Lentiviral Nef Proteins Utilize PAK2-mediated Deregulation of Cofilin

JOURNAL OF VIROLOGY, Apr. 2010, p. 3935–3948 Vol. 84, No. 80022-538X/10/$12.00 doi:10.1128/JVI.02467-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilinas a General Strategy To Interfere with Actin Remodeling�†

Bettina Stolp,1 Libin Abraham,1,2 Jochen M. Rudolph,1 and Oliver T. Fackler1*Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany,1 and

The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, Heidelberg University,Im Neuenheimer Feld 282, 69120 Heidelberg, Germany2

Received 23 November 2009/Accepted 31 January 2010

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simianimmunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2(HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellularkinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severingfactor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, theefficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting usto evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis ofa total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actinremodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note,even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrityof a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These resultsestablish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highlyconserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical forNef’s activity in vivo.

The Nef protein is a 25- to 35-kDa myristoylated accessorygene product that is exclusively expressed by the lentiviruseshuman immunodeficiency virus type 1 (HIV-1), HIV-2, andsimian immunodeficiency virus (SIV). Largely dispensable forvirus replication in ex vivo cell cultures, Nef is critical forproduction of high virus titers and disease progression in theinfected host (7, 14, 16). Moreover, isolated expression of Nefin transgenic mice is sufficient to establish AIDS-like depletionof CD4� T lymphocytes (13). Nef manipulates host cell vesic-ular transport and signal transduction processes via numerousprotein interactions with host cell factors, including compo-nents of the endocytic sorting and T-cell receptor (TCR) sig-naling machineries (22). Together, these activities presumablyprevent immune recognition of virally infected cells and boostviral replication (8, 34, 42). While many protein interactionshave been described for Nef and the respective interactionmotifs were mapped (11), the in vivo relevance of individualNef ligands remains to be demonstrated.

One of the first protein interactions described for Nef was aserine-threonine kinase activity that can be immunoisolatedwith Nef (37) from Nef-expressing cells, but also from HIV-1-infected T lymphocytes and macrophages, as well as fromHIV-1 provirus transgenic mice (5, 37, 43). This activity wasidentified as p21-associated kinase 2 (PAK2) (27, 33), a mem-

ber of the PAK kinase family. PAKs are effectors of RAC andCDC42 GTPases, whose physiological roles include the reg-ulation of cytoskeletal rearrangements, cell motility, tran-scription, and cell cycle progression as well as cell death andsurvival (4). Nef-PAK2 association results in the autophos-phorylation of the kinase and the phosphorylation of a yet-unidentified 72-kDa substrate (27, 37). The association of Nefwith PAK2 occurs within a highly active kinase subpopulationin the context of a labile multiprotein complex about onemegadalton in size (10) that is specifically assembled in lipidmicrodomains (18, 32). The full composition of this complexremains to be determined; however, besides Nef, PAK2, andthe unidentified substrate, the RAC1 GTPase and its guanineexchange factor VAV1 are known components of this proteincomplex (20, 31, 36).

Even though residues in Nef known to be important for itsinteraction with PAK2, such as Nef’s N-terminal myristoyl-ation site (G2; numbering according to HIV-1SF2 Nef) and thePxxP (P76/P79) motif within the SH3 binding domain arelargely conserved among primary lentivirus isolates (see Fig.S1 in the supplemental material), it remains unclear whetherthis interaction plays a physiological role for Nef action in theinfected host. One reason for this controversy is the conflictingresults obtained in studies with SIV-infected macaques, whichrevealed no or strong selective pressure on the PxxP motif inSIV Nef in vivo (6, 15, 19, 36). Moreover, although the inter-action of Nef with PAK2 activity is conserved for many Nefproteins derived from HIV-1, HIV-2, and SIV strains, theefficiency of Nef-PAK2 association varies greatly and is difficultto detect for some nef alleles (2, 17, 21, 30, 32). Finally, intro-ducing mutations into Nef determinants for PAK2 associationgenerally affects Nef activities beyond PAK2 association, ren-

* Corresponding author. Mailing address: Department of InfectiousDiseases, Virology, University of Heidelberg, INF 324, 69120 Heidel-berg, Germany. Phone: 49-6221-561322. Fax: 49-6221-565003. E-mail:[email protected].

† Supplemental material for this article may be found at http://jvi.asm.org/.

� Published ahead of print on 10 February 2010.

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dering the interpretation of results obtained with such Nefmutants difficult. This situation was significantly improved bythe identification of a complex PAK2 recruitment surface inHIV-1 Nef (1, 28). Mutation of a critical phenylalanine (F191and F195 in Nef from HIV-1 NL4-3 and SF2, respectively; seeFig. S1 in the supplemental material) specifically disruptsPAK2 association, presumably due to its role in the recruit-ment of VAV1 into Nef-PAK2 containing membrane microdo-mains. This Nef mutant also revealed that PAK2 association isnot involved in Nef activities in receptor transport (28, 31) andis largely dispensable for effects of HIV-1 Nef on enhancementof virion infectivity and virus replication (39). The equivalentof this HIV-1 Nef phenylalanine motif HIV-2 and SIV Nefproteins remains to be identified. In line with the cellularfunction of PAKs, Nef was suggested to modulate HIV-1 tran-scription, cell survival, and actin organization via its associationwith PAK2 (12, 20, 44), but a specific underlying mechanismhas not been described.

Based on results with Nef F195A as well as upon RNA inter-ference (RNAi)-mediated depletion of endogenous PAK2, werecently demonstrated that HIV-1SF2 Nef uses its associationwith PAK2 to induce a potent block to T-lymphocyte actinpolymerization after chemokine treatment and thereby impairschemotaxis of Nef-positive cells (40). Nef achieves this by re-directing PAK2 toward phosphorylation of the tightly con-trolled actin-severing factor cofilin that contributes to actinremodeling by providing F-actin monomers as substrate fornew filaments (3, 40). Hyperphosphorylation of cofilin in thepresence of Nef thus inactivates cofilin, resulting in markedreduction of actin turnover (40). Given the high variability ofNef-PAK2 association efficiency among Nef proteins, we won-dered whether use of the mechanism we established for Neffrom HIV-1SF2 is a general feature of lentiviral Nef proteins.In the present study, we therefore performed a comprehensiveanalysis for a total of 17 nef alleles and tested their ability tointerfere with chemokine-induced actin remodeling and withthe deregulation of cofilin and the potential requirement forPAK2 association in these processes.

MATERIALS AND METHODS

Expression constructs, reagents, and antibodies. Expression constructs forgreen fluorescent protein (GFP) fusion proteins of HIV-1SF2 Nef and its mutantsand yellow fluorescent protein (YFP)-tagged Nef variants were already describedand were constructed by cloning PCR fragments of the respective nef genes intopEGFP-N1 (Clontech) under the control of the cytomegalovirus (CMV) pro-moter (12, 35, 40). The scrambled (5�-AGGUAGUGUAAUCGCCUUGUU-3�)and PAK2-specific (1, 5�-AGAAGGAACUGAUCAUUAAUU-3�; 2, 5�-GAAACUGGCCAAACCGUUAUU-3�) small interfering RNAs (siRNAs) were al-ready described (12, 35, 40). The expression construct for HIV-1NA7 NefF191I.GFP was generated by quick-change PCR from the wild-type (wt) plasmidusing a QuikChange II XL site-directed mutagenesis kit (Stratagene) accordingto the manufacturer’s instructions using the following primers: 5�-GACAGCCGCCTAGCATTACATCACATGGCCCGA-3� and 5�-TCGGGCCATGTGATGTAATGCTAGGCGGCTGTC-3�. The proviral constructs used are based onHIV-1NL4-3 and lack Nef expression (�Nef) or encode Nef from HIV-1SF2/NA7

(9, 40). The proviral constructs encoding the NA7 Nef or its F191I mutant weregenerated in analogy as described previously (9). The proviral constructs encod-ing HIV-1 NL4-3 internal ribosome entry site (IRES) GFP harboring the variousNef variants in their nef gene locus or lacking Nef expression (�Nef) weredescribed earlier (35, 38).

Reagents used were as follows: [�-32P]ATP (Hartmann Analytic), recombi-nant HIV-1 capsid (CA) (gift from Vanda Bartonova and Hans-Georg Krauss-lich), CCL-19 and CCL-21 (R&D Systems), recombinant cofilin (tebu-bio),Hoechst 33258 (Invitrogen), human recombinant interleukin-2 (IL-2; biomol),

Polybrene (Sigma), poly-L-lysine (Sigma), Mowiol 4-88 reagent (Calbiochem),phalloidin-TRITC (tetramethyl rhodamine isocyanate; Sigma), phytohemagluti-nin (Sigma), and stromal-derived factor 1� (SDF-1�; Immunotools).

Antibodies were as follows: rabbit anti-Ser3 phosphorylated cofilin (77G2)(Cell Signaling), fluorescein isothiocyanate (FITC)-labeled mouse anti-HIV-1p24CA KC57FITC (Beckman Coulter), allophycocyanin (APC)-labeled mouseanti-human CD184 (CXCR4) (BD Pharmingen), mouse anti-GFP antibodyclone GFP-20 (Sigma), rabbit anti-GFP antibody (12), mouse anti-myosin lightchain (anti-MLC) clone MY-21 (Sigma), sheep anti-Nef (9), rabbit anti-PAK2(abcam), mouse anti-transferrin receptor (TfR) clone H68.4 (Zymed), mouseanti-alpha-tubulin antibody clone B-5-1-2 (Sigma), and goat anti-rabbit IgGAlexa Fluor 568 (Invitrogen).

Cell lines, culture, transfection, immunostaining, and microscopy. Jurkat TAgand Jurkat-CCR7 represent both Jurkat E6.1-derived cell lines. The easy-to-transfect Jurkat TAg cells express the large T antigen of simian virus 40 (SV40)and were grown in RPMI 1640 medium supplemented with 10% fetal calf serum(FCS), 100 U/ml penicillin, and 100 �g/ml streptomycin (all from Gibco). Jurkat-CCR7 cells were a gift from Scott R. Struthers (University of California, SanDiego, CA) (29) and were maintained in Dulbecco’s modified Eagle (DME)medium supplemented with 10% FCS, 100 U/ml penicillin, 100 �g/ml strepto-mycin, 1� nonessential amino acids, 1� sodium pyruvate (all from Gibco), and0.65 �M �-mercaptoethanol (Sigma). Compared to Jurkat TAg cells, Jurkat-CCR7 cells exhibit low background actin remodeling and chemotaxis activity inthe absence of chemokine and thus increased responsiveness to chemokinetreatment. 293T cells were maintained in DME medium with high glucose,supplemented with 10% FCS, 100 U/ml penicillin, and 100 �g/ml streptomycin(all from Gibco). For transfection, 1 � 107 Jurkat T lymphocytes were electro-porated using 30 to 60 �g of plasmid DNA (960 �F for Jurkat TAg and 850 �Ffor Jurkat E6-1 and Jurkat CCR7, 250 V; Bio-Rad Genepulser). Immunofluo-rescence stainings were performed as previously described (40). Briefly, forstaining of F-actin (0.5 �g/ml phalloidin-TRITC), cells were fixed for 15 min with3% paraformaldehyde (PFA)/phosphate-buffered saline (PBS), permeabilizedwith 0.1% Triton X-100/PBS for 2 min and subsequently blocked for unspecificbinding with 1% bovine serum albumin (BSA)/PBS for 15 min. HIV-1-infectedcells were fixed for 90 min and additionally stained against HIV-1 p24CA (1:100)in cases in which no IRES GFP viruses were used. Hoechst 33258 was used in aconcentration of 1 ng/ml and added to the secondary antibody (1:2,000). Forstaining of phosphocofilin (p-cofilin) (1:50), all solutions, dilutions, and washsteps were done with Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl [pH7.5]). Blocking was performed for 30 min, and incubation was performed with thefirst antibody overnight at 4°C.

Confocal pictures of p-cofilin in Jurkat T lymphocytes were acquired using aZeiss LSM 510 Axiovert microscope and LSM Meta software. Relative meanpixel intensities of p-cofilin levels for single cells were quantified as describedbefore (40) using ImageJ. Pixels of sum intensity projections of single cells werequantified after background subtraction. Confocal stacks (0.3-�m stack size) ofF-actin in Jurkat T lymphocytes were acquired using a Leica TCS SP5 mi-croscope and LAS AF software. Images were processed using Adobe Photo-shop CS3.

T-lymphocyte chemotaxis assay. Chemotaxis assays were performed with threeindependent transfections per experiment. At 24 h posttransfection, cells werestarved in medium containing 0.5% FCS and incubated for another 4 h. Trans-well inserts (5-�m pores, 24-well plates, Costar3421; Corning) were equilibratedovernight. The bottom chamber of the transwell was filled with 450 �l of starvingmedium containing 10 ng/ml or no SDF-1�. Transfected cells (1 � 106) wereresuspended in 100 �l starving medium and were loaded to the upper side ofeach transwell. Total cell numbers and transfection efficiencies were determinedfrom another 100-�l aliquot of the identical cell suspension. Cells were allowedto chemotax for 2 h at 37°C before cells in the lower chamber were collected andanalyzed by flow cytometry (FACScalibur; BD) for 1 min with monitoring ofGFP-expressing cells. Typically, 30 to 60% or 1 to 5% of all cells chemotaxed inthe presence or absence of SDF-1�, respectively. The percentage of GFP-posi-tive cells migrated relative to transfection efficiency was calculated to addressinhibitory effects.

Flow cytometry analysis of cell surface CXCR4. Flow cytometry analysis wasperformed at 24 h posttransfection. T lymphocytes were stained for 1 h on iceusing APC-conjugated anti-human CXCR4 antibody (1:20 in PBS) and washedwith PBS once, prior to analysis of 5,000 GFP/YFP-positive cells by flow cytom-etry (FACScalibur; BD). Cell surface levels of CXCR4 were calculated relativeto those in untransfected cells in each sample, and the GFP control was set to100%.

IVKA. Standard in vitro kinase assays (IVKA) were essentially performed asdescribed previously (31). Briefly, Jurkat T lymphocytes were transfected with

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expression plasmids for Nef.GFP/YFP. After 24 h, cells were lysed in KEB (137mM NaCl, 50 mM Tris/HCl [pH 8], 2 mM EDTA, 0.5% Nonindet P-40, andprotease inhibitors) supplemented with Na3VO4, and cleared lysates were im-munoprecipitated with a rabbit anti-GFP antibody. After intensive washing inKEB, the immunoprecipitates were resuspended in KAB (50 mM HEPES [pH8], 150 mM NaCl, 5 mM EDTA, 0.02% Triton X-100, 10 mM MgCl2) containing10 �Ci of [�-32P]ATP per reaction. After incubation for 10 min, samples werewashed, and bound proteins were separated by SDS-PAGE and subjected toautoradiography. The kinase signal was quantified relative to the SF2 nef alleleusing Quantity One (Bio-Rad). In the IVKA for cofilin phosphorylation by theNef-associated PAK2 complex, anti-GFP immunoprecipitates were coincubatedwith 3 �g recombinant cofilin or HIV-1 CA and 10 �Ci of [�-32P]ATP inrecombinant PAK2 buffer (60 mM HEPES [pH 7.4], 1.2 mM dithiothreitol[DTT], 50 �g/ml PEG 20000, 3 mM MgCl2, and 3 mM MnCl2 [pH 7.4], withNaOH) for 30 min at 30°C and separated by SDS-PAGE without having beenwashed previously. Gels were fixed, stained with Coomassie brilliant blue, dried,and subjected to autoradiography. In order to optimize assay conditions for thedetection of kinase signals for weakly PAK2-associating nef alleles, IVKA wasperformed using either KAB or recombinant PAK2 buffer with or without wash-ing after the kinase reaction in the presence or absence of cofilin as a substrateat different temperatures and incubation times. Best results were obtained byperforming the kinase reaction using recombinant PAK2 buffer for 30 min at30°C in the absence of an exogenous substrate and without subsequent washing.

Lymphocyte ruffling assay, virus production, and infection. Lymphocyte ruf-fling assay, virus production, and infections were essentially performed as de-scribed previously (12, 23, 40). Jurkat-CCR7 cells were spin-infected (1 h, 2,300rpm) with 1 �g virus per 5 � 106 cells in the presence of Polybrene (1:1,000).Medium was exchanged after 6 h, and cells were harvested after another 36 h.Transfected Jurkat T lymphocytes were harvested 24 h after transfection withNef expression plasmids. Cells were seeded onto poly-L-lysine-coated coverglasses for 5 min at 37°C and either fixed directly for staining of p-cofilin orincubated with 200 ng/ml SDF-1� or solvent control for another 20 min at 37°Cfor the analysis of membrane ruffling before fixation. Infected peripheral bloodlymphocytes (PBLs) were incubated in fresh complete RPMI for 60 min at 37°Con day 4 postinfection, and cells were subsequently seeded onto poly-L-lysine-coated cover glasses and treated either as described above or with 200 ng/mlCCL-19/CCL-21. In the absence of a chemokine, usually 5 to 20% of the cellsshowed membrane ruffling. Quantifications were performed using an OlympusIX81 microscope with cellM software. At least 100 GFP/p24-CA-positive cellswere manually counted for each condition. Cells were counted as ruffling if atleast one lamellipodium was present on the cell surface and as containing highp-cofilin levels when they were visibly brighter than untransfected neighboringcells.

Statistical evaluation and software. Statistical significance was calculated byperforming Student’s t test (***, P 0.0005; **, P 0.005; *, P 0.05).Correlations were calculated using a Pearson correlation analysis. Alignment ofNef protein sequences was performed using Geneious Pro 4.8.3 (Biomatters)software.

RESULTS

Inhibition of actin remodeling and cell motility as well asderegulation of cofilin is conserved among lentiviral Nefproteins. We recently reported that Nef from HIV-1SF2 (SF2Nef) inhibits SDF-1�-induced actin remodeling/membraneruffling and chemotaxis as well as induces the hyperphosphor-ylation of cofilin (40). In a first series of experiments, we set outto test whether these represent generally conserved Nef activ-ities. To this end, we made use of a panel of previously exten-sively characterized GFP/YFP fusion proteins consisting of atotal of 17 Nef proteins from HIV-1, HIV-2, and SIV (for thesequence alignment, see Fig. S1 in the supplemental material)(35, 38). As described before (40), transient expression of SF2Nef.GFP but not a GFP control potently inhibited the ability ofJurkat T lymphocytes to undergo dynamic actin rearrange-ments and membrane ruffling in response to SDF-1� (Fig. 1Aand B). In the absence of a chemokine (ctrl), membrane ruf-fling was significantly less pronounced, and Nef also reduced

this background ruffling activity (Fig. 1B) (for images, see Fig.S2 in the supplemental material). Most Nef variants analyzedwere similar to SF2 Nef.GFP in potency for interfering withSDF-1�-induced membrane ruffling, with the exception of theNef protein from SIVcpz Tan3 (Tan3 Nef), which displayed noinhibitory activity (Fig. 1A and B). Very similar results wereobtained when the ability of Nef to inhibit chemotaxis towardSDF-1� was analyzed in a transwell chemotaxis assay. All nefalleles potently inhibited chemotaxis, with the exception ofTan3 Nef and SIVcpz sab1 Nef, the latter only marginally miss-ing statistical significance (P 0.051) (Fig. 1C). In parallel, theability of these nef alleles to downregulate CXCR4 from thecell surface was analyzed in order to test whether downregu-lation of this chemokine receptor determines the ability of Nefto block the response to chemokine treatment. In line with ourprevious findings (23, 24), comparably moderate reduction ofCXCR4 cell surface exposure was induced by all nef alleles butnot by the SF2 Nef AxxA (prolines 76 and 79 mutated toalanine) negative control (Fig. 1D). The overall correlationbetween CXCR4 downregulation and inhibition of membraneruffle formation (R2 0.4381) or chemotaxis (R2 0.3436)was weak. In turn, inhibition of membrane ruffling and ofchemotaxis exhibited strong correlation with each other (R2 0.7208).

We next analyzed the ability of Nef variants to induce highlevels of phosphorylated, inactive cofilin using a single cell-based immunofluorescence quantification established previ-ously (40) (for low-magnification micrographs, see Fig. S3 inthe supplemental material). These analyses revealed that ex-pression of all Nef proteins except Tan3 Nef markedly aug-mented the frequency of Jurkat T lymphocytes with high levelsof p-cofilin (Fig. 2A and B). Again, a strong correlation be-tween frequency of cells with high p-cofilin levels and inhibi-tion of membrane ruffling (R2 0.8194) or chemotaxis (R2 0.7417), but not CXCR4 downregulation (R2 0.4826), wasobserved. To validate these findings in the context of HIVinfection and thus with nonfusion Nef proteins expressed atphysiological levels, Jurkat T lymphocytes were infected withHIV-1NL4-3 IRES GFP viruses harboring the various nef allelesin their nef gene locus or lacking Nef expression due to pointmutations in the nef gene (�Nef). Analysis of membrane ruffleformation upon SDF-1� treatment (Fig. 3A and B), CXCR4downregulation (Fig. 3C), and deregulation of cofilin (Fig. 3Dand E) closely matched the results obtained by transient trans-fection. These results establish inhibition of SDF-1�-inducedactin remodeling and chemotaxis, as well as cofilin deregula-tion as highly conserved activities of lentiviral Nef proteins.

Detection of PAK2 association is variable among lentiviralNef proteins. Since efficient inhibition of actin remodeling aswell as cofilin deregulation by SF2 Nef requires its ability toassociate with the cellular kinase PAK2 (12, 40), we next an-alyzed the degree of PAK2 association for our panel of Nefproteins. Following standard protocols (31, 37), Nef was im-munoisolated from transiently transfected Jurkat T lympho-cytes and subsequently subjected to an IVKA to analyze asso-ciated PAK2 activity (Fig. 4A). Expectedly, SF2 Nef wild-type(wt) but not its AxxA (P76/79A) mutant efficiently associatedwith autophosphorylating PAK2 (62 kDa; p-PAK2) as well aswith variable amounts of the phosphorylated 72-kDa substrate(p72). However, Nef-PAK2 association was highly variable

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FIG. 1. Inhibition of chemokine-induced membrane ruffling and chemotaxis is a conserved feature of lentiviral Nef proteins. (A) SDF-1�-induced actin ruffle formation is inhibited by various nef alleles. Representative micrographs of Jurkat T lymphocytes (Jurkat CCR7) transientlyexpressing the indicated GFP- or YFP-fused Nef proteins were fixed 20 min after treatment with 200 ng/ml SDF-1� and stained for F-actin. For


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among our panel of Nef variants, including some that failed todisplay any detectable association with PAK2 activity underthese experimental conditions. Furthermore, the extent ofPAK2 association did not correlate with Nef’s effects on actinremodeling and cofilin deregulation. Several nef alleles (HIV-1NL4-3 Nef, HIV-1NA7 Nef [NA7 Nef], SIVmon Val Nef, HIV-2Ben Nef, HIV-2Cbl-23 Nef, and SIVblue Nef) showed no or onlyweak PAK2 association yet were highly active in inhibition ofF-actin-rich membrane ruffle formation and induction of cofi-lin hyperphosphorylation (Fig. 1, 2, and 4A). Some of these nefalleles were previously analyzed for PAK2 association, withlargely consistent results (2, 17, 21, 30). For some of the allelesthat displayed marginal PAK2 association in our experimentalsystem, such as HIV-2Cbl23 Nef, robust PAK2 association wasdetected when Nef was coexpressed with a constitutive activevariant of CDC42 (30). Since this approach failed to increasespecific detection of Nef-associated PAK2 activity in our ex-perimental system (data not shown), we sought to enhance thesensitivity of our assay system by alternative means. NA7 Nefwas selected as an example for non-PAK2-associating Nefs andcompared to SF2 Nef. By extending the time for the kinasereaction, we were able to detect a slight association of NA7 Nefwith PAK2 activity (Fig. 4B). This association was, however,much less pronounced than for SF2 Nef even though both nefalleles were comparably active in inhibiting actin remodeling(Fig. 1A and Fig. 4B). However, the low levels of PAK2 asso-ciation of NA7 Nef required, in analogy to SF2 Nef, the phe-nylalanine recruitment motif in the Nef C terminus (Fig. 4B,NA7 Nef F191I mutant).

We next sought to address whether low levels of PAK2association as observed with NA7 Nef could be functionallyrelevant. Since SF2 Nef-associated PAK2 complexes can phos-phorylate cofilin (40), we compared SF2 and NA7 Nef immu-noprecipitates for their ability to phosphorylate cofilin in vitro.Surprisingly, we found that the two nef alleles were comparablypotent in mediating cofilin phosphorylation (Fig. 4C). Therespective F195/F191 Nef mutants served as specificity controlsand did not display cofilin hyperphosphorylation. Importantly,we also noted a pronounced accumulation of higher-molecu-lar-weight phosphoproducts under these experimental condi-tions for both SF2 and NA7 Nef (Fig. 4C, bracket). Eventhough individual bands were not resolved on these gels, whichwere designed to detect p-cofilin, this accumulation encom-passed the area where p-PAK2 is typically detected, raising thepossibility that these kinase assay conditions could be suitablefor detection of low-affinity Nef-PAK2 associations (Fig. 4C,compare lanes 3 and 5). Systematic comparison between theassay conditions used in Fig. 4A and C revealed that ratherthan the presence of cofilin, the nature of the buffer, the

incubation time, the temperature, and the omission of exten-sive washing steps caused the increase in sensitivity of detec-tion of Nef-associated PAK2 activity (Fig. 4D). Even thoughthe omission of a washing step resulted in a marked increase ofunspecific background signal, the typical specific kinase reac-tion products (p-PAK2 and p72) could be detected for NA7Nef. Consistently, when theses modified in vitro kinase condi-tions were applied to Nef immunocomplexes containing sev-eral Nef variants that previously displayed poor or undetect-able PAK2 association (Fig. 4A), moderate and variable levelsof PAK2 association could be observed (Fig. 4E). We con-cluded that all Nef proteins analyzed here are in principle ableto associate with PAK2 activity, although with greatly variedlevels of efficiency.

HIV-1NA7 Nef requires a PAK2 recruitment motif for inhi-bition of actin remodeling and deregulation of cofilin. In thecase of SF2 Nef, inhibition of actin remodeling and inductionof cofilin hyperphosphorylation are a direct consequence ofPAK2 association (40). Since these activities were well con-served among lentiviral Nef proteins, we next asked whetherPAK2 association is functional even for those nef alleles withlow detectable PAK2 association. We selected NA7 Nef as anexample for a Nef variant that potently disrupts SDF-1�-in-duced actin remodeling and induces cofilin hyperphosphoryla-tion despite weak PAK2 association and again made use of theF195/191I mutation, which specifically disrupts the ability ofNef to associate with PAK2 activity (Fig. 4B). A comparison ofwt and F/I mutant SF2 and NA7 Nef proteins in the SDF-1�-induced membrane-ruffling assay revealed that both wt Nefproteins potently interfered with actin ruffle formation andinduced high p-cofilin levels. In contrast, both of the F/I mu-tants failed to interfere with actin remodeling (Fig. 5A and B)and were impaired in inducing cofilin hyperphosphorylation(Fig. 5C and D). For cofilin, this effect was most apparent ona single-cell level, where pixel quantification demonstrated a 3-to 4-fold reduction in p-cofilin levels compared to those for wtNef proteins (Fig. 5E). To extend these findings to the contextof HIV infection, primary human PBLs were infected withHIV-1NL4-3 harboring either HIV-1SF2 or HIV-1NA7 nef intheir nef gene locus or lacking Nef expression due to pointmutations in the nef gene (�Nef). Membrane ruffling inducedby treatment with the CXCR4 ligand SDF-1� or the CCR7ligands CCL-19 and CCL-21 was potently blocked by infectionwith viruses expressing wt, but not mutant, Nef proteins orlacking Nef expression. Nef proteins from HIV-1NA7 and HIV-1SF2 were indistinguishable in these analyses (Fig. 6A and B),and cofilin deregulation correlated well with the inhibition ofactin remodeling (Fig. 6C and D). These results highlight thatthe two nef alleles depend to a comparable degree on the

representative micrographs of control cells treated with medium instead of SDF-1�, see Fig. S2 in the supplemental material. Shown are mergepictures of the GFP/YFP and F-actin channel. Scale bar 10 �m. (B) Frequency of the cells shown in panel A that display membrane rufflingin response to treatment with SDF-1� or a medium control (ctrl). Depicted are mean values from three independent experiments � standarddeviations (SD) with at least 100 cells analyzed per condition. P values were calculated relative to the GFP control. (C) Chemotaxis toward SDF-1�is inhibited by various nef alleles. The cells shown in panel A were subjected to a transwell chemotaxis assay. Depicted is the percentage ofGFP-positive cells that migrated toward 10 ng/ml SDF-1� over 2 h. Values are the means with standard errors of the means (SEM) from threeexperiments performed in triplicate. (D) CXCR4 is downregulated from the cell surface by various nef alleles. The cells shown in panel A werestained for cell surface CXCR4 and analyzed by flow cytometry. Shown are mean values from three experiments � SD, with 5,000 GFP- orYFP-positive cells analyzed in each experiment. ***, P 0.0005; **, P 0.005; *, P 0.05.


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conservation of the PAK2 recruitment motif encompassing theF195/191 residue for interference with actin remodeling andcofilin deregulation.

Lentiviral Nef proteins depend on endogenous PAK2 forefficient interference with actin dynamics and cofilin activity.To directly address the involvement of endogenous PAK2 inthe effects of Nef on the actin cytoskeleton, we used two in-dependent RNAi oligonucleotides to knock down PAK2 ex-pression in Jurkat T lymphocytes. We previously establishedthat potent reduction of PAK2 protein levels in these cells

caused a marked, but incomplete, decrease in Nef-associatedPAK2 activity in the presence of SF2 Nef that correlated wellwith the partial rescue of membrane ruffling and cofilin dereg-ulation under these conditions (31, 40) (Fig. 7). PAK2 knock-down did not affect membrane ruffling or cofilin phosphoryla-tion in the absence of Nef, confirming that PAK2 per se isdispensable for these cellular functions (40). In stark contrast,depletion of PAK2 markedly enhanced membrane ruffling inthe presence of SF2 and NA7 Nef (Fig. 7A to C). Similarly, thefrequency and magnitude of cofilin hyperphosphorylation in

FIG. 2. Induction of cofilin hyperphosphorylation is a conserved feature of lentiviral Nef proteins. (A) Representative micrographs ofJurkat T lymphocytes (Jurkat TAg) transiently expressing the indicated GFP- or YFP-fused Nef proteins. Cells were plated onto coverglasses, fixed, and stained for p-cofilin. Identical fields of view are shown for GFP/YFP, p-cofilin, and merge channels. Arrows indicateGFP/YFP positive cells. Scale bar 10 �m. (B) Frequency of the cells shown in panel A with high p-cofilin levels. Depicted are mean valuesfrom three independent experiments � SD, with at least 100 cells analyzed per transfection; cells were scored as containing high p-cofilinlevels when they were visibly brighter than untransfected neighboring cells. P values are calculated relative to the GFP control.


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the presence of SF2 and NA7 Nef were significantly reducedupon PAK2 knockdown (Fig. 7D to G), confirming that thetwo nef alleles equally require PAK2 to exert their inhibitoryeffects on actin remodeling. With HIV-1NL4-3 Nef, SIVmon Val

Nef, HIV-2Cbl23 Nef, SIVmac239 Nef, SIVagm tan1 Nef, andSIVagm sab1 Nef, very similar findings were made for severaladditional nef alleles with mostly weak PAK2 association butpotent inhibition of actin remodeling: upon PAK2 knockdown,all of these nef alleles displayed a reduction in their ability tointerfere with actin ruffle formation and to induce cofilin hy-perphosphorylation that was comparable to that of SF2 Nef(Fig. 8). In contrast, silencing of PAK2 expression did notaffect CXCR4 cell surface levels, ruling out elevated receptorsurface exposure as a reason for this increase in SDF-1� re-sponsiveness (data not shown). On the other hand, no effect ofthe PAK2 knockdown could be observed for SIVcpz Tan3 Nef,which does associate with PAK2 activity but is defective in all

PAK2-dependent Nef functions (Fig. 8). Together, these re-sults demonstrate that irrespective of the magnitude of de-tected PAK2 association in vitro, Nef requires endogenousPAK2 to efficiently block membrane ruffling and to inducecofilin inactivation in intact HIV-1 target cells. We concludethat hijacking PAK2 function is a highly conserved activity oflentiviral Nef proteins.

DISCUSSION

This study reveals that the ability of Nef to hijack PAK2 forits purposes is highly conserved among Nef proteins fromHIV-1, HIV-2, and SIV. In total, 17 nef alleles were comparedfor their ability to interfere with chemokine-induced mem-brane ruffle formation and chemotaxis, deregulation of cofilin,downregulation of CXCR4 from the cell surface, and associa-tion with PAK2 activity. With the exception of PAK2 associa-

FIG. 3. Various Nef variants inhibit membrane ruffle formation and induce cofilin hyperphosphorylation in the context of an HIV-1 infection.(A) Representative maximum projections of confocal Z stacks of Jurkat T lymphocytes (Jurkat-CCR7) infected with HIV-1NL4-3 IRES GFPcarrying the indicated nef alleles in its nef gene locus or its nef-deleted counterpart (�Nef). At 48 h postinfection, cells were treated with 200 ng/mlSDF-1� for 20 min, fixed, and stained for F-actin. Infected cells appear green due to IRES-driven GFP expression. Shown are merge pictures ofthe GFP and F-actin channels. Scale bar 10 �m. (B) Frequency of cells shown in panel A with membrane ruffles. Depicted are mean values fromthree experiments � SD, with at least 100 cells analyzed per condition. P values are calculated relative to HIV-1�Nef-infected cells. (C) CXCR4is downregulated from the cell surface by all nef alleles. The cells shown in panel A were stained for cell surface CXCR4 and analyzed by flowcytometry. Shown are mean values from three experiments � SD, with 5,000 GFP-positive cells analyzed in each experiment. (D) Cofilinderegulation in HIV-1 infection. Representative confocal micrographs of Jurkat T lymphocytes (Jurkat-CCR7) infected with HIV-1NL4-3 IRESGFP carrying the indicated nef alleles in its nef gene locus or its nef-deleted counterpart. At 48 h postinfection, cells were seeded onto cover glasses,fixed, and stained for p-cofilin. Infected cells appear green due to IRES-driven GFP expression and are indicated by arrows. Scale bar 10 �m.The p-cofilin and GFP panels show identical fields of view. (E) Frequency of cells shown in panel D with high p-cofilin levels. Depicted are meanvalues from three experiments � SD, with at least 100 cells analyzed per condition. Cells were scored as containing high p-cofilin levels when theywere visibly brighter than uninfected neighboring cells.


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tion, all these activities were found to be well conserved amonglentiviral Nef proteins. However, use of modified kinase assayprotocols revealed that all Nef variants analyzed associate withPAK2 activity, albeit to widely varied extents. Despite thesedifferences in in vitro PAK2 association, siRNA-mediatedknockdown of endogenous PAK2 and mutations in Nef thatdisrupt PAK2 association demonstrated that lentiviral Nef pro-teins require PAK2 to efficiently block actin cytoskeleton re-modeling and to inactivate cofilin. These data indicate thatlentiviruses have evolved the viral factor Nef to modify theactin cytoskeleton of their host cells, and the high conservationof this function supports the notion that Nef-PAK2-mediatedderegulation of cofilin may be an important aspect of Nef’sbiological properties in vivo.

In the panel of 17 nef alleles analyzed here, the SIVcpz Tan3

Nef protein behaved differently than all other Nefs analyzed.Surprisingly, this Nef protein was defective in interfering withactin remodeling despite robust association with PAK2 activ-ity. PAK2 association is therefore necessary but not sufficient

for Nef’s ability to interfere with host cell actin remodeling.This conclusion is also supported by the partial nature of therescue observed for other Nef proteins upon PAK2 silencing ormutation of the PAK2 recruitment motif. Even though residualNef-associated PAK2 activity persists upon PAK2 knockdown(12, 20, 44), these results suggest the involvement of additional,PAK2-independent mechanisms in the Nef-mediated inhibi-tion of T-lymphocyte chemotaxis. These may directly involvethe phenylalanine motif for which additional protein interac-tions beyond PAK2 complexes cannot be excluded. Moreover,SIVcpz Tan3 Nef failed to induce cofilin hyperphosphorylationdespite its ability to efficiently associate with PAK2 activity.Since this particular nef allele is also defective in lymphocyte-specific protein tyrosine kinase p56 (LCK) accumulation, inhi-bition of immunological synapse formation, and concomitanttyrosine phosphorylation events (35), this might indicate, e.g.,a mislocalization of this Nef protein per se or when in complexwith PAK2. Alternatively, the idea that kinases other thanPAK2 are typically present in Nef immunoisolates and mediate

IP: R

FIG. 4. Nef-PAK2 association in vitro for lentiviral Nef proteins. (A to E) Jurkat T lymphocytes (Jurkat TAg) expressing the indicated GFP- orYFP-fused Nef proteins were subjected to anti-GFP immunoprecipitation and subsequent in vitro kinase assay (IVKA). Nef-associated PAK2 activity isrevealed by the phosphorylated 62-kDa band (IVKA, p-PAK2). (A) Characterization of Nef proteins analyzed in Fig. 1 and 2 in IVKA. Nef-PAK2association was quantified relative to that of SF2 Nef (arbitrarily set to 1). Shown are results representative of those of three independent experimentsin which wt SF2Nef and SF2NefAxxA were always included as positive and negative control, respectively. (B) Nef-PAK2 association depends onF195/191. By increasing the incubation time of the IVKA to 10 min, weak but F191-specific Nef-PAK2 association can be detected for NA7 Nef. (C) SF2and NA7 Nef-associated PAK2 can phosphorylate cofilin. IVKA was performed using recombinant PAK2 buffer for 30 min at 30°C in the presence ofrecombinant cofilin or HIV-1 CA as substrate, and phosphorylated proteins were detected by autoradiography. Input levels of Nef, recombinant cofilin,and p24CA were detected by Western blotting and Coomassie staining, respectively. p-cofilin designates phosphorylated cofilin, and the bracket indicateshigh-molecular-weight phosphoproducts. (D) Optimization of kinase assay conditions to allow detection of a kinase signal for weakly PAK2-associatingNefs. IVKA conditions for every lane are indicated. (E) Weak association of all Nefs with PAK2 activity can be detected by performing the IVKA reactionin recombinant PAK2 buffer for 30 min at 30°C without subsequent washing.


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FIG. 5. NA7 Nef blocks actin remodeling and induces cofilin hyperphosphorylation in an F191-dependent manner. (A) Representativemicrographs of Jurkat T lymphocytes (Jurkat CCR7) transiently expressing the indicated GFP-fused wt or F195/191I Nef proteins were fixed 20min after treatment with 200 ng/ml SDF-1� and stained for F-actin. Scale bar 10 �m. F-actin and GFP panels show identical fields of view.Arrows indicate GFP-positive cells. (B) Frequency of the cells shown in panel A that display membrane ruffling in response to treatment withSDF-1�. Depicted are mean values from three independent experiments � SD, with at least 100 cells analyzed per condition. (C) Representativesum-intensity projections of confocal Z stacks of Jurkat T lymphocytes (Jurkat TAg) transiently expressing the indicated GFP-fused wt orF195/191I Nef proteins. Cells were plated onto cover glasses, fixed, and stained for p-cofilin. Scale bar 10 �m. p-cofilin and GFP panels showidentical fields of view. Arrows indicate GFP-positive cells. (D) Frequency of the cells shown in panel C with high p-cofilin levels. Depicted aremean values from three independent experiments � SD, with at least 100 cells analyzed per transfection; cells were scored as containing highp-cofilin levels when they were visibly brighter than untransfected neighboring cells. (E) Relative mean pixel intensity of the cells shown in panelC. Depicted are mean values � SD from at least 10 representative cells.


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FIG. 6. Inhibition of actin remodeling and induction of cofilin hyperphosphorylation are dependent on the Nef phenylalanine motif in the contextof HIV-1 infection. (A) Representative maximum projections of confocal Z stacks of PBLs infected with HIV-1NL4-3 encoding wt SF2 or NA7 Nef ortheir F195/191I mutants or its nef-deleted counterpart (�Nef). Cells were treated with 200 ng/ml SDF-1�, CCL-19, or CCL-21 for 20 min, fixed, andstained for intracellular p24CA and F-actin. The arrows indicate infected cells. Scale bar 10 �m. (B) Frequency of cells shown in panel A withmembrane ruffles. Depicted are mean values from triplicate infections � SD for two independent donors with at least 100 cells analyzed per condition.(C) Representative confocal micrographs of PBLs infected with HIV-1NL4-3 carrying either wt SF2 or NA7 Nef or their F195/191I mutants or itsnef-deleted counterpart (�Nef). Cells were seeded onto cover glasses, fixed, and stained for p24CA and p-cofilin. The arrows indicate infected cells. Scalebar 10 �m. (D) Frequency of cells shown in panel C with high p-cofilin levels. Depicted are mean values from triplicate infections � SD for twoindependent donors, with at least 100 cells analyzed per condition; cells were scored as containing high p-cofilin levels when they were visibly brighterthan uninfected neighboring cells. P values are calculated relative to results for HIV-1�Nef-infected cells.


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FIG. 7. Inhibition of membrane ruffle formation and induction of cofilin hyperphosphorylation by SF2 and NA7 Nef are similarly dependenton PAK2 expression. (A) Representative micrographs of Jurkat T lymphocytes (Jurkat CCR7) transfected with siRNA oligonucleotides specificfor PAK2 or a nonspecific scrambled siRNA (scr.) together with expression plasmids for GFP or wt SF2/NA7 Nef.GFP. Cells were fixed 20 minafter treatment with 200 ng/ml SDF-1� and stained for F-actin. Scale bar 10 �m. F-actin and GFP panels show identical fields of view.Arrows indicate GFP-positive cells. (B) Frequency of the cells shown in panel A that display membrane ruffling in response to treatmentwith SDF-1�. Depicted are mean values from three independent experiments � SD, with at least 100 cells analyzed per condition.(C) Western blot analysis of lysates of the cells used in panel A. TfR, transferrin receptor as loading control. (D) Representativesum-intensity projections of confocal Z stacks of Jurkat T lymphocytes (Jurkat TAg) transfected with siRNA oligonucleotides specific forPAK2 or a nonspecific scrambled siRNA (scr.) together with expression plasmids for GFP or wt SF2/NA7 Nef.GFP. Cells were plated ontocover glasses, fixed, and stained for p-cofilin. Scale bar 10 �m. p-cofilin and GFP panels show identical fields of view. Arrows indicateGFP-positive cells. (E) Western blot analysis of lysates of the cells used in panel D. (F) Frequency of the cells shown in panel D with highp-cofilin levels. Depicted are mean values from three independent experiments � SD, with at least 100 cells analyzed per transfection; cellswere scored as containing high p-cofilin levels when they were visibly brighter than untransfected neighboring cells. (G) Relative mean pixelintensity of the cells shown in panel D. Depicted are mean values � SD from at least 10 representative cells.


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cofilin phosphorylation but are not recruited by Tan3 Nefcannot be excluded. Finally, SIVcpz Tan3 Nef downmodulatedcell surface CXCR4 as potently as most other nef alleles with-out showing significant inhibition of membrane ruffle forma-tion or chemotaxis toward the CXCR4 ligand SDF-1�, high-lighting that the moderate and PAK2-independent cell surfacedownregulation of CXCR4 induced by Nef is insufficient toblock reactivity to this ligand. SIVcpz Tan3 Nef will serve as auseful tool in future analyses of the requirements for down-stream signaling of Nef-PAK2 to its substrates.

All other Nef variants tested inhibited chemokine-induced

membrane ruffle formation and induction of cofilin hyperphos-phorylation downstream of PAK2 to comparable extents de-spite variable efficiencies in PAK2 association in vitro. Thisapparent lack of correlation between functionality and avidityof Nef-PAK2 complexes indicates that individual Nef proteinsassemble Nef-PAK2 complexes in intact cells with comparableefficiency, but the stability of these complexes after cell lysisvaries dramatically. This implies that analyzing p-cofilin levelsin intact cells rather than in vitro association of Nef with PAK2activity may serve as a more-reliable mirror of functional Nef-PAK2 interactions. While differences obtained for Nef-PAK2

FIG. 8. PAK2 dependency of actin remodeling inhibition and deregulation of cofilin are conserved for several Nef variants with weak PAK2association. (A) Representative micrographs of Jurkat T lymphocytes (Jurkat CCR7) transfected with siRNA oligonucleotides specific for PAK2or a nonspecific scrambled siRNA (scr.) together with the indicated expression plasmids. Cells were fixed 20 min after treatment with 200 ng/mlSDF-1� and stained for F-actin. Shown are merge pictures of the GFP and F-actin channels. Scale bar 10 �m. (B) Western blot analysis of lysatesof the cells used in panel A. MLC, myosin light chain as loading control. (C) Frequency of the cells shown in panel A that display membrane rufflingin response to treatment with SDF-1�. Depicted are mean values from three independent experiments � SD, with at least 100 cells analyzed percondition. (D) Representative micrographs of Jurkat T lymphocytes (Jurkat CCR7) transfected with siRNA oligonucleotides specific for PAK2 ora nonspecific scrambled siRNA (scr.) together with the indicated expression plasmids. Cells were plated onto cover glasses, fixed, and stained forp-cofilin. Scale bar 10 �m. p-cofilin and GFP panels show identical fields of view, and the arrows point at transfected cells. (E) Frequency ofthe cells shown in panel D with high p-cofilin levels. Depicted are mean values from three independent experiments � SD, with at least 100 cellsanalyzed per transfection; cells were scored as containing high p-cofilin levels when they were visibly brighter than untransfected neighboring cells.


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association by IVKA should thus be interpreted with caution,this result does not exclude the possibility that in an isogeniccontext, detection of differences in Nef-PAK2 association canbe predictive for the functional outcome of this association.The reasons for the variable efficiency of detection for Nef-PAK2 association in vitro are not clear but could reflect intrin-sic differences in complex stability or turnover rates. Sinceneither inhibition nor activation of kinase or GTPase activityduring complex isolation significantly affected the detection ofPAK2 association (data not shown), resolving this question willrequire a more-detailed understanding of the composition andregulation of Nef-PAK2 complexes. Irrespective of the deter-minants of Nef-PAK2 complex stability, this parameter is notlimiting in intact cells where Nef-PAK2 association results inefficient phosphorylation and thus inactivation of the down-stream target cofilin.

Our analyses of the block of T-lymphocyte actin remodelingfollowing T-cell receptor and chemokine stimulation establishNef as a potent inhibitor of the formation of lamellipodium-like membrane protrusions (12, 35, 40). In sharp contrast, Nefwas recently reported to induce, rather than inhibit, filopodia-like, F-actin-rich protrusions in macrophages and T lympho-cytes (26, 45). This may indicate that Nef generally exertsopposing effects on lamellipodia and filopodia, reflecting theinvolvement of distinct signaling pathways that govern thesetwo types of cell protrusions. In line with this scenario, weoccasionally observed filopodia on T lymphocytes for whichNef blocked lamellipodium formation (e.g., Fig. 5A for HIV-1Nef wt). Finally, we cannot exclude the possibility that theeffects of Nef on cell morphology are distinct when analyzed invivo in their physiological environment.

Our previous (40) and the current study identify actin re-modeling by Nef as one critical effect involved in blockingT-lymphocyte chemotaxis. Based on observations of the SIVmonkey model and of B-cell dysfunction in AIDS (25, 41), weproposed a role of this inhibition in B-cell dysfunction ob-served for HIV-infected individuals, by inhibition of intra-lymph node motility of infected T cells. The herein-identifiedconservation of this Nef function across various lentivirusessupports such a model and indicates that cofilin-dependentinhibition of actin remodeling represents a major activity ofNef that may provide the virus with a powerful means tocircumvent the host’s humoral immune response. Such an ef-fect may not have been noted in previous infections of ma-caques with SIV viruses harboring nef genes with disruptedPAK2 interaction motives since they (i) primarily investigatedvirus replication, a parameter that is not directly affected byNef-PAK2 association, and (ii) employed SIV Nef mutantsthat carried pleiotropic defects rather than specific loss-of-function in PAK2 association. This warrants further in vivoanalysis using an equivalent of the F195A HIV-1SF2 Nef mu-tant that will include additional parameters, such as antibodyquantity and affinity as well as lymph node morphology andcomposition. With cofilin deregulation via PAK2 emerging asa conserved and critical activity of Nef, increased future effortswill be devoted to the analysis of complete regulation of thiscomplex and the pathophysiological consequences of this as-sociation in the HIV-infected host.

ACKNOWLEDGMENTS

We are grateful to Vanda Bartonova, Hans-Georg Krausslich, ScottR. Struthers, Michael Schindler, and Frank Kirchhoff for the kind giftof reagents and to Nadine Tibroni for expert technical help.

This project was supported financially by the Deutsche Forschungs-gemeinschaft (SFB638, project A11 to O.T.F.; GRK1188 fellowshipsto B.S.; Cellular Networks Ph.D. fellowship to L.A.).

O.T.F is a member of the CellNetworks Cluster of Excellence(EXC81).

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Documents

Lentiviral Nef Proteins Utilize PAK2-mediated Deregulation of Cofilin