HIV-1 Nef Hijacks Lck and Rac1 Endosomal Traffic To Dually Modulate Signaling-Mediated and Actin Cytoskeleton–Mediated T Cell Functions

Antigen recognition triggers T cell polarization toward the APC. This process involves the reorganization of the actin and microtubule cytoskeleton, the reorientation of intracellular vesicle traffic, and the generation of dynamic signaling and adhesion complexes at the T cell/APC contact site, termed the immunological synapse. Altogether, these processes control immunological synapse formation and function, ensuring T cell activation leading to T cell proliferation and differentiation and T cell effector functions like polarized secretion of cytokines and cytotoxic granules (1, 2).

TCR signal transduction involves the CD3γ, δ, ε, and ζ subunits, which contain, in their intracellular regions, ITAMs that are phosphorylated soon after TCR engagement. ITAM phosphorylation by the Src family protein tyrosine kinase Lck facilitates the recruitment of downstream effectors, including the tyrosine kinase ZAP70, which is recruited to phosphorylated ITAMs of TCRζ via its SH2 domains. This induces ZAP70 tyrosine phosphorylation and activation, the subsequent tyrosine phosphorylation of the signaling adaptors LAT and SLP76, and the recruitment of effectors to these adaptors. Altogether, these proteins form a signalosome necessary to proceed to downstream activation events, including the activation of phospholipase C–γ1 (PLCγ1), the generation of lipid second messengers, calcium flux, and the activation of serine/threonine kinases like MAP and protein kinase C (PKC) kinases. The coordinated action of these different signaling molecules drives the activation of transcription factors (e.g., NFAT, NF-κB, and AP1), which in turn, activate the expression of genes involved in T cell growth and differentiation and the production of cytokines, crucial for the development of adaptive immune responses (3, 4).

HIV-1 infects CD4⁺ T cells, subverting a variety of T cell physiological mechanisms. This facilitates the production of viral particles and their transmission to other cells, eventually leading to chronic viral infection while reducing the impact of the host immune defenses. HIV-1 genome encodes several “accessory” proteins that are key for the HIV-1 subversion of infected cell processes. Among them, Nef is crucial for in vivo viral replication and AIDS pathogenesis. Nef is abundantly expressed early postinfection and optimizes the intracellular environment to improve virus replication and reduce host immunity by modulating endosomal traffic, actin cytoskeleton components, and cell activation in infected T cells. As a consequence, HIV-1 infection modifies the expression of several T cell surface molecules, including CD4, CD28, and MHC class I and II (5); it alters cytoskeleton remodeling and its associated cellular events (6–14), and it modulates T cell activation by affecting various signaling pathways (15, 16). HIV-1 Nef interferes with Lck and LAT endosomal traffic to the immunological synapse, altering T cell activation (17–19). The action of Nef on these processes occurs through the presence of specific motifs in its primary sequence, allowing interactions with a number of cellular proteins. Nef may also modify some intracellular traffic pathways, resulting in the modulation of processes regulated by proteins transported through those pathways.

We and others have shown that the TCR and the signaling molecules Lck and LAT are associated with distinct endosomal and Golgi intracellular compartments. Their traffic to the immunological synapse is differentially regulated and is crucial for TCR signal transduction (20–30). Interestingly, we also observed that the GTPase Rac1, a key actin cytoskeleton regulator, is also associated with recycling endosomes that control Rac1 subcellular localization, its targeting to the immunological synapse, and its ability to regulate actin remodeling in T cells (31, 32).

In this study, we performed a systematic analysis of the potential interplay between HIV-1 Nef and the T cell activation molecular machinery by analyzing its capacity to control the subcellular localization and activation of signaling molecules downstream of the TCR and its consequence for T cell physiology. Our results show that Nef exerts a refined control of signaling and cytoskeleton regulators to modulate T cell activation and cytoskeleton mediated events.

Vectors encoding GFP, wild type (WT), and mutant GFP-tagged HIV-1 NL4-3 Nef (Nef/GFP and Nef PXXP/AXXA-GFP) were previously described (33, 34). WT and Nef-deleted (NL4-3–based) proviral plasmids (HIV-1 WT and HIV-1 ΔNef, respectively) have already been described (35, 36). pCMV/vesicular stomatitis virus (VSV)–G was a gift from R. Weinberg (37) (Addgene plasmid no. 8454).

Rab11 family interacting protein 3 (FIP3) was depleted with small interfering RNA (siRNA) duplexes based on human FIP3 (siFIP3) sequence described elsewhere: siFIP3-1 (5′-AAGGGATCACAGCCATCAGAA-3′) and siFIP3-2 (5′-AAGGCAGTGAGGCGGAGCTGTT-3′) (28, 31).

Virions were produced by the transient calcium phosphate DNA precipitation technique. HEK293T cells were transfected with 20 μg of proviral DNA. Seventy two hours later, supernatant was recovered, centrifuged, and cell free virion stocks were stored at −80°C. The concentration of p24 Ag in viral stocks was measured by a quantitative ELISA (PerkinElmer).

Abs and primer sequences are described in detail in the Supplemental Tables I and II.

Human peripheral blood T cells from healthy volunteers were obtained from the French National Blood Bank (Etablissement Français du Sang) and through the Institut Pasteur Clinical Investigation and Access to Biological Resources core facility (NSF96-900 certified, from sampling to distribution, reference BB-0033-00062/ICAReB platform/Institut Pasteur, Paris, France/BBMRI AO203/1 distribution/access: 2016, May 19th, [BIORESOURCE]), under the CoSImmGEn protocol approved by the Committee of Protection of Persons, Île de France-1, Paris, France (no. 2010-dec-12483). Informed consent was obtained from all subjects. PBMCs were isolated by centrifugation through Ficoll–Hypaque from healthy donors. PBMCs were cultured in RPMI 1640 medium containing 10% FCS, 1 mM sodium pyruvate, and 1% penicillin/streptomycin. For HIV-1 infection assays, PBMCs were cultured with 5 μg/ml PHA for 2 d, then infected with the equivalent of 2 μg/ml capside protein of 24 kDa (p24) of either WT or ΔNef HIV-1 virions during 16 h. Cells were then washed and resuspended in RPMI 1640 medium supplemented with 10% FCS and 10 U/ml IL-2 for 3 d before been used for immunofluorescence assays.

For transfection assays of primary cells, CD4⁺ T cells were further purified using the CD4⁺ T Cell Isolation Kit (Miltenyi Biotec) and cultured in RPMI 1640 medium containing 10% FCS, 1 mM sodium pyruvate, and nonessential amino acids. Isolated CD4⁺ T cells were transfected with 10 μg plasmid DNA using the Amaxa Nucleofector System and the Human T Cell Nucleofector Kit (Lonza). Cells were harvested and used for immunofluorescence analysis 24 h after transfection.

The human T cell line Jurkat clone J77cl20 was previously described (17). Jurkat were cultured in RPMI 1640 containing 10% FCS. For HIV-1 infection assays, 5 × 10⁶ Jurkat cells were infected with 2 μg of cell-free HIV-1 virions or VSV-pseudotyped virions during 16 h. Cells are then washed and resuspended in RPMI 1640 medium supplemented with 10% FCS for 3 d (or 36 h for the VSV-pseudotyped virions) before being harvested.

For siRNA, a total of 2 nmol of control or FIP3 siRNA were used per 10⁷ Jurkat cells. Two transfections were performed at 24-h interval with a Neon Transfection System (Life Technologies) using the following protocol: 1400 V, 10 ms, and three pulses. Seventy two hours after the first transfection, cells were harvested and processed for analysis. In the case of plasmid transfection, the Neon Transfection System was used in the same conditions to electroporate 10⁷ Jurkat cells with 10 μg plasmid DNA. Cells were harvested and processed for analysis 24 h after the transfection. When both infection and FIP3 depletion conditions were applied, Jurkat cells were previously transfected with siRNA and consecutively infected with VSV-pseudotyped virions for 36 h, with a total of 72 h from the first transfection, before the cells were harvested and processed for analysis.

Immunofluorescence and confocal imaging was performed as previously described (38, 39). Coverslips were coated with poly-l-lysine 0.002% (w/v) in water (Sigma-Aldrich). Cells were plated onto the coverslips for 3 min (if not otherwise indicated), then fixed with 4% paraformaldehyde for 20 min at room temperature (RT), washed in PBS, and incubated 30 min in PBS with 1% BSA (w/v) to prevent unspecific binding. Coverslips were then incubated 1 h at RT in PBS with 1% BSA with 0.1% Triton X-100 and the indicated dilution of primary Ab. Coverslips were rinsed three times in PBS with 1% BSA and then incubated with the corresponding fluorescent-coupled secondary Ab for 1 h at RT. After three washes in PBS with 1% BSA, coverslips were mounted on microscope slides using 8 μl of ProLong Gold Antifade mounting medium with DAPI (Life Technologies).

Confocal images were acquired with a LSM 700 confocal microscope (ZEISS) using the Plan-Apochromat 63× objective. Optical confocal sections were acquired using ZEN software (ZEISS) by intercalating green and red laser excitation to minimize channel cross-talk. Confocal optical sections were acquired at 0.2-μm depth intervals, and images were treated by deconvolution with the Huygens Professional Software (version 14.10; Scientific Volume Imaging). A two-dimensional visualization of three consecutive confocal sections (cut of 0.4-μm depth) centered on the Nef-induced endosomal compartment, when visible (or on a midsection of the cell), was generated from a sum intensity projection using Fiji software (40). Images showing the density gradient fluorescent intensities were obtained with the mpl-Inferno LUT of Fiji software.

Colocalization analyses were performed on the whole compartment (Nef, Lck, or Rac1) of deconvoluted images using Fiji software and the JACoP plugin (41). Threshold was automatically determined using the Costes method autothreshold determination (42). Analysis plots show the Pearson correlation coefficient. Colocalization scatter plot images show one representative colocalization analysis of the whole analysis and were obtained using the Colocalization Threshold plugin of the Fiji software. Statistical analyses were carried out using the nonparametrical Mann–Whitney U test of Prism software (GraphPad).

Images to quantify phospho-protein accumulation in the Lck compartment were acquired at 1-μm depth intervals in the z-axis to avoid fluorescence overlap. Fluorescence intensity in the area corresponding to the Lck compartment was calculated in percentage of the total fluorescence of the cell. Statistical analyses were carried out using the nonparametrical Mann–Whitney U test of Prism software (GraphPad).

For densitometry profile analysis, a z-stack of 1-μm confocal optical sections was acquired for each cell. Fluorescence intensity of pY319-ZAP70 or Rac1 was measured across cells, including plasma membrane and the Nef pericentrosomal compartment.

Cells were plated on poly-l-lysine–coated coverslips, incubated at RT for the indicated times, and fixed with 4% paraformaldehyde for 20 min. Coverslips were then treated as previously described in the Immunofluorescence section above using a Texas Red–labeled phalloidin to stain F-actin. For the measurement of cell spreading, z-stacks of 0.5-μm confocal optical sections were acquired. Two contiguous sections starting from the coverslip surface were stacked, and cell surface was measured on the phalloidin staining using the Fiji Analyze Particles tool on GFP-positive particles larger than 20 μm². Statistical analyses were carried out using the nonparametrical Mann–Whitney U test of Prism software (GraphPad).

Cells were stimulated by incubation with 10 μg/ml soluble CD3 mouse Ab (UCHT1) and 10 μg/ml CD28 at 37°C. At the indicated times, cells were plated on poly-l-lysine–coated coverslips as previously described, fixed with 4% paraformaldehyde for 20 min at RT, washed in PBS, and incubated 30 min in PBS with 1% BSA before immunofluorescence was performed.

Infection levels were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). Cells were isolated, fixed with 4% paraformaldehyde for 20 min at RT, washed in PBS, and incubated with the appropriate dilution of fluorescent-labeled Ab in PBS with 1% BSA. For fluorescence intensity levels of intracellular phospho-proteins, fixed cells were incubated in PBS with 1% BSA with 0.1% Triton X-100 and the indicated dilution of primary Ab and secondary fluorescent-labeled Ab. Flow cytometry data were analyzed with FlowJo software (FlowJo), restricting the analyses to single cells using forward scatter height/width signals.

Cells were lysed for 30 min in ice-cold buffer composed of 150 mM NaCl, 20 mM Tris (pH 7.4), 0.25% lauryl-β-maltoside, 4 mM orthovanadate, 1 mM EGTA, 50 mM NaF, 10 mM Na₄P₂O₇, 1 mM MgCl₂, and protease inhibitors (1 mM AEBSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Cell lysates were centrifuged at 20,800 × g for 10 min at 4°C. Equal amount of protein extract was loaded in NuPAGE 4–12% Bis-Tris gels (Life Technologies) by using the BCA Assay Kit (Thermo Fisher Scientific). Protein transfer to nitrocellulose blots (LI-COR Biosciences) was performed using the Bio-Rad Laboratories system and a transfer buffer composed of 25 mM Tris, 192 mM glycine, 20% EtOH, and 0.1% SDS. Membranes were saturated with blocking buffer (Rockland Immunochemicals) and incubated with primary Abs for 1 h at RT or overnight at 4°C in blocking buffer. After incubation with secondary Abs, an Odyssey scanner (LI-COR Biosciences) was used to detect and image near-infrared fluorescence. Images of blots were quantified using Fiji software.

Total RNA was extracted using the RNeasy Mini Plus Kit (Qiagen), following the manufacturer’s instructions. cDNA was prepared from 1 μg of total RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories). Gene products were quantified by quantitative PCR using the FastStart Universal SYBR Green PCR Master Mix (Roche) and the ABI PRISM 7900HT technology. For most of the cases, quantitative PCR quantifications were performed at least in three replicates, and its quantity values were calculated by the relative standard curve method and normalized to the mRNA expression of the B2M housekeeping gene.

Primer sequences used to target the different genes are described in Supplemental Table II.

Statistical analyses were carried out using Prism software (GraphPad V.7). Details about the data presentation, the experimental replication, and the adequate statistical tests used are included in the individual figure legends. Data met the assumptions of the statistical tests, and their distribution was previously checked using the Shapiro–Wilk normality test. Horizontal bars in plots represent the mean ± SEM. The p values are represented as follows: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. NS, p ≥ 0.05.

We and others have shown that HIV-1 Nef subverts the intracellular traffic of Lck and its function as a TCR-signaling molecule at the immunological synapse (17–19, 43). The molecular mechanism involved in Lck retention by Nef is not completely elucidated and may involve, at least in part, the Lck traffic regulatory protein Unc119 and the transferrin-recycling endosomal compartment (17, 19, 34) that are regulated by the Rab11 GTPase (22, 44).

We have recently shown that Lck is associated with the Rab11⁺ endosomal compartment, and its function in TCR signaling is regulated by the Rab11 effector FIP3 (28). Interestingly, T cells overexpressing FIP3 retain Lck in their Rab11⁺ endosomal compartment in a similar manner to Nef (17, 28). Moreover, we have shown that Rac1 is also associated with Rab11⁺ endosomes, and its subcellular localization and function are regulated by FIP3 (31). These findings prompted us to hypothesize that Nef could jointly alter Lck and Rac1 endosomal traffic, modulating their functions concomitantly. Therefore, we analyzed the intracellular localization of Lck and Rac1 upon HIV-1 infection by WT and Nef-deficient (ΔNef) viruses as well as in T cells expressing an Nef/GFP chimeric protein.

We indeed observed that HIV-1 infection in both primary and Jurkat T cells induced the accumulation of Lck and Rac1 in an Nef-dependent manner. Lck and Rac1 concentrate in the pericentrosomal area while partially disappearing from their cortical localization (Fig. 1A–D). Moreover, expression of Nef alone was sufficient for the intracellular relocalization of both Lck and Rac1 (Fig. 2A, 2C). Both molecules colocalized with Nef, although the overlap of Rac1 and Nef was more extensive than that of Nef and Lck (Fig. 2A–C). The Nef-induced Lck compartment colocalized with Rab11, and not with TGN46, indicating that Lck is mainly located in recycling endosomes (Fig. 2D, 2E).

Hence, HIV-1 Nef is necessary and sufficient to induce the concomitant intracellular relocalization of Lck and Rac1 in intracellular compartments, prompting us to investigate the characteristics and functional consequences of these compartments.

Lck kinase activity is regulated by the balanced phosphorylation of two tyrosine residues: Tyr394, which favors kinase activity and substrate interaction, and Tyr505, which prevents it by stabilizing Lck in a folded conformation. The two species are present at equilibrium in resting T cells, and their phosphorylation ratio does not change upon TCR engagement (45). This suggests that TCR signal transduction may be triggered by changes in localization of active forms of Lck that facilitate Lck contiguity to its substrates (e.g., delivery of endosomes carrying Lck to the immunological synapse). In this line, we have recently shown that modifying the endosomal localization of Lck changes the activation capacity of T cells, as assessed by the phosphorylation status of Lck and ZAP70 substrates (28).

To investigate the potential functional effects of Nef-induced endosomal accumulation of Lck, we analyzed whether active forms of Lck were present in that compartment, and if so, whether there was an effect on Lck substrates and downstream effectors. To this end, we performed a systematic analysis in Jurkat T cells expressing GFP-tagged Nef protein. This experimental setup provides sufficient spatial resolution to analyze these vesicular compartments while eliminating the influence of other viral proteins. The use of specific Abs directed to phosphorylated tyrosine (pTyr) residues allowed us to distinguish whether signaling molecules were in their signaling-competent phosphorylated state (e.g., pTyr residues described to interact with other signaling molecules).

We observed that active phosphorylated Lck (pTyr394) concentrates in the Nef-induced Lck compartment (Fig. 2B). Moreover, the phosphorylated forms of TCRζ (pTyr142) and ZAP70 (pTyr319), both substrates of Lck and interacting with each other upon TCRζ phosphorylation, are enriched within the Lck endosomal compartment where they significantly colocalize with Lck (Figs. 3A, 3B, 4A). Interestingly, the signaling adaptors LAT and SLP76 that interact with each other upon LAT phosphorylation by ZAP70 (3) are differentially concentrated at the Nef-induced Lck compartment; pLAT (pTyr191) is neither concentrated nor colocalized with Lck, whereas pSLP76 (pTyr128) accumulates and colocalizes with Lck, but to a lesser extent than pTCRζ and pZAP70 (Figs. 3C, 3D, 4A). Therefore, these findings show that Nef sequesters the initial triggering complex formed by Lck, TCRζ and ZAP70, apparently separating the two components of the signal amplification complex formed by the phosphorylated adaptors LAT and SLP76 (3, 4).

Importantly, concentration of phosphorylated Lck substrates in the pericentrosomal compartment was related to Lck accumulation, because overexpression of the Nef P₇₂XXP₇₅/AXXA mutant, which does not induce Lck accumulation (46), does not result in pZAP70 accumulation (Fig. 4B, 4C). Moreover, the capacity of Nef to increase total pZAP70, as assessed by flow cytometry, was significantly reduced in cells expressing the Nef P₇₂XXP₇₅/AXXA mutant (Fig. 4D).

In addition, we found phosphorylated Vav1 (pTyr174) significantly concentrated at the Nef-induced Lck endosomal compartment, colocalizing with Lck (Figs. 3E, 4A). Vav1 is involved in both the TCR/CD3 and CD28 signaling pathways, acting as signaling adaptor molecule (47, 48). Moreover, Vav1 is associated with the CD28 intracellular region via the signaling adaptor Grb2 (48). CD28 is downregulated during HIV-1 infection in an Nef- and Vpu-dependent manner, inducing CD28 internalization and degradation (5, 49, 50). In agreement with this, we observed that Nef expression induces the accumulation of CD28 in a pericentrosomal compartment that colocalizes with Nef (Fig. 5A, 5B). However, we did not find significant colocalization between CD28 and pVav1 in this compartment (Fig. 5C, 5D). In contrast, we observed significant colocalization between intracellularly accumulated CD28 and Rac1 (Fig. 5E, 5F). This is consistent with the extensive colocalization in the pericentrosomal compartment of Nef and CD28 and of Nef and Rac1. Therefore, Nef-induced pVav1 accumulation in the Nef-induced Lck compartment seems not to be the consequence of recruitment of CD28-associated Vav1, but of a possible soluble fraction. Finally, Vav1 is a regulator (guanine exchange factor [GEF]) of Rac1, controlling its activity on actin cytoskeleton dynamics (51). Its accumulation in the Nef-induced Lck compartment under its GEF active form (pTyr174) could be relevant, at least in part, for the Nef influence on Rac1 activity.

Altogether, these data show that Nef expression induces the concentration of activation-competent signaling molecules belonging to the TCR signaling pathways, leaving other molecules unperturbed.

We next investigated whether the subcellular localization of signaling proteins downstream of Lck, ZAP70, Vav1, and Rac1 was modified in Nef-expressing cells. To this end, we analyzed the intracellular localization of a number of signaling molecules of the TCR/CD28 signaling pathways leading to the activation of NFAT, NF-κB, and AP1 (Fos and Jun) transcription factors that, together, drive T cell differentiation, cytokine production, and eventually T cell proliferation (1). Among these downstream signaling proteins, PLCγ1 is activated through Lck and ZAP70-mediated tyrosine phosphorylation to give rise to two key phospholipids second messengers: inositol trisphosphate and diacylglycerol. Inositol trisphosphate regulates calcium flux from the endoplasmic reticulum, whereas diacylglycerol activates PKC serine threonine kinases. Increase in intracellular calcium concentration then activates the serine phosphatase calcineurin that dephosphorylates NFAT transcription factor, inducing its translocation to the nucleus. In turn, PKCθ may phosphorylate and activate the kinase Ikkβ, inducing the NF-κB signaling pathway and NF-κB nuclear translocation. Moreover, binding of Grb2/SOS to phosphorylated LAT facilitates Ras activation and the triggering of the MAP kinase cascade involving Raf, MEK, Erk1/2, and Elk serine/threonine kinases that activate the Fos transcription factor. Finally, Vav1 cooperates with PI3K in the CD28 cosignaling pathway to activate the GTPase Rac1 that, in turn, activates the JNK and the Jun transcription factor (1).

Therefore, we analyzed the effect of Nef/GFP expression in the subcellular localization of proteins and active forms of a number of these key signaling molecules including Fyn, AKT, pPLCγ1, NEMO, BCL10, CARMA1, mTOR, NF-κB (p105 and p65 subunits), PI3K, PKCθ, pT538-PKCθ, PKCζ, pThr202pY204-Erk1/2, and pY185-JNK. We did not find any of these proteins relocalized to the Nef-induced Lck endosomal compartment (Supplemental Fig. 1A–D).

These data indicate that Nef effects on the localization of T cell signaling molecules mainly concerns the TCR initiation signaling complex but does not affect downstream signaling molecules.

The accumulation of signaling-competent forms of Lck, TCRζ, ZAP70, and Vav1 suggests that HIV-1 infection may generate, via Nef, an autonomous endosomal signaling compartment that delivers constitutive activation signals to the infected T cell. Therefore, we investigated the effect of Nef on activation pathways downstream of those signaling molecules. To this end, we analyzed the expression of a battery of early- and late-activation genes in T cells infected with HIV-1 WT or ΔNef viruses or in Nef-expressing cells.

Jurkat T cells were infected with HIV-1 WT and ΔNef viruses, and retrotranscription quantitative PCR analysis was performed on nonstimulated cells to estimate the potential effects of Nef on constitutive T cell activation, independent of TCR stimulation. Among the genes investigated, the immediate, early-activation genes FOS and JUN (52) were upregulated upon HIV-1 infection with respect to their basal state in cells infected by WT virus, but to a lesser extent, in those infected by ΔNef virus (Fig. 6A). In contrast, MYC basal expression was equally inhibited by both WT and ΔNef viruses (Fig. 6A). However, the expression of two target genes regulated by the NF-κB signaling pathway IκBα (NFKBIA) and TNF-α–induced protein 3 (TNFAIP3) (53, 54) was barely changed (Fig. 6B). In addition, we found the expression of several late T cell activation genes, namely IL2, IL2RA (CD25), and IFNG (IFN-γ), significantly increased in cells infected by HIV-1 WT and, to a lesser extent, in those infected with ΔNef viruses (Fig. 6C).

To further investigate whether Nef expression was sufficient to induce those effects, Jurkat cells were transfected with GFP or Nef/GFP expression vectors and sorted by FACS. GFP and Nef/GFP-expressing cells were then assayed for their expression of some of the early- and late-activation genes referred above (Fig. 6D). The activation trend was confirmed; JUN and FOS were upregulated by Nef, whereas TNFAIP3 and NFKBIA gene expression were not significantly changed. Finally, no significant variation of MYC expression was observed in Nef-expressing cells in agreement with the Nef-independent inhibitory effect of HIV-1 virus infection.

Therefore, HIV-1 infection enhances the expression of several early- and late-activation genes in a Nef-dependent manner. The effect of Nef was different from gene to gene, suggesting a variable influence of Nef and other viral proteins.

To test whether the activation events induced by HIV-1 Nef were due to the formation of the Nef-induced endosomal Lck compartment, we took advantage of our previous findings showing that Lck is associated with Rab11⁺ endosomes, whose centripetal movement and localization in the pericentrosomal zone are regulated by the Rab11/FIP3 effector protein (FIP3) (28). Moreover, modification of Rab11 endosomal traffic by the depletion of FIP3 inhibits Lck-mediated T cell signaling events, even in nonstimulated cells (28). Interestingly, FIP3 overexpression induces Lck intracellular accumulation in a similar fashion as Nef (17, 28). In contrast, FIP3 depletion has the opposite effect, dispersing Rab11 endosomes carrying Lck all over the cytoplasm (28). Therefore, we tested whether FIP3 silencing could overcome Lck intracellular accumulation induced by Nef, as well as its potential functional effects on early and late gene expression.

As observed previously on the Lck pericentrosomal compartment in noninfected cells (28), FIP3 silencing dispersed the Nef-induced Lck/pZAP70 pericentrosomal accumulation, which became more fragmented and spread over the cytoplasm, as assessed by the distribution of Lck and pZAP70 in the pericentrosomal cytoplasmic area (Fig. 7A, 7B).

We next investigated the effect of FIP3 silencing on HIV-1 Nef-induced upregulation of some of the genes mentioned above. We used two siRNA oligonucleotides directed to distinct sequences and displaying different silencing efficiencies, siFIP3-2 being more efficient than siFIP3-1 (Fig. 8A, top), as we previously reported (28, 31). Note that FIP3 has two isoforms of slightly different electrophoretic mobility (Fig. 8A, double arrow head): siFIP3-1 preferentially silenced (although partially) the one of higher molecular mass, whereas siFIP3-2 silenced both. In cells transfected with small interfering control RNA (si-control), infection with HIV-1 WT viruses led to upregulation of JUN, FOS, IFNG, and IL2 genes, as described above, that was significantly lower in cells infected with ΔNef virus (Fig. 8B, 8C). Interestingly, FIP3 silencing counteracted HIV-1 WT–induced gene upregulation, reducing the differences between HIV-1 WT and ΔNef viruses to lower or nonsignificant levels in cells silenced with siFIP3-1 and siFIP3-2, respectively (Fig. 8B, 8C, white and gray histogram). Of note is that FIP3 silencing did not alter the percentage of infected cells (Fig. 8D) or Nef levels in cell extracts, but slightly reduced the level of the p24 capsid protein (Fig. 8A, 8D).

These data indicate the following: 1) the expression of several of the genes analyzed depends, in part, on the appropriate regulation of the recycling endosomal compartment controlled by Rab11/FIP3; 2) HIV-1 exacerbates the function of this physiological compartment in an Nef-dependent manner, increasing the expression of these various genes; and 3) perturbation of this compartment by FIP3 silencing counteracts the effects of Nef in a dose-dependent manner, reducing gene expression of HIV-1 WT–infected cells to the levels of cells infected with ΔNef viruses.

In addition to Lck and several of its substrates, we found the GTPase Rac1 concentrated in a pericentrosomal compartment in an Nef-dependent fashion (Fig. 1B, 1D). To better define the intracellular compartment in which Nef retains Rac1, we expressed Nef/GFP in Jurkat and primary CD4⁺ T cells and analyzed its relative localization to endogenous Rac1. In control cells expressing GFP, Rac1 is localized at the plasma membrane in the cytosol and, to a lesser extent, in pericentrosomal endosomes, likely corresponding to Rab11⁺ endosomes, as we previously described (31) (Fig. 9A, top). In Nef/GFP–expressing cells, Rac1 was massively localized in an intracellular compartment that extensively colocalized with Nef (Fig. 9A, bottom; 9C). Similar results were found in primary human CD4⁺ T cells transfected with Nef/GFP (Fig. 9B, 9C), although the intracellular compartment was less spatially resolved because of the smaller cytoplasmic volume of primary T cells.

T cell membrane protrusions formed during T cell spreading, and immunological synapse formation are reminiscent of lamellipodium structures observed in migrating cells (55), whose formation depends on Rac1 (56). Moreover, we have recently shown that Rac1 is associated with Rab11⁺ endosomes, and its subcellular localization is controlled by FIP3. Thus, FIP3-silenced T cells lose the Rac1/Rab11 pericentrosomal compartment, which gets fragmented and spreads all over the cytoplasm. Conversely, FIP3 overexpression induces the accumulation of Rac1 in the Rab11⁺ pericentrosomal compartment (31), somehow reminiscent of Nef effect on Rac1 intracellular localization (Figs. 2C, 9A, 9B). Rab11-mediated Rac1 traffic controls Rac1 functions in T cells. In particular, we showed that T cell capacity to spread on surfaces was exacerbated, both in the presence and absence of TCR stimulation or integrin adhesion (i.e., T cells spreading on polylysine). Therefore, we analyzed to what extent FIP3 silencing could compensate Nef-induced Rac1 concentration in the pericentrosomal compartment, and whether Nef was still capable to sequester Rac1 in FIP3-silenced cells. We observed that Rac1 intracellular dispersion in FIP3-silenced/Nef-expressing cells was less efficient than the one we previously observed in control cells not expressing Nef (31) and did not significantly alter Nef and Rac1 colocalization (Fig. 9D–F).

We then investigated whether Nef has an effect on T cells spreading on polylysine, which we showed was enhanced in FIP3-silenced cells (31). Therefore, we transfected Jurkat T cells with GFP or Nef/GFP together with si-control or siFIP3 oligonucleotides and analyzed the capacity of cells to spread on polylysine. We used polylysine as adhesion substrate to analyze the effects due to Rac1, and not those potentially involving Lck signaling in T cells spreading on anti-CD3, a process reported to be affected by Nef (10). We observed that Nef expression did not have a significant effect on si-control–treated T cell spreading on polylysine, but significantly inhibited the enhancing effect of FIP3 silencing, especially at late times (Fig. 9G, 9H, 15 min).

Altogether, these data indicate that the Nef subversion of Rac1 endosomal traffic and Rac1 sequestering in the Nef intracellular compartment modulates Rac1-mediated actin remodeling that supports TCR signaling–independent T cell spreading.

Under physiological conditions, the endosomal traffic of TCR and several of its proximal signaling molecules is crucial for immunological synapse formation and T cell activation. At least three main functions for this molecular traffic have been proposed: first, the targeting of TCR and signaling molecules to the immunological synapse, allowing the generation of signaling complexes at the synaptic plasma membrane (2, 20, 21, 23, 25, 26, 30, 57); second, the delivery of signaling complexes to endosomes to sustain T cell activation (58); and third, the removal of TCRs and signaling molecules from the synaptic plasma membrane to downregulate T cell activation (59–61). During HIV-1 infection, the viral protein Nef appears to specifically hijack some of these endosomal pathways, perturbing Lck intracellular traffic with several opposite effects reported: first, to limit Lck clustering and tyrosine phosphorylation of signaling adaptors at the immunological synapse (17, 18); and second, to increase the sensitivity of the Ras/Erk and calcium signaling pathways, leading to NFAT activation and IL2 production in response to TCR and CD28 stimulation (16, 19, 62–67). The influence of HIV-1 infection and Nef expression on other components of the T cell activation molecular machinery remains, however, poorly defined.

In this study, we show that HIV-1 infection induces the accumulation of Lck and Rac1 in a pericentrosomal vesicular compartment. Nef is necessary to induce this double accumulation, but the differences between WT and ΔNef infected cells do not allow us to ensure a full dependence on Nef expression. Other viral proteins might also be involved. Nevertheless, Nef expression alone is enough to induce these effects. Indeed, Nef appears to finely assemble an autonomous, endosomal signaling compartment that gathers activation-competent forms (i.e., phosphorylated at key Tyr residues) of several TCR/CD3 and CD28 proximal signaling molecules. This compartment includes the phosphorylated active form of Lck together with pTCRζ, pZAP70, pSLP76, and pVav1 but not pLAT. Interestingly, Lck, TCRζ, and LAT are associated with spatially adjacent, but distinct, endosomal compartments (21, 25, 26, 28), whereas ZAP70, SLP76, and Vav1 are not associated with endosomes (68) (I. del Rio-Iñiguez and J. Bouchet, unpublished observations). Therefore, the Nef-induced signaling compartment seems not to be just the consequence of the general perturbation of endosomal traffic. Rather, our data are consistent with Nef specifically altering Lck and TCRζ endosomal traffic, concentrating both proteins in pericentrosomal endosomes and favoring TCRζ phosphorylation. In addition, the reported interactions of Nef with Lck (69) and TCRζ (70) might also contribute to generate this compartment. In turn, locally phosphorylated TCRζ would bind ZAP70, favoring its concentration in that compartment, ZAP70 autophosphorylation, and ZAP70 phosphorylation and activation by Lck (4). It is tempting to speculate that Nef could stabilize a transient physiological intermediate signaling compartment (58), providing steady activation signals to the infected cell. To test this hypothesis, we cross-linked CD3 and CD28 and followed the potential generation of an enhanced Lck endosomal compartment containing phosphorylated Lck substrates. We could not detect increased accumulation of Lck or pZAP70 in the pericentrosomal area at activation time points between 5 and 30 min in which pZAP70 and TCRζ phosphorylation occurs (28) together with TCR/CD3 internalization (20) (Supplemental Fig. 2A, 2B). Rather, the Lck endosomal compartment appeared to lose intensity, as we previously reported (25). This indicates that a translocation of Lck to the plasma membrane occurs instead of Lck endosomal enrichment.

In contrast to what occurs at the immunological synapse plasma membrane, endosome-associated active ZAP70 seems not to be able to phosphorylate endosomal LAT. Nevertheless, pSLP76 concentrates there. Therefore, pSLP76 recruitment to the Nef-induced signaling compartment would not occur via pLAT (3). The lack of local formation of optimal amplification complex formed by pLAT and pSLP76 (3) may explain why the constitutive activation of genes induced by Nef is relatively weak (1.5–4-fold higher than the expression of noninfected cells) as compared with that induced by TCR/CD28 stimulation, which generally leads to higher gene expression levels (4–100-fold higher than the expression of nonstimulated cells) (28). Instead of pLAT, the interaction of SLP76 with Vav1 (71) could account for SLP76 recruitment, but how these two molecules are recruited remains unknown. It has been reported that Nef interacts with Vav1 in cholesterol and sphingolipid-enriched (detergent insoluble) membrane microdomains (8, 9, 11). These membrane microdomains also continuously cycle between the plasma membrane and the endosomal compartment and could help Vav1 concentration in the pericentrosomal region close to Lck. It is tempting to speculate that although TCR and CD28 induce a strong response leading to proliferation, Nef does not generate a proliferation signal, but rather a mild survival signal that may favor virus replication.

The kinetics of Lck and Rac1 accumulation in the pericentrosomal compartment upon HIV-1 infection remain unresolved. The difficulty to detect low levels of Nef expression by immunofluorescence and confocal microscopy, together with intrinsic variability of Lck and Rac1 concentration in the pericentrosomal compartment in noninfected cells, makes these quantitative measurements at present poorly reliable (I. del Rio-Iñiguez and J. Bouchet, unpublished observations).

We have identified several early- and late-activation genes that are differentially regulated during HIV-1 infection in a partly Nef-dependent fashion, even in the absence of TCR stimulation. Thus, FOS and JUN were upregulated by HIV-1 infection in a partly Nef-dependent manner. In contrast, MYC expression was inhibited, although in an Nef-independent manner. Finally, NFKBIA and TNFAIP3 remained nonsignificantly changed. This is consistent with previous reports proposing an Nef-mediated modulation of the Ras/Erk, calcium, and NFAT signaling pathway with no effect on NF-κB (19, 62–64, 72). In addition, we observed that several late-activation genes like IL2, IL2RA, and IFNG were upregulated in HIV-1–infected cells in a partly Nef-dependent manner. The extent of upregulation and Nef dependence varies among the different genes, suggesting that other HIV-1 proteins may contribute to these effects by this or different mechanisms. Further evidence for Nef dependency of HIV-1–infected cells was the observations that Nef expression by itself increased JUN and FOS gene expression without affecting MYC, NFKBIA, or TNFAIP3.

Although our data in this study show that HIV-1 infection increases the expression of these various genes independently of TCR stimulation, other authors have shown that Nef expression enhances TCR/CD28 stimulation (16, 19, 62–67). In our case, we cannot rule out that TCR-independent costimulation via T cell/T cell interaction occurs and contributes to the observed effects. Under physiological conditions, infected cells might get mild stimulations from other cells in lymphoid organs that may cooperate with the Nef-induced compartment to activate infected cells.

We have previously shown that Rab11 endosomal traffic is key for Lck and Rac1 regulatory functions in T cells. This mechanism modulates TCR signaling and Rac1-mediated actin cytoskeleton remodeling, both important during immunological synapse formation and T cell activation, leading to cytokine production (28, 31, 32). Interestingly, Nef mimics FIP3 overexpression in its ability to accumulate Lck and Rac1 in the pericentrosomal compartment. The resemblance of effects of Nef and FIP3 overexpression on Lck and Rac1 endosomal traffic is striking and suggests a common mechanism involving Rab11-driven endosomal traffic. For instance, both proteins affect transferrin and transferrin receptor endosomal traffic (34, 73). Moreover, both proteins interact with members of the exocyst complex that regulates vesicle tethering to the plasma membrane and may affect endosomal traffic (74, 75). Conversely, Nef has the opposite effect of FIP3 silencing, enhancing the phosphorylation of some Lck substrates and counteracting the effect of FIP3 silencing on T cell spreading. Altogether, these data indicate that Nef hijacks the endosomal traffic of Lck and Rac1 to modulate, in an opposite manner, signaling- and actin cytoskeleton–mediated T cell functions. These combined mechanisms may account, in part, for the reported multiple, and sometimes contradictory, effects of HIV-1 infection and Nef expression on T cell activation (16) as well as Nef effects on T cell actin cytoskeleton, influencing T cell spreading, cell shape changes, and migration (9, 10, 13, 14, 76). Additionally, the Nef effects we describe in this study may complement other reported mechanisms involving Nef interactions with some signaling and cytoskeleton regulators (16). Finally, Pan et al. (19) reported that Nef-induced intracellular retention of Lck could be counteracted by the overexpression of Unc119, a protein also involved in Lck traffic via the Rab11 endosomal compartment and in Lck activation (22, 77), further supporting the effect of Rab11 traffic in the effects of Nef on Lck.

FIP3 silencing affects gene expression in both noninfected and HIV-1–infected cells and balances the differences between cells infected with HIV-1 WT and ΔNef viruses. This is consistent with the idea that HIV-1 hijacks via Nef signaling mechanisms existing in noninfected T cells, exacerbating them to produce higher basal levels of some transcription factors (i.e., Fos and Jun) and cytokines (i.e., IFN-γ and IL2). This may contribute to an equilibrium between the virus and the infected cell, favoring virus replication. In this line, Nef-induced modulation of T cell endosomal and Lck traffic was reported to contribute to virus replication (19, 34). Finally, the subtle relocalization of part of the TCR and CD28 signaling machinery might be important for specific HIV-1 effects on apoptosis or in the process of reprogramming infected cells to quiescence, but this will need further investigation (78).

We observed that Nef recruits Rac1 in a compartment that overlaps and colocalizes with Nef more extensively than Lck and the other recruited signaling molecules. This is consistent with an interaction between Nef and Rac1 as shown by others (11, 79). Rac1 and its GEF activator Vav1 have two related functions in T cells: to transduce TCR and CD28 activation signals and to regulate actin cytoskeleton dynamics. In this line, previous reports proposed that Vav1 and Rac1 are involved in Nef-mediated activation of PAK2 (8, 11) and may enhance Ras/Erk and JNK signaling pathways (9, 64). In some of these reports, detergent-insoluble membrane microdomains were suggested to be involved, but the two mechanisms would not be exclusive because membrane microdomains transit through recycling endosomes. Interestingly, we did not observe relocalization of active phosphorylated forms of Erk or JNK. However, according to the reports cited above, these kinases are more prone to be activated in Nef-expressing cells when the TCR is engaged. Therefore, we could speculate that the Nef-induced endosomal signaling compartment facilitates the delivery of activation signals to these kinases without concentrating them in this compartment, leading to increase transcription of cytokine genes, as we show in this study.

We could not elucidate in our study whether Nef can relocalize other Rho-family GTPases, such as Rho and Cdc42, because of lack of performing Abs able to detect endogenous proteins. Worth noting, Rauch et al. (11) reported that Rac1, Cdc42, and Vav1 are recruited to Nef/PAK2 complexes within detergent-insoluble membrane microdomains. They proposed that this recruitment may contribute to Nef effects on T cell activation. However, the subcellular localization of the reported complexes appears very different from the Rac1/Nef corecruitment we describe in this report. Our data indicate that Rac1 sequestering by Nef is inhibitory for Rac1-mediated cytoskeletal rearrangements, leading to T cell spreading. This could also explain the modulatory effect of Nef on T cell shape and migration reported by others (12–14, 76). Therefore, different localizations of Rac1 with respect to Nef might lead to distinct opposite effects. Localization in membrane microdomains might favor signaling, whereas endosomal sequestering might have a dual effect: enhancing the Vav1-Rac1 signaling pathway and inhibiting Rac1-mediated actin cytoskeleton reorganization, as shown in this study on T cell spreading.

In conclusion, this work provides new insights into the understanding of HIV-1 host T cell interactions via the subtle modulation of the endosomal traffic of signaling and cytoskeleton regulators. The two mechanisms are likely different and lead to opposite outputs. Our findings may be the reflection of Nef effects at various steps of the virus life cycle: first, during early phases of infection in which virus-borne Nef enters the target cell and may cause subcellular local effects on signaling or cytoskeleton; second, during HIV-1 genome transcription in which Nef is expressed from the integrated viral genome, causing more general effects in the infected cells together with other viral proteins; and third, Nef could affect bystander cells to which it could be transferred from infected cells, causing Nef specific effects. Finally, although our work largely focused on Nef-dependent effects, our data show that other viral proteins may have significant effects on T cell signaling using, perhaps, complementary mechanisms. Nef induced reorganization of the recycling endosomal compartment, and as a consequence, the proteins transported by these endosomes may contribute to the effect of Nef in virus replication as indicated by previous reports (19, 34).

We thank the Photonic BioImaging UTechS microscopy core facility at the Institut Pasteur for microscopy and technical support, the Cytometry and Biomarkers UTechS core facility at the Institut Pasteur for flow cytometry, cell sorting, and technical support, and the French Blood Bank (Etablissement Francais du Sang) and the Clinical Investigation and Access to Biological Resources core facility team for providing blood from healthy donors and primary T cell samples, respectively. The following reagents were obtained through the National Institutes of Health AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID): mAb to HIV-1 p24 (AG3.0) from Dr. Jonathan Allan (80), anti–HIV-1 Nef mAb (6.2) from Dr. Kai Krohn and Dr. Vladimir Ovod (81), and HIV-1SF2 p24 antiserum from Division of AIDS, NIAID, produced by BioMolecular Technologies. We thank Drs. S. Benichou and F. Niedergang (Institut Cochin, Paris), H. Moreaux, A. M. Lennon-Dumenil and S. Agüera-Gonzalez (Institut Curie, Paris), and A. Echard, R. Weil, and S. Etienne-Manneville (Institut Pasteur, Paris) for Abs, expression vectors, and methodological help and advice.

The authors have no financial conflict of interest.