The HIV-1 Pathogenicity Factor Nef Interferes with Maturation of Stimulatory T-lymphocyte Contacts by Modulation of N-Wasp Activity

Abstract

The Nef protein is a key determinant of human immunodeficiency virus (HIV) pathogenicity that, among other activities, sensitizes T-lymphocytes for optimal virus production. The initial events by which Nef modulates the T-cell receptor (TCR) cascade are poorly understood.TCRengagementtriggersactinrearrangementsthatcontrol receptor clustering for signal initiation and dynamic organization of signaling protein complexes to form an immunological synapse. Here we report that Nef potently interferes with cell spreading and formation of actin-rich circumferential rings in T-lymphocytes upon surface-supported TCR stimulation. These effects were conserved among Nef proteins from different lentiviruses and occurred in HIV-1-infected primary human T-lymphocytes. This novel Nef activity critically depended on its Src homology 3 domain binding motif and required efficient association with Pak2 activity. Notably, whereas overall signaling microcluster formation immediately following TCR engagement occurred normally in Nef-expressing cells, the viral protein inhibited the concomitant activation of the actin organizer N-Wasp. During the subsequent maturation phase of the stimulatory contact, Nef interfered with the translocation of N-Wasp to the cell periphery, the overall induction of tyrosine phosphorylation, and the selective recruitment of phosphorylated LAT to stimulatory contacts. Consistent with such a critical role of N-Wasp in this process, Nef also blocked morphological changes induced by the known N-Wasp regulators Rac1 and Cdc42. Together, our results demonstrate that Nef alters both the amount and composition of signaling microclusters. We propose modulation of actin dynamics as an important mechanism for Nef-induced alterations of TCR signaling.The Nef protein of the primate lentiviruses HIV-1/-2 2 and simian immunodeficiency virus is a crucial pathogenicity factor and substantially increases virus replication in vivo (1-4). Whereas the underlying molecular mechanisms remain to be fully elucidated, Nef facilitates immune evasion of infected cells and directly boosts virus spread. These effector functions probably reflect the ability of Nef to serve as a protein-interaction adaptor that modulates cellular vesicle transport and signal transduction machineries (5, 6). In CD4 ϩ T-lymphocytes, one of the major HIV-1 target cell populations in vivo, Nef lowers the threshold of TCR activation possibly to induce an intermediate activation state that is permissive for HIV-1 replication (7-12). Several protein assemblies, including the association with the Nef-associated kinase complex, the guanine exchange factors Vav and DOCK2-ELMO1, and the TCR chain, are involved in the modulation of TCR signaling by Nef (13-16). However, it has not been addressed how Nef affects early events of TCR signal initiation. Physiologically, TCR triggering occurs in the context of a close contact between a T-cell and antigenpresenting cell referred to as the immunological synapse (IS). Within this highly dynamic structure, spatial redistribut...

HIV-1 Nef Limits Communication between Linker of Activated T Cells and SLP-76 To Reduce Formation of SLP-76–Signaling Microclusters following TCR Stimulation

“…At this time point, control cells displayed hallmarks of early T cell activation, including cell spreading and formation of circumferential F-actin rings, both of which were potently inhibited by the presence of Nef, as reported earlier (Fig. 1A) (37)(38)(39)48). Coexpression of the TCR z-chain fused to GFP (TCR.GFP) in these cells revealed the formation of numerous MCs at cell-cover glass stimulatory contacts that were distributed across the entire cell diameter, including its periphery (see schematic in Supplemental Fig.…”

“…T cell spreading and actin ring assay T cell activation using anti-CD3ε-coated stimulatory surfaces for TCRmediated cell spreading was essentially carried out as described (37,45). Briefly, microscope cover glasses (Marienfeld) were placed on a parafilmcoated petri dish and incubated with a 0.01% poly-lysine (Sigma-Aldrich) solution at room temperature for 10 min.…”

“…Subsequently, cells were fixed for 20 min (Jurkat T lymphocytes) or 90 min (infected PBL) by adding TBS-3% paraformaldehyde. After permeabilization with TBS-0.1% Triton X-100 for 1-5 min, the cells were blocked with TBS-1% BSA for 30 min and stained, as previously described (37). Cover glasses were mounted in LinMount (Linaris).…”

“…Two days after transfection, culture supernatants were harvested and the HIV-1 p24 Ag concentration was determined by a p24 Ag ELISA. Isolation, activation, and HIV-1 infection of PBL were carried out, as described (37). A total of 1000 ng p24 was used to spin-infect 5 3 10 6 Jurkat cells or PBLs.…”

“…In contrast, HIV-1 Nef severely impairs the formation and organization of IS structures between Nef-expressing T lymphocytes and APCs by reducing the frequency of IS formation, blocking Factin polarization at cell-cell contacts, and inducing mislocalization of the TCR itself as well as its effector kinase Lck (28,(35)(36)(37)(38)(39). These morphological alterations at the IS were accompanied by interference with early TCR signaling such as induction of tyrosine phosphorylation (36,37,39). We recently reported that Nef achieves enhanced T cell activation responses despite this disruption of early signaling events by the assembly of active signaling complexes at the trans Golgi network (TGN) (40).…”