Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molec
ular 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 fac
tor of activated T cells (NFAT), a transcription factor that plays a centra
l role in coordinating T cell. activation. Expression of Nef did not signif
icantly alter the basal level of NFAT activity in Jurkat cells nor the incr
eased activity following T cell receptor stimulation by anti-CD3 or anti-CD
3 + anti-CD28. We also mimicked NFAT induction by TCR triggering by simulta
neous activation of the Ras and calcium signaling pathways with phorbol 12-
myristate 13-acetate and ionomycin, respectively, Strikingly, whereas activ
ation 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 expre
ssion. Experiments with different dominant negative mutant signaling protei
ns, inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this
effect was mediated via activation of calcineurin by Nef-induced changes i
n calcium metabolism, but was independent of TCR-associated signaling event
s. This ability of Nef to substitute for triggering of the calcium pathway
in induction of NFAT could promote activation of human immunodeficiency vir
us (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.