AEGiS-14IAC: Hiv-1 nef protein downregulates lipid rafts and mediated a defective activation of Ca2+ signaling.

14th International AIDS Conference

Barcelona, Spain - July 7-12, 2002

Hiv-1 nef protein downregulates lipid rafts and mediated a defective activation of Ca2+ signaling.

Int Conf AIDS 2002 Jul 7-12; 14:(abstract no. WeOrA1258)

Tuosto L, Marinari B, Andreotti M, Federico M, Piccolella E
Dept. Cell. and Devel. Biol., University La Sapienza, Rome, Italy

BACKGROUND: Although it is widely accepted that HIV-1 Nef protein interfers with T cell receptor machinery, less is known about its effects on the first events regulating T cell activation such as the organisation of the immunological synapses. We hypothesised that Nef could firstly interact with the specialised membrane microdomains known as rafts and subsequently with the signalling cascades that ultimately induce the transcription of genes controlling either the activation and/or the inhibition of T cell proliferation. Therefore we investigated the effect of Nef on organisation of membrane rafts as well as on TCR-mediated activation of the MAP kinases and the transcription factors regulating cytokine gene expression.

METHODS: Jurkat cells were transiently transfected with CD8-Nef and CD8 constructs and: i) the expression of membrane rafts was evaluated by GM1 staining using cholera toxin B; ii) NFAT, NF-kB, AP-1 and Fos activation by TCR were analysed by luciferase assay and MAP kinase activity by in vitro kinase assays. Raft expression was also analysed in Jurkat cells infected with a VSV-G pseudotyped Denv HIV virus and its variant lacking nef.

RESULTS: We show that both transient expression of Nef protein in Jurkat as well as Jurkat infection with HIV induced the downregulation of membrane rafts. Furthermore, Nef expression significantly inhibited TCR-induced NFAT activity, without affecting NF-kB, AP-1 and Fos. Indeed, neither JNK nor ERK kinase activities were affected in Nef expressing cells. Ionomycine and constitutively active calcineurin restored NFAT activation in Nef expressing cells thus suggest that Ca2+ pathway was impaired by Nef expression.

CONCLUSIONS: The results suggest that Nef may modify raft-associated proteins not only quantitatively but also qualitatively. Likely consequences of this effect may be a differential recruitment of signalling proteins by TCR and costimulatory molecules, thus resulting in a defective activation of Ca 2+.

Keywords: AEGIS, Gene Products, nef, HIV-1, HIV Long Terminal Repeat, Receptors, Antigen, T-Cell, NF-kappa B, Transcription Factors, Transcription Factor AP-1, Jurkat Cells, Membrane Microdomains, HIV, Down-Regulation, Mitogen-Activated Protein Kinases, Transcription, Genetic, Human, In Vitro, genetics

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WeOrA1258