HIV‐1 Nef induces p47^phox phosphorylation leading to a rapid superoxide anion release from the U937 human monoblastic cell line

Abstract

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.

HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

“…Finally, Nef is known to regulate generation of superoxide anions from human macrophages [81], [82], [83] and increase astrocyte sensitivity towards exogenous hygrogen peroxide [83], [84], [85]. However, it has not been known that Nef is a responsible viral element in regulation of ROS production in hepatocytes.…”

At the crossroads of homoeostasis and disease: roles of the PACS proteins in membrane traffic and apoptosis

“…For example, binding of the SFK Hck to Nef at the Golgi region of macrophages is required for down-regulation of the M-CSF (macrophage colony stimulating factor) receptor [136]. In addition, Nef interaction with SFKs and PI3K induces superoxide release from macrophages to enhance HIV-1 pathogenesis [137], and Nef stimulates class I PI3K to drive the TLR4 (Toll-like receptor 4)-mediated suppression of the innate immune system, possibly contributing to secondary mycobacterial infections suffered by AIDS victims [138]. In addition, Nef-mediated ZAP-70 activation is required for PAK2 (p21-activated kinase 2) activation, Fas-ligand up-regulation and formation of virological synapses [139–142].…”

HIV-1 Nef Impairs Key Functional Activities in Human Macrophages through CD36 Downregulation

“…Nef influences cellular signaling pathways leading to the enhancement of viral replication, immune elusion and enhanced survival in T-cells and macrophages [12]. It also widely affects the innate immune system impairing oxidative burst response and phagocytosis in monocytes/macrophages from HIV-1 patients [14]–[16]. In this regard, studies on human alveolar macrophages from HIV-1 infected individuals demonstrate an impaired phagocytosis of Pneumocystis Carinii [44] that is also associated to a reduced oxidative burst response to the pathogen in vitro challenge [45].…”

“…Nef protein can affect the innate immune system impairing oxidative burst response and phagocytosis in monocytes/macrophages from HIV-1 patients [14]–[16]. Moreover, Nef induces the secretion from primary human monocyte/macrophages of chemotactic factors like the CC-chemokines CCL2 and CCL4 [17], which correlate with the activation of AP-1, NF-κB, STAT1 and STAT3 transcription factors [18]–[22].…”