primary T lymphocytes and MDM. Despite the reported high-affinity interaction of Nef with Src kinase Hck in vitro, we were unable to detect an interaction between these proteins in infected primary macrophages where both are abundantly expressed and in which a Nef phenotype is manifested. Furthermore, activation of Hck as measured by its phosphorylation state (Fig. 2) and ability to phosphorylate an exogenous substrate (enolase; data not shown) was not detected over background level. These findings therefore call into question the physiological relevance of a Nef-Hck interaction. Nevertheless, the possibility that Nef and Hck may interact transiently and/or at a very early step to facilitate viral infectivity, making such an event difficult to capture by the type of experiments performed in this study, cannot be excluded.

Under the same infection conditions, however, Nef interaction with and activation of the PAK-related kinase can be readily detected in primary macrophages as well as primary T lymphocytes (Fig. 3). Importantly, Nef-PAK interaction and activation result in the phosphorylation of a complex containing the PAK-interacting partner PIX (Fig. 4). PIX belongs to a new class of Rho-p21 GEFs that have been shown to bind with high affinity through their N-terminal SH3 domains to a conserved proline-rich sequence of PAK (42). It has been suggested that PIX binding is required for the translocation of PAK to focal complexes. The formation of focal complexes is regulated by the p21 GTP-binding proteins Cdc42 and Rac1 (GTPases) (2, 28, 40, 41, 43) and are important sites for the transduction of signals mediated through the actin cytoskeleton.

The PAK-PIX complex has been shown to contain additional proteins that include the adaptor Nck and a p95 PAK substrate whose identity has recently been revealed (61). Nck and PIX bind to distinct domains of PAK (6, 42), while p95 binds specifically to PIX (61). Several findings suggest that p95 is also present in the Nef-PAK-PIX complex in HIV-1-infected cells. First, similar to Manser et al. (42), who reported that several proteins coimmunoprecipitate with PAK and serve as substrates, we find that in Nef or PAK IPs from infected T lymphocytes, the PAK substrate runs as a doublet (Fig. 3B). Immunoblot analysis demonstrated that PIX is present as a single isoform in these cells and migrates as the faster protein within the substrate (Fig. 4A and B). The slower-migrating band, therefore, is likely to be p95. Second, data from reimmunoprecipitation analyses support the idea that p95 is tightly bound to PIX (Fig. 4). Protein-protein interactions between PAK and PIX could be efficiently disrupted, while under the same conditions, PIX and p95 binding could not be dissociated. Lastly, preliminary studies involving transient cotransfection of Nef, PIX, and p95 expression plasmids into 293T cells support the idea of the formation of a complex by these proteins (data not shown).

The mechanism by which Nef mediates PAK activation remains undefined. Recently, Vav has been reported to bind directly to HIV-1 Nef both in vitro and in vivo, resulting in the activation of Vav GEF activity (17). Since PAK activity is potentiated through binding to the active forms of Rac1 or Cdc42, and Vav bears GEF activity for these GTP-binding proteins, it is conceivable that Nef-mediated activation of Vav may stimulate PAK. However, Vav expression was barely detectable in the T-cell-B-cell hybrid CEMx174 cell line, and in primary macrophages where it is abundantly expressed, Vav was not the phosphorylated protein in the Nef immune complex (Fig. 5). Thus, the activation of PAK observed in infected macrophages and in CEMx174 cells is most likely not due to Vav activity. In this regard, it has been reported that PAK activity can be modulated as a consequence of physical interaction with PIX by mechanisms that require or are independent of exchange factor binding (14). Thus, it will be important to determine whether Rac1 or Cdc42 is recruited within the Nef macromolecular complex and if activation of PAK is mediated through PIX.

In vitro kinase experiments with Nef expressed in 293T (human embryonic kidney cells) (64) and COS cells (simian fibroblasts) (53) led to the conclusion that p85 is present only in the T-cell lineage (53, 54). We have now demonstrated that this PAK substrate, which is composed of PIX and p95, is also present in primary PBMCs and macrophages and that it can coimmunoprecipitate with Nef in infected cells. Nevertheless, Western analyses show that PIX is also present in 293T cells (data not shown) and COS cells (42). Thus, it is surprising that Nef-mediated activation only of PAK and not of PIX was observed in 293T and COS cells. It could be argued that different isoforms of PIX may be expressed in fibroblast and lymphoid or myeloid cells and that the Nef-PAK complex fails to interact with PIX present in 293T or COS cells or induce its phosphorylation. Alternatively, the Nef-PAK complex may require another essential component to recruit PIX that is specifically expressed in T lymphocytes and macrophages and which is absent from 293T and COS cells. Additional studies will be required to address these different possibilities. Since antibody against p95 is currently unavailable, its tissue and cell distribution is as yet unknown. However, Nef⁺ virions produced in 293T and COS cells still exhibit enhancement of infectivity properties (36, 44, 63), suggesting that PAK activation of another downstream target besides PIX or p95 might be involved in mediating this effect of Nef. As PAK has been shown to participate in multiple signaling pathways, it is not surprising that depending on upstream signals and specific downstream effectors, the binding and activation of this kinase by Nef may lead to pleiotropic effects.

Indeed, our results show that although both PIX and p95 serve as substrates for PAK that is activated as a result of binding to Nef in infected T lymphocytes, p95 appears to be preferentially phosphorylated by PAK in infected macrophages expressing the same nef allele (Fig. 3). These findings of differential phosphorylation of the PAK substrates further support the notion that the regulation of the downstream effector activity of PAK is tightly controlled and is likely to be cell type dependent. Nevertheless, the possibility that another kinase present in the Nef immune complex in infected macrophages specifically phosphorylates p95 cannot be excluded at present.

Nef is synthesized at high levels early in infection and yet is also packaged into the virion at the end of the infectious cycle. Furthermore, Nef exerts its functions in both the producer and target cells at various stages of the viral replication cycle. With the identification of downstream effectors of PAK that are recruited as a result of Nef binding and activation, their role in mediating Nef function can now be addressed. Whether overexpression of transdominant PIX or p95 mutant proteins, or peptides derived from these proteins, affects receptor down-modulation, early postentry events that would include uncoating and/or activation of gene transcription, and virion morphogenesis, can now be assessed. Identification of additional proteins in the Nef-PAK complex should further advance our understanding of Nef function at the molecular level. Although there are major challenges to performing molecular analyses in primary cells, attempts to interfere with the function of the PIX-p95 complex in such cell types will be necessary to understand how the activation and regulation of Nef effectors results in the enhanced ability of HIV-1 virions to infect and spread within the host.