The
Nef protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in
AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the
NADPH oxidase complex, a key
enzyme involved in the generation of
reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV-1 Nef in human macrophages cell line modulates in bi-phasic mode the
superoxide anion release by
NADPH oxidase, inducing a fast increase of the
superoxide production, followed by a delayed strong inhibition mediated by Nef-induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef-mediated activation of
NADPH oxidase and
superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3-binding domain are needed to induce
superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor,
PD98059, demonstrated that Nef-induced
superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of
NADPH oxidase complex p47(
phox) to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the
Src family kinases on Nef-mediated
NADPH oxidase activation. In addition,
LY294002, a specific inhibitor of
phosphoinositide 3-kinase (PI3K) inhibited both the Nef-induced p47(
phox) phosphorylation and the
superoxide anion release. These data indicate that Nef regulates the
NADPH oxidase activity through the activation of the
Src kinases and PI3K.