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Functional Analysis of HIV Type 1 Nef Reveals a Role for PAK2 as a Regulator of Cell Phenotype and Function in the Murine Dendritic Cell Line, DC2.4¹

Jelena Mann^*, Claire N. Patrick, Mark S. Cragg^,, Jamie Honeychurch, Derek A. Mann^* and Mark Harris^2,

^* Liver Group, Division of Infection, Inflammation and Repair, University of Southampton, Southampton General Hospital, Southampton, United Kingdom; Cancer Sciences Division, University of Southampton, Southampton General Hospital, Southampton, United Kingdom; Molecular Genetics of Cancer Division, Walter and Eliza Hall Institute, Victoria, Australia; and Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom

	Abstract

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The HIV-1 Nef protein plays a critical role in viral pathogenesis. Nef has been shown to modulate dendritic cell (DC) function, in particular perturbing their ability to present Ag. To further characterize the effects of Nef on DCs, we established a panel of transfectants of the murine DC line, DC2.4, stably expressing differing levels of either wild-type Nef, or a number of Nef mutants lacking key functional motifs. Transfectants expressing increasing levels of wild-type Nef demonstrated a dose-dependent shrinkage and loss of dendrites. Nef expression levels also correlated with increased proliferative ability but did not confer resistance to proapoptotic stimuli. Importantly, Nef expression resulted in an impairment of Ag presentation to T cells correlating with a reduction in the cell surface expression of molecules involved in Ag presentation such as MHC class I, CD80/86, and ICAM-1. Nef expression also rendered DC2.4 cells resistant to the maturation stimulus provided by an anti-CD40 Ab. Mutations in either the myristoylation site or Src homology 3-domain binding polyproline motif of Nef abolished these effects. Previous studies had shown that these mutations also abolished the ability of Nef to activate the p21-activated kinase, PAK2. Consistent with this, stable expression of constitutively active PAK2 in DC2.4 mimicked the effects of Nef. We conclude that Nef, acting via activation of PAK2, inhibits both DC maturation and Ag presentation. These data have clear implications for the role of Nef in early stages of HIV-1 infection and validate Nef as a valid target for development of antiviral chemotherapeutics.

	Introduction

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The HIV type 1 (HIV-1)³ Nef protein is a polypeptide of 205 residues that is both myristoylated and phosphorylated, the latter modification is reported to be performed by protein kinase C (1, 2). Numerous studies over the past two decades have demonstrated that Nef plays a critical role in both virus replication and pathogenesis. Despite this intensive research effort, there is still much confusion and ambiguity in the literature regarding both the functions of this enigmatic protein and the underlying biochemical mechanisms. An additional area of controversy relates to the effects of Nef on the function of dendritic cells (DC). Immature DC (iDC) are the first cells to be infected by the virus during mucosal transmission and have been shown to play a key role in the dissemination of virus from the periphery (e.g., rectal or vaginal mucosa) to the lymph nodes (3), where they can transmit the virus to naive T cells during the process of Ag presentation.

An early study (4), using wild-type HIV-1 and an isogenic Nef-deleted (Nef) derivative, showed that Nef inhibited endocytosis of the DC-specific lectin, DC-specific ICAM-3-grabbing nonintegrin, promoting clustering of DCs with lymphocytes and enhancing HIV-1 transmission. A separate study used adenovirus vectors to demonstrate that Nef activated iDC, enhancing release of inflammatory cytokines (e.g., IL-6, IL-12) and chemokines (e.g., CXCL8) but did not result in maturation of the iDC (5). More recently, Nef expressed from vesicular stomatitis virus-G-pseudotyped HIV-1 was shown to down-regulate the Ag-presenting molecules CD1a and MHC class I (6). Other groups have shown that iDC are able to take up exogenous recombinant Nef (expressed in Escherichia coli), resulting in up-regulation of a range of molecules involved in Ag presentation including CD1a, CD40, and HLA-DR (7).

Exogenous Nef was shown to induce actin rearrangements and differentiation in the study; it was also observed that Nef enhanced GTPase activity of the Rho family p21-GTPase, Rac1, by increasing tyrosine phosphorylation of the Rac1 guanine nucleotide exchange factor, Vav1, providing a possible molecular mechanism for the phenotype (8). Activation of Rac1 results in activation of PAK2, one member of the family of p21-activated kinases; in this regard, PAK2 is one of the key cellular targets of Nef. Binding of the GTP-bound GTPases Cdc42 and Rac1 leads to PAK2 autophosphorylation (on Thr⁴⁰²); however, many studies have shown that the interaction of Nef with PAK2 also results in stimulation of autophosphorylation (9, 10, 11). Recent data have shown that Nef associates with a subpopulation of active PAK2 within lipid rafts (12, 13). The activation of PAK2 by Nef thus requires membrane association of Nef; consistent with this, a myristoylation-defective mutant (Gly²-Ala) failed to associate with, or activate, PAK2 (14). Furthermore, the association between Nef and PAK2 requires a conserved polyproline (PxxP) motif within Nef, previously characterized as binding to the Src homology 3 (SH3) domains of members of the Src family of tyrosine kinases, in particular Hck. Because PAK2 does not contain an SH3 domain, this suggests that the Nef-PAK2 interaction is not direct and is mediated via a (as yet unidentified) cellular SH3 domain-containing protein. Intriguingly, and consistent with the effects of exogenous Nef on Vav1 in DC discussed above (8), the C-terminal SH3 domain of Vav has been shown to interact with Nef, resulting in Vav activation with concomitant cytoskeletal rearrangements and activation of the JNK pathway in NIH3T3 cells (15). Dominant-negative Vav blocked the activation of PAK2, leading to the suggestion that Nef participates in a multiprotein complex including Vav, Rac1/Cdc42, and PAK2.

In an attempt to reconcile the literature pertaining to the effects of Nef on DC function, we have established stable transfectants of the murine DC cell line DC2.4 (16), expressing either wild-type Nef, or a series of mutants designed to abrogate specific functions of the protein. We show in this study that expression of wild-type Nef resulted in increased proliferation of DC2.4 cells in a dose-dependent fashion. Nef expression was also linked to changes in morphology—specifically the loss of dendrites and a reduction in size. This phenotype corresponded with a decreased ability to present Ag to autologous T cells and a reduction in cell surface levels of molecules involved in Ag presentation such as MHC class I and CD80/86. This was consistent with the maintenance of an immature phenotype, indeed Nef-expressing DC2.4 cells were refractory to stimulation with an anti-CD40 Ab. Mutations within the myristoylation sequence or in the PxxP motif abolished the effects of Nef on DC morphology and function. Because these mutants were predicted not to activate PAK2, we established that these effects of Nef were mimicked by stable expression of a constitutively active mutant of PAK2, but not by a kinase-inactive mutant. These data are consistent with a role for Nef-mediated activation of PAK2 in perturbing DC function and further suggest that PAK2 is an important regulator of DC maturation.

	Materials and Methods

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Cell culture

The DC2.4 parental cell line (16) and all DC2.4/Nef-expressing stable lines were cultured in RPMI 1640 medium, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10% FBS (Invitrogen Life Technologies). Transfected cell lines were pulsed with 0.5 mg/ml G418 at monthly intervals for 1 wk to prevent loss of stable-transfected protein expression.

Transfections and selection of stable clones

DC2.4 cells were transfected by electroporation using 5 x 10⁶ cells and 30 µg of expression vector DNA per transfection reaction. Briefly, cells were placed in a 4-mm diameter electroporation cuvette and mixed with DNA in a total volume of 0.7 ml of Optimem buffer. The mixture was incubated on ice for 10 min, then electroporated at 280 V and 960 µF with a single pulse. Following sequential 5-min incubations on ice and then room temperature, the cells were plated out on 96-well plates in complete medium supplemented with 0.5 mg/ml G418 added 24 h later. Clones were selected on the basis of their resistance to G418 and expression of wild-type or mutant Nef proteins assessed by Western blot. For generation of DC2.4-expressing PAK2 mutants, the cells were electroporated (as described above) with 30 µg of pPAK2-K278R or pPAK2-T402E (gifts from Kalle Saksela, University of Tampere, Tampere, Finland) and 5 µg of pCIpuro vector DNA. Electroporated cell populations were selected in 1 µg/ml puromycin, and the resulting population was tested for expression of hemagglutinin (HA)-tagged PAK2 mutants by Western blot.

Plasmid DNA

All plasmid DNA was prepared using a commercial DNA extraction and isolation kit (Maxiprep; Qiagen) per the manufacturers instructions. Nef from the HIV-1 strain SF2 (17) was amplified by PCR and cloned into the eukaryotic expression vector pEF/myc/cyto (Invitrogen Life Technologies) as a NcoI-NotI fragment. Mutant derivatives were generated by PCR-mediated mutagenesis; these mutants were produced in the laboratories of Kalle Saksela (University of Tampere, Tampere, Finland) and Andreas Barr (University of Miami, Miami, FL) as part of an European Union Fifth Framework Consortium (QLK2-CT-2000-01630, Targeting Nef). All of the constructs were verified by sequencing, primer sequences are available on request. Mutations and their abbreviated numerical names are listed in Table I.

One hundred microliters of DC2.4 cells at a concentration of 1 x 10⁶/ml were incubated with 15 µl of 100 µg/ml FITC-labeled primary Ab for 30 min at 4°C and washed with PBS containing 1% BSA and 20 mM sodium azide. Cells were resuspended in 150 µl of the same buffer, and analysis was conducted by flow cytometry using the CellQuest program on a FACSCalibur (BD Biosciences).

SDS-PAGE and immunoblotting

Whole cell extracts were prepared, and protein concentration of samples was determined using Bradford DC assay kit (Bio-Rad). Thirty or 50 µg of whole cell extracts were then fractionated by electrophoresis through a 9% SDS-polyacrylamide gel. Gels were run at 100 V for 1.5 h before transfer onto nitrocellulose as described previously (18). Following overnight blocking of nonspecific protein binding in TBS/0.075% Tween 20 (TBS-T) containing 5% (w/v) nonfat skimmed milk powder, nitrocellulose blots were incubated for 1 h with primary Abs diluted in TBS-T containing 5% (w/v) nonfat skimmed milk powder. Mouse mAb recognizing Nef (obtained from the Centralized Facility for AIDS Reagents, National Institute of Biological Standards and Control, Potters Bar, U.K.; catalog no. ARP3026) was used at a final concentration of 1 µg/ml, whereas mouse anti-HA-tag Ab was used at 1/5000. Mouse mAb recognizing -actin was used at a dilution of 1/1000 (Santa Cruz Biotechnology). Blots were then washed three times in TBS-T before incubation for 1 h with rabbit anti-mouse HRP Ab at a 1/2000 dilution in TBS-T containing 5% (w/v) nonfat skimmed milk powder. After extensive washing in TBS-T, the blots were processed for detection of Ag using the ECL system (Amersham Biosciences).

Detection of apoptosis: DNA fragmentation assay

Treated cells (5 x 10⁵) were centrifuged for 5 min at 500 g and then washed once in PBS. The cells were then resuspended in hypotonic fluorochrome solution (50 µg/ml propidium iodide, 0.1% (w/v) sodium citrate, 0.1% (w/v) Triton X-100) and incubated in the dark at 4°C overnight or 4 h at room temperature. The DNA content of at least 7.5 x 10³ cells was analyzed by flow cytometry, and the proportion giving fluorescence below the G₁/G₀ peak was taken as a measure of apoptosis.

[³H]Thymidine incorporation assay

All of the cell lines tested were set up in triplicate in 96-well plates. Proliferation was measured at different time points by adding 0.5 µCi of [³H]thymidine per well during the last 16 h of culture. Incorporated [³H]thymidine was measured by harvesting cells onto UniFilter-96, GF/C plates (PerkinElmer) using a 96-well plate harvester, and the plates were analyzed for [³H]thymidine incorporation using a Packard Topcount Microplate Scintillation counter (Packard Instrument). Incorporated radioactivity is expressed as counts per minute.

IL-2 release assay

Empty vector-transfected DC2.4 cells or cell lines expressing Nef were pulsed with the synthetic OVA peptide SIINFEKL at 1 µg/ml. After a 2-h incubation at 37°C, the cells were washed, irradiated, and resuspended at 5 x 10⁴ cells/ml in complete media on a 12-well plate. T cell hybridoma (1 x 10⁴) RF33.70 responder cells were added to stimulators in triplicate wells. These cultures were incubated at 37°C for 48 h, and supernatant was harvested. It was then assayed for the presence of IL-2 by MCTLL proliferation assay. Briefly, 10⁵ MCTLL cells were starved of IL-2 for 2 h before the addition of 100 µl of supernatant from the stimulated RF33.70 cell culture. The cells were then incubated for 24 h at 37°C and pulsed with 0.5 µCi/well [³H]thymidine for 18 h. Following harvesting of the cells, the amount of incorporated radioactivity as determined as already described.

Luciferase assays

Cells were transfected by the nonliposomal Effectene protocol (Qiagen) according to the manufacturer’s instructions. Cells were harvested 48 h after transfection, and luciferase assays were performed using a dual luciferase kit (Promega) according to the manufacturer’s instructions. IL-6 promoter-driven expression of firefly luciferase was normalized for differences in transfection efficiency by measurement of the activity of a cotransfected Renilla vector pRLTK.

Total RNA isolation and RT-PCR

Total RNA was isolated from 5 x 10⁶ empty vector-transfected pEF2 and Nef1D cell line using the Total RNA purification kit (Qiagen) following the manufacturer’s instructions. First-strand cDNA was generated using 500 ng of total RNA, 1 µl of random hexamer primer (p(dN)₆), and RNase-free water (Qiagen), heated at 70°C for 5 min, and then placed on ice. RNasin (RNase inhibitor), 100 U of Moloney murine leukemia virus-reverse transcriptase, 1x Moloney murine leukemia virus reverse-transcriptase buffer, and 0.4 mM dNTPs were added, and the mix was incubated at 42°C for 1 h. PCR amplification of PAK2 cDNAs was conducted using specific oligonucleotide primers selected within the coding regions of the genes. The primers used were as follows: 5'-TCTACGACTCCAACACTG-3' (sense) and 5'-ACAACTCATCTCCTACCA-3' (antisense) designed to produce a 573-bp PCR product. PCRs were composed of 25 ng cDNA template, 100 ng each of sense and antisense oligonucleotide primers, 2.5 µl of optimized Taq PCR buffer (Promega), 0.4 mM dNTP mixture, and 2 U of Taq polymerase in a total reaction volume of 25 µl. Following an initial 5-min incubation at 94°C, PCRs were performed using a 1-min annealing step at 50°C followed by a 1-min elongation step at 72°C and a 45-s denaturation step at 94°C. A total number of 32 PCR cycles were conducted for amplification, followed by a final elongation reaction for 10 min at 72°C. PCR products were separated by electrophoresis at 50 V for 90 min through a 1% agarose gel and were detected by ethidium bromide staining. Expected sizes of specific PCR products were verified by reference to a 1-kbp DNA ladder.

	Results

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Expression of wild-type Nef in DC2.4 cells changes their morphology and increases their rate of cell proliferation

To examine the effects of Nef expression on DCs, we stably transfected DC2.4 cells with either an expression vector for wild-type Nef (pEF-Nef1) or the control empty vector pEF/myc/cyto. Seven cell lines were selected (Nef1A–G), which together provided a range of Nef expression levels as confirmed by Western blot (Fig. 1a), along with two control cell clones not expressing Nef (pEF1, pEF2). It was noted that the increase in Nef expression was linked to a change in DC morphology relating to the loss of dendrites and acquisition of a rounded shape (Fig. 1b). This effect appeared to be dose-dependent, because both the loss of processes and cell rounding were more pronounced in clones with higher Nef expression (Fig. 1b). The change in morphology was observable even at low levels of Nef expression, although clones Nef1A, B, and C still had a proportion of cells that exhibited normal DC morphology comparable to that of mock-transfected cells (Fig. 1b). Four clones (Nef1D–G) expressing higher levels of Nef were characterized by the complete absence of cells with normal morphology. The change in shape and size of the Nef1 cell clones compared with the control pEF1 and pEF2 clones was confirmed on forward/side scatter using flow cytometry (Fig. 1c). This analysis demonstrated a progressive decline in side scatter with increasing levels of Nef expression.

View larger version (50K): [in this window] [in a new window]   FIGURE 1. a, Expression of increasing levels of wild-type Nef in DC2.4 cells. Western blot analysis was conducted using 30 µg of whole cell protein isolated from DC2.4 cell lines expressing wild-type Nef (Nef1) designated Nef1A–G. Nef and -actin were detected using 1 µg/ml mouse anti-Nef Ab (ARP3026) or 1/1000 mouse anti--actin Ab, respectively; HRP-conjugated rabbit-anti-mouse Ig was used as the secondary Ab. b, A dose-dependent effect of Nef expression on the morphology of DC2.4 cells. DC2.4 clones were photographed using a Zeiss microscope at x20 magnification. The images show a decrease in the number and length of dendritic processes correlating with an increased level of Nef wild-type expression (A–G). c, Nef expression causes shape and size changes in DC2.4 cells. DC2.4 clones were assessed for their shape and size using flow cytometry. Differences in forward (FSC-H) and side (SSC-H) scatter show that Nef-expressing cells are smaller in size and less granular.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. a, Nef expression provides DC2.4 cells with a growth advantage reflected in an increased rate of proliferation. Cells (1 x 105) from clones transfected either with empty vector (pEF1 or pEF2) or expressing wild-type Nef were seeded in triplicate and incubated in growth medium for 2 h before measuring proliferation at different time points by adding 0.5 µCi of [3H]thymidine per well for 16 h. Incorporated [3H]thymidine was measured as outlined in Materials and Methods and is expressed as counts per minute. All experiments shown represent n = 3, where each separate experiment was conducted in triplicate. White bars are empty vector-transfected cells, whereas cell lines expressing wild-type Nef are in black bars. Statistical analysis of this data is presented in Table II. b, Nef expression in DC2.4 cells does not alter their susceptibility to apoptosis. Empty vector (pEF2)-transfected or Nef1D cells were incubated with ceramide, etoposide, cycloheximide, or nocodazole at the indicated concentrations. Cells were resuspended in hypotonic fluorochrome solution and incubated in the dark at 4°C overnight. The DNA content of 7.5 x 103 cells was analyzed by flow cytometry, and the proportion giving fluorescence below the G1/G0 peak was taken as a measure of apoptosis. The graph was plotted from mean percentage apoptosis taken from three separate experiments ±SEM.

A key role of DC is to present foreign Ag to T cells. It was therefore of interest to examine the effects of Nef on the ability of DC2.4 cells to present Ag, particularly because previous studies had demonstrated that DC are the first cell type in the body to be infected with HIV. As such, any perturbation of DC Ag presentation mediated by Nef during this early phase of immune response might significantly alter the course and outcome of the disease. The Nef wild-type-expressing clones Nef1A–G or control clones pEF1/pEF2 were incubated with the synthetic OVA peptide SIINFEKL and the RF33.70 T cell hybridoma. Results of these experiments, as shown in Fig. 3a, revealed that ability of DC2.4 to present Ag to T cell hybridoma decreased with an increase in Nef expression, although this effect was not absolutely dose dependent. We found that expression of low levels of Nef caused a marked perturbation in ability of DC to present Ag, which was further diminished in cells expressing higher levels of Nef. However, increased expression of Nef beyond the levels measured in Nef1D failed to have any further repressive activity on Ag presentation. All of the Nef-expressing cells showed a statistically significant reduction in presentation compared with the control pEF1 clone (see Table III). However, although clearly exhibiting a reduction, the differences between Nef1A or Nef1C and the other control clone, pEF2, were not statistically significant.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. a, Nef expression in DC2.4 cells decreases their ability to present Ag in vitro to a T cell hybridoma. Empty vector-transfected control DC2.4 cells (pEF1/2), or cell lines expressing increasing levels of wild-type Nef were pulsed with the synthetic OVA peptide SIINFEKL for 2 h at 37°C. The cells were washed, irradiated, and mixed with the RF33.70 T cell hybridoma as outlined in Materials and Methods. Successful Ag presentation was reflected in production of IL-2 by RF33.70, which was assayed for by MCTLL proliferation assay. The counts per minute were converted into units of IL-2 produced using a standard curve within the experiment. The graph shows data from n = 3 experiments ±SEM. Statistical analysis of this data is presented in Table III. b, Expression of wild-type Nef leads to reduced surface expression of costimulatory markers by DC2.4 cells. Empty vector-transfected and two cell lines expressing wild-type Nef (Nef1D and Nef1G) were harvested and resuspended at a concentration of 1 x 106/ml. One hundred microliters of cell suspension were incubated with 15 µl of 100 µg/ml FITC-labeled Abs to CD40, CD80, CD86, ICAM-1, or MHC class I for 30 min at 4°C. Cells were washed with 3 ml of PBS containing 1% BSA and 20 mM azide, resuspended in 150 µl of buffer, and analyzed by flow cytometry on FACSCalibur using the CellQuest program. The shaded graphs correspond to cells incubated with the test Abs, and the empty graphs correspond to an isotype-matched control Ab. The numbers in the top right corner of each graph refer to mean fluorescence of the cell population.

The reduction in presentation and expression of costimulatory markers suggested that Nef expression might render DC2.4 cells resistant to maturation stimuli. DC can be stimulated to mature by treatment with a mAb to CD40; in addition, we have previously shown that such treatment induces IL-6 gene transcription (22). We therefore transfected pEF1 and Nef1D cells with an IL-6 promoter luciferase reporter plasmid, treated the cells with an anti-CD40 mAb (3/23), and measured luciferase levels as an indication of the response to CD40 ligation. As shown in Fig. 4, treatment of the control pEF1 cells with 3/23 resulted in a 5-fold increase in IL-6 promoter activity, whereas Nef1D cells failed to respond to this treatment. This result is consistent with the hypothesis that Nef expression impairs the maturation of DC2.4 cells.

View larger version (14K): [in this window] [in a new window]   FIGURE 4. Expression of wild-type Nef renders DC2.4 cells resistant to a maturation stimulus. Empty vector-transfected (pEF1), or cells expressing wild-type Nef (Nef1D) were transfected with 100 ng of pRLTK and 1 µg of pIL6wt-Luc (16 ). Anti-mouse CD40 mAb 3/23 (30 µg/ml) was added 24 h later. The cells were harvested at 48 h after transfection. Luciferase activity was determined, normalized to pRLTK activity, and expressed as mean ± SE of three independent triplicate transfections.

To characterize the residues or motifs within Nef responsible for the effects on DC shape, proliferation, and Ag presentation, we next generated a panel of DC2.4 cell lines expressing a variety of different Nef mutant proteins, characterized in other systems (Table I). These included the following: a myristoylation-negative mutant (Nef2), mutations in the SH3-binding PxxP motif (Nef6, Nef7, Nef8, and Nef13), a mutation reported to abolish Nef oligomerization (Nef9), and a mutation of a residue identified as required for the dominant-negative activity of the Nef F12 allele (Nef12) (23). DC2.4 cell clones were selected on the basis of expressing comparable levels of Nef mutants to that of the Nef1D clone (Fig. 5a). Morphological assessment of the lines revealed that DC2.4 cells expressing Nef2, Nef6, and Nef13 behaved in a similar manner to control, empty vector-transfected lines, with a flattened appearance complete with dendrite extensions (Fig. 5b). By contrast, expression of Nef9 and Nef12 caused a similar change in shape and adherence as observed for wild-type Nef. Lines expressing Nef7 and Nef8 appeared to have an intermediate morphological phenotype, with a significant number of cells in the cultures showing signs of rounding and loss of dendrites. The size and shape of the Nef mutant-expressing DC2.4 cell lines was confirmed on forward/side scatter using flow cytometry (Fig. 5c). Nef9 and Nef12 showed reduced side scatter compared with Nef2, Nef6, Nef7, and Nef13. Nef8 exhibited an intermediate phenotype. Proliferation assays produced similar results with DC2.4 cells expressing Nef9 and Nef12 proliferating at a similar rate to those expressing wild-type Nef, whereas Nef2, Nef6, Nef7, and Nef13 had only a minor effect on proliferation (Fig. 5d). DC2.4 cells expressing Nef8 had a rate of proliferation intermediate between Nef9, Nef12, and wild-type, which was in accordance with the intermediate phenotype in the morphological analysis (Fig. 5, b and c).

View larger version (57K): [in this window] [in a new window]   FIGURE 5. a, Expression of Nef mutants in DC2.4 cells. Western blot analysis was conducted using 30 µg of whole cell protein isolated from DC2.4 cell lines expressing either wild-type Nef (Nef1D, for comparison with other clones), or the Nef mutants Nef2, Nef6, Nef7, Nef8, Nef9, Nef12, and Nef13. Nef and -actin were detected as described in Fig. 1a. b, Alterations in DC2.4 cell morphology upon expression of Nef mutants. Clones expressing levels of mutant Nefs comparable to that of Nef1D were photographed using a Zeiss microscope at x20 magnification. DC2.4 clones expressing Nef7, Nef8, Nef9, and Nef12 had similar morphology to those expressing wild-type Nef (Nef1D), whereas Nef2, Nef6, and Nef13 were similar to mock-transfected cells. c, Alterations in DC2.4 cell shape and size upon expression of Nef mutants. DC2.4 clones were assessed for their shape and size by measuring forward (FSC-H) and side (SSC-H) scatter using flow cytometry. d, Alterations in DC2.4 cell proliferation rates upon expression of Nef mutants. Cells (1 x 105) from each clone were seeded in triplicate and incubated in growth medium for 2 h before measuring proliferation by adding 0.5 µCi of [3H]thymidine per well for 16 h. Incorporated [3H]thymidine was measured as outlined in Materials and Methods and is expressed as counts per minutes. All experiments shown represent n = 3, where each separate experiment was conducted in triplicate. Empty vector-transfected cells are shown in white bars, Nef1D in black, cell lines with similar proliferation capacity to that of empty vector-transfected cells (Nef2, Nef6, Nef7, Nef8, and Nef13) are in light gray, whereas Nef9 and Nef12 (similar to Nef1D) are in dark gray.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. a, Ag presentation by DC2.4 cell lines expressing Nef mutants. Empty vector-transfected control DC2.4 cells or cell lines expressing Nef1D or mutant Nef alleles as indicated were pulsed with the synthetic OVA peptide SIINFEKL at 1 µg/ml. Following a 2-h incubation at 37°C, the cells were washed, irradiated, and mixed with the RF33.70 T cell hybridoma as outlined in Materials and Methods. Successful Ag presentation was reflected in production of IL-2 by RF33.70, which was assayed for by the MCTLL proliferation assay. The counts per minute were converted into units of IL-2 produced using standard curve within the experiment. The graph shows data from n = 3 experiments ±SEM. b, Surface expression of costimulatory markers in DC2.4 cell lines expressing Nef mutants. Nef6 and Nef9 were used as representative cell lines with the characteristics of empty vector-transfected or wild-type Nef-expressing cells, respectively. The cells were harvested and resuspended at a concentration of 1 x 106/ml. One hundred microliters of cell suspension were incubated with 15 µl of 100 µg/ml FITC-labeled Abs to CD40, CD80, CD86, ICAM-1, or MHC class I for 30 min at 4°C. Cells were washed with 3 ml of PBS containing 1% BSA and 20 mM sodium azide, resuspended in 150 µl of buffer, and analyzed by flow cytometry on FACSCalibur using the CellQuest program. The shaded graphs correspond to cells incubated with the test Abs, and the empty graphs correspond to an isotype-matched control Ab. The numbers in the top right corner of each graph refer to mean fluorescence of the cell population.

Our data demonstrated that mutations of either the myristoylation site (Nef2) or the PxxP motif (Nef6, Nef7, Nef8, and Nef13) significantly disrupted the ability of Nef to attenuate DC function, whereas the mutations that were predicted not to affect either myristoylation or PxxP function (Nef9 and Nef12) exhibited a wild-type phenotype. We noted with interest that the phenotype of these mutants correlated well with their predicted effects on the interaction with, and activation of, PAK2 (24, 25). We reasoned, therefore, that Nef might influence DC function via activation of PAK2. To investigate this possibility, in the absence of a PAK2-specific Ab, we used RT-PCR to determine whether DC2.4 cells expressed endogenous PAK2, and, if so, whether expression was modulated by Nef. As shown in Fig. 7a, both control and wild-type Nef-transfected DC2.4 cells (Nef1D) expressed similar high levels of PAK2 mRNA. To determine the effects of PAK2 activation on DC function, we generated stable DC2.4 cell lines expressing HA-tagged versions of either a constitutively active PAK2 mutant (containing a phospho-mimic mutation at the site of autophosphorylation, T402E) or a kinase-inactive mutant (K278R). Stable expression of the mutant PAK2 proteins in DC2.4 cell lines was verified by Western blotting with an HA-specific Ab (Fig. 7b). Morphological analysis revealed that DC2.4 expressing the constitutively active PAK2-T402E had a similar rounded appearance to that observed with Nef-expressing lines; by contrast, DC2.4 cells expressing the kinase-inactive PAK2-K278R retained the normal DC-like appearance of control cells (Fig. 7c). The attenuated morphology of PAK2-T402E-expressing DC2.4 cells was associated with an elevated rate of proliferation again as observed with DC2.4 cells expressing wild-type Nef. In contrast, overexpression of the kinase-inactive PAK2-K278R did not enhance proliferation of DC2.4 cells (Fig. 7d).

View larger version (35K): [in this window] [in a new window]   FIGURE 7. a, DC2.4 cells constitutively express PAK2. Total RNA was isolated from pEF1 or Nef1D cells, and first-strand cDNA synthesis was performed using 500 ng of total RNA as template. Primers specific for PAK2 were used in PCR using the first-strand cDNA products as template. The 590-bp PCR product (indicated by an arrowhead) shows that DCs express PAK2, which does not appear to change in cells expressing wild-type Nef. b, Expression of HA-tagged PAK2 mutants in stably transfected DC2.4 cells. Western blot analysis was conducted using 50 µg of whole cell protein isolated from DC2.4 cell lines expressing HA-tagged PAK2-K278R or PAK2-T402E kinase mutants. Both PAK2 mutants were detected using a mouse anti-HA-tag Ab (Sigma-Aldrich; HA7, 1/5000), and HRP-conjugated rabbit-anti-mouse Ig was used as the secondary Ab (the arrowhead indicated the appropriate HA-PAK2 band). Both PAK2 mutant proteins show a similar level of expression in selected populations. c, Expression of the PAK2-T402E constitutively active mutant results in loss of dendrites and rounding up of DC2.4 cells. Cell populations expressing kinase-inactive (PAK2-K278R) or constitutively active (PAK2-T402E) were photographed using a Zeiss microscope at x20 magnification. Constitutively active PAK2-T402E-expressing cells show morphology similar to that found in cell lines expressing wild-type Nef. d, DC2.4 cells expressing constitutively active PAK2-T402E proliferate faster than cells expressing kinase-inactive or untransfected cells. Naive DC2.4 cells or populations expressing either PAK2-K278R or PAK2-T402E (1 x 105 cells) were seeded in triplicate and incubated in growth medium for 2 h before measuring proliferation by adding 0.5 µCi of [3H]thymidine per well for 16 h. Incorporated [3H]thymidine was measured as outlined in Materials and Methods and is expressed as counts per minute. All of the experiments shown represent n = 3, where each separate experiment was conducted in triplicate. Untransfected DC2.4 population is shown as a black bar, PAK2-K278R is represented with a white bar, and PAK2-T402E in gray.

	Discussion

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Analysis of a panel of Nef mutants revealed a striking correlation between the effects on DC phenotype and the reported ability to interact with and activate PAK2, suggesting that the effects of Nef might be mediated via PAK2. In further support of this hypothesis, we also show that stable expression of a constitutively active PAK2 mutant protein mimicked the effect of Nef in terms of increasing proliferation and loss of dendrites (Fig. 7). These data implicate a role of PAK2 as a negative regulator of DC maturation, a proposition supported by a recent publication showing that maturation of DC2.4 cells driven by LPS treatment resulted in a dramatic reduction in the levels of activated Rac1 (26). Interestingly, the activation of Rac1 in DC2.4 cells required DOCK180, and recent data suggest that Nef activation of Rac1 in T cells is mediated via interactions with the related protein DOCK2 (complexed to ELMO1) (27). This interaction required both myristoylation and the PxxP motif of Nef, the latter presumably interacting with the SH3 domain of DOCK2. It is tempting to speculate that in DC2.4 cells Nef associates with the SH3 domain of DOCK180, resulting in activation of Rac1 and PAK2 and leading to negative effects on DC maturation. However, this hypothesis remains to be tested.

A further implication of the above scenario is that Nef-expressing iDC would be resistant to maturation stimuli such as LPS or CD40 ligation. Our preliminary analysis (Fig. 4) shows that this is indeed the case, because the maturation of DC2.4 cells expressing wild-type Nef in response to treatment with an anti-CD40 Ab was indeed inhibited in comparison to cells transfected with the control, empty vector.

What are the implications of our data for the process of HIV-1 infection? There is a clear advantage to the virus in reducing the ability of infected DC to present Ag; however, in terms of spread of virus from the periphery, it would seem counterintuitive to maintain infected iDC in a state of immaturity. However, it is possible that the increased proliferative capacity combined with reduced surface expression of costimulatory markers such as CD80, CD86, and ICAM-1 could lead to induction of T cell tolerance to viral Ags in the early stages of infection. This may be of paramount importance in preventing acute clearance of the virus and thus allowing establishment of chronic infection. Clearly, the interactions between HIV and DC are complex, the virus must both reduce the Ag presentation ability of infected DC while at the same time exploiting the ability of DC to traffick from the periphery to lymph nodes. Our data, coupled with studies from other laboratories, (4, 6, 28) are consistent with a key role for Nef in this process.

	Acknowledgments

 
We thank Kalle Saksela (University of Tampere, Tampere, Finland) for the gift of the PAK2 expression constructs. We are grateful to the Centralized Facility for AIDS Reagents at National Institute of Biological Standards and Control (Potters Bar, U.K.) for the provision of the anti-Nef mAb, ARP3026.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from the European Union Fifth Framework (QLK2-CT-2000-01630; to M.H. and D.A.M.).

² Address correspondence and reprint requests to Dr. Mark Harris, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K. E-mail address: mharris{at}bmb.leeds.ac.uk

³ Abbreviations used in this paper: HIV-1, HIV type 1; DC, dendritic cell; iDC, immature DC; PxxP, conserved polyproline; SH3, Src homology 3; HA, hemagglutinin.

Received for publication March 30, 2005. Accepted for publication September 6, 2005.

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