Synergistic activation of NFAT by HIV-1 nef and the Ras/MAPK pathway

Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molecular mechanisms of action remain incompletely understood. Here we report a novel effect of Nef on lymphocyte signaling, which is mediated via a T cell receptor (TCR)-independent contribution of Nef to induction of nuclear factor of activated T cells (NFAT), a transcription factor that plays a central role in coordinating T cell activation. Expression of Nef did not significantly alter the basal level of NFAT activity in Jurkat cells nor the increased activity following T cell receptor stimulation by anti-CD3 or anti-CD3 + anti-CD28. We also mimicked NFAT induction by TCR triggering by simultaneous activation of the Ras and calcium signaling pathways with phorbol 12-myristate 13-acetate and ionomycin, respectively. Strikingly, whereas activation of either of these pathways individually did not induce NFAT activity in control cells, in Nef-expressing cells phorbol 12-myristate 13-acetate treatment alone resulted in a 100-fold increase in NFAT-directed gene expression. Experiments with different dominant negative mutant signaling proteins, inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this effect was mediated via activation of calcineurin by Nef-induced changes in calcium metabolism, but was independent of TCR-associated signaling events. This ability of Nef to substitute for triggering of the calcium pathway in induction of NFAT could promote activation of human immunodeficiency virus (HIV)-infected T cells in response to stimuli mediated via TCR or other cell surface receptors under conditions when activation of Ras rather than calcium signaling would otherwise predominate.