Nef-Mediated Lipid Raft Exclusion of UbcH7 Inhibits Cbl Activity in T Cells to Positively Regulate Signaling

Simmons, Alison; Gangadharan, Bevin; Hodges, Ashleigh; Sharrocks, Katherine; Prabhakar, S.; García, Ángel; Dwek, Raymond A.; Zitzmann, Nicole; McMichael, Andrew J.

doi:10.1016/j.immuni.2005.11.003

Cited by 36 publications

(34 citation statements)

References 55 publications

(59 reference statements)

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“…Such effects are generally consistent with previous reports on the disassembly of F-actin structures by Nef in fibroblasts (14,33). Interestingly, Nef is known to activate Rac1 and Cdc42 in T-lymphocytes (15,44) but, as shown herein, disrupts cytoskeletal structures induced by these GTPases. This might reflect that both, seemingly opposing, activities of Nef are independently exerted by distinct Nef subpopulations in cells.…”

Section: Discussionsupporting

confidence: 93%

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

Haller

Rauch

Michel

et al. 2006

Journal of Biological Chemistry

148

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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...

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Section: Discussionsupporting

confidence: 93%

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

Haller

Rauch

Michel

et al. 2006

Journal of Biological Chemistry

148

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“…Importantly, other mem- brane-associated HIV-1 proteins, including Env, Nef, Vpu, and Tat, may interact with membrane microdomains. In particular, Nef has been proposed to alter lipid rafts and their constituent lipids and proteins (2,99,127,143,144). To control for the effects of other HIV-1 proteins on microdomains, we compared the copatching of microdomain markers in cells transfected with a WT HIV-1 molecular clone or cells transfected with a molecular clone containing multiple MA mutations (1GA/6A2T).…”

Section: Resultsmentioning

confidence: 99%

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

Hogue

Grover

Soheilian

et al. 2011

J Virol

107

116

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The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.

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“…In addition, Nef affects the basal states of T-cell activation and the responsiveness of T lymphocytes to TCR signaling. [19][20][21][22] Initial studies with overexpression strategies in cell lines reached contradicting conclusions about the effects of Nef on TCR signaling. More recent work with viral infection and/or primary target T cells revealed that Nef moderately enhances distal responses to exogenous TCR stimulation by mitogens or anti-TCR Abs.…”

mentioning

confidence: 99%

HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network–associated Lck signaling

Pan

Rudolph

Abraham

et al. 2012

Blood

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IntroductionReplication of HIV-1 in primary human T lymphocytes is tightly coupled to their activation state. Whereas HIV-1 undergoes early replication events in quiescent CD4 ϩ T lymphocytes, subsequent steps in the viral life cycle require cell activation. 1 T lymphocyte activation is primarily governed by signaling through the TCR complex after engagement in a tight contact with APCs; this is referred to as the immunological synapse (IS).TCR engagement by specific MHC-presented peptides launches highly dynamic and coordinated transport events that recruit specific factors to the IS and exclude others from it. This signal initiation triggers a broad cascade of downstream signaling that include dynamic F-actin remodeling at the IS, tyrosine phosphorylation, release of calcium flux, and activation of transcription. These events increase production of the T-cell survival cytokine IL-2 and are coordinated by the TCR proximal tyrosine kinase Lck, a master switch of TCR signaling. Immediately after TCR engagement, active Lck is recruited to the IS. 2-4 Whereas signal diversification and enhancement occur at the plasma membrane (PM), subsequent TCR signaling is compartmentalized and also occurs at intracellular membranes. An important intracellular arm of the TCR response is regulated by the N-Ras GTPase that is activated at Golgi membranes downstream of Lck. [5][6][7][8][9] T-cell activation is thought to be beneficial to HIV-1 because it allows transcriptional activation of latent provirus and progression of the life cycle. However, activation-induced cell death after TCR engagement runs the risk of limiting the lifespan of productively infected cells and thus the amount of viral progeny produced.Consequently, HIV-1 encodes gene products such as Nef to fine-tune the activation states of infected T lymphocytes. 10,11 Nef is a 25-to 34-kDa myristoylated accessory protein encoded by HIV-1, HIV-2, and SIV. Ex vivo, Nef enhances the single-round infectivity of virus particles and moderately accelerates virus spread over multiple rounds. 12 In vivo, Nef strongly boosts virus replication, particularly during primary infection, when the presence of Nef can elevate virus titers by more than 2 logs, and is critical for rapid disease progression. [13][14][15] This role of Nef as a pathogenicity factor is also revealed in transgenic mice, in which Nef expression induces AIDS-like depletion of CD4 ϩ T lymphocytes. 16 Delineating the mechanisms of Nef action has been hampered by the multitude of interactions with host T-cell proteins suggested to modulate various intracellular transport and signaling pathways. 17,18 This includes modulating exposure of cell-surface receptors such as MHC-I and II, CD4, and chemokine receptors to evade immune recognition and to prevent superinfection of infected cells, respectively (reviewed in Laguette et al 12 ). In addition, Nef affects the basal states of T-cell activation and the responsiveness of T lymphocytes to TCR signaling. [19][20][21][22] Initial studies with overexpression strategies ...

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Nef-Mediated Lipid Raft Exclusion of UbcH7 Inhibits Cbl Activity in T Cells to Positively Regulate Signaling

Cited by 36 publications

References 55 publications

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

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

Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane

HIV-1 Nef compensates for disorganization of the immunological synapse by inducing trans-Golgi network–associated Lck signaling

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