We explored the effect of HIV-1 on the shape and motility of CD4+
T cells by combining various techniques (scanning electron microscopy, immunofluorescence confocal microscopy, automated flow cytometry imaging with the Amnis apparatus, and live-cell imaging). We reported that HIV-1, through expression of Nef, profoundly alters the morphology of infected T cells. Nef decreases ruffle protrusions but promotes the formation of filopodium-like structures. The total amount of F-actin is apparently not modified by Nef, as assessed by Amnis analysis, suggesting that the viral protein does not inhibit polymerization but rather alters actin remodeling. Nef likely modulates regulators of actin assembly pathways, leading to the formation of these long, thin membrane extensions instead of ruffles. It has been suggested that ruffle formation involves Arp2/3 complex-generated dendritic networks while filopodia assemble by “convergent elongation” of actin filaments (6
). We can speculate that Nef alters the formation of ruffles through interaction with Arp2/3, promoting the formation of unbranched actin filaments, leading to filopodium extension. The phenotype induced by Nef is indeed reminiscent of that observed in Arp2-depleted lymphocytes (35
), which exhibit defects in ruffle assembly and remain covered with finger-like protrusions. To better understand how these morphological changes are induced, we used HIV-1 expressing Nef proteins mutated in different functional domains (15
). The effect of Nef is dependent on its myristoylated motif and SH3-binding domain. Myristoylation, and hence membrane association, is required for most known activities of Nef. The proline-rich SH3-binding domain is involved in Nef association with Vav, DOCK2-ELMO1, and Pak2 (p21-activated kinase 2) and Nef-induced activation of Rac and Pak2 (14
). Pak2 activation promotes phosphorylation of cofilin (54
) and has been proposed to mediate Nef-induced inhibition of actin rearrangements (22
). It will be worthwhile to determine, by using a large panel of Nef mutants, the domains of Nef responsible for filopodium induction, as well as to identify the actin regulators involved, such as Pak2 and Arp 2/3.
The filopodium development induced by Nef may enhance communication and the exchange of cellular or viral materials between infected lymphocytes and bystander cells and may facilitate viral transfer to other cells. HIV-1 efficiently propagates through cell-to-cell contacts, mainly through virological synapse and polysynapse formation, and also by establishing remote connections via filopodial bridges or nanotubes (25
). Cell-to-cell viral spread is dependent on actin rearrangements (26
). HIV-1 infection of macrophages also enhances filopodium formation and transfer of Nef to neighboring B cells via long-range intercellular conduits (12
). We showed here that Nef is also found within T-cell filopodia, suggesting it could be similarly delivered from infected lymphocytes to bystander immune or nonimmune cells. Interestingly, Nef transfer between T cells may also occur through exchange of microvesicles or patches of plasma membrane (33
). Altogether, these results indicate that Nef uses various means to perturb intercellular communication networks.
We further documented the consequences of Nef expression for actin-dependent processes by studying adhesion to extracellular matrix. We showed that Nef expression reduces T-cell adhesion to fibronectin-coated surfaces. After binding to fibronectin, Nef-expressing T cells displayed impaired ruffling activity and spreading, as visualized by real-time imaging. Nef does not alter surface expression of α4β1 and α5β1 integrins that bind fibronectin. Adhesion of T cells to the extracellular matrix is controlled by a modulation of the affinity of integrins and involves actin cytoskeleton rearrangements and cell spreading (43
). Rac and Lck regulate integrin-mediated spreading and adhesion of T cells (9
). The intracellular distribution of Lck is modified by Nef (57
). The adhesion defect of T cells may thus in part result from Nef-induced perturbation of Lck and Rac proteins (4
). The impaired adhesion to fibronectin suggests that infected lymphocytes may not correctly bind to HEV, the first step of the extravasation process, and helps to explain why transendothelial migration of Nef-expressing cells is decreased in culture systems (39
). It will be worthwhile to further study the interaction of infected lymphocytes with endothelial cells.
We demonstrated here that Nef inhibits the intrinsic motility of infected T cells. Lymphocytes move by a mechanism that involves contractility of the actomyosin cortex (38
). Our results suggest that Nef globally alters this process, probably as a consequence of the morphological changes described above. We also reported, using Nef-transduced or HIV-infected Jurkat cells, that Nef inhibits T-cell chemotaxis toward CXCL12, thus confirming previous reports (7
). We extended these observations by showing that primary lymphocyte motility toward a variety of chemokines controlling homing to LNs (CXCL12, CCL19, and CCL3) is also impaired. A recent work indicated that Nef inhibits morphological changes induced by chemokines, providing a link between actin rearrangements induced by Nef and inhibition of motility (54
The viral envelope glycoprotein (gp120) also interferes with chemokine-receptor or CD4 signaling pathways (2
). The combined effect of Nef and Env on the behavior and motility of lymphocytes in culture experiments is probably highly relevant to the in vivo
situation. Studies of the dynamics of HIV-1 infection in lymphoid tissues, after initiation of antiretroviral therapy, identified two populations with different turnovers: activated CD4+
T cells (with a half-life [t1/2
] of 1 to 2 days) and resting or low-level-proliferating T cells (t1/2
= 14 days) (8
), which constitute a long-term reservoir. Expression of Nef in acutely infected activated cells, but also in these long-living T cells, could modulate their migration capacity. During chronic HIV infection, LNs are often enlarged and inflammatory and are characterized by dramatic lymphocyte sequestration (27
). In monkey lymphoid tissues, WT simian immunodeficiency virus (SIV)-infected and Δnef
SIV-infected cells accumulate in different zones (55
), suggesting that Nef may affect cell migration in vivo
. T cells, once infected by HIV within LNs, might display defective migration properties, which could explain their sequestration, as well as their different localization, within these organs. In summary, Nef displays complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impact the capacity of infected cells to recirculate and to encounter and communicate with antigen-presenting cells (APCs) and other cells and to disseminate infection.