In Vitro Treatment of Human Monocytes/Macrophages with Myristoylated Recombinant Nef of Human Immunodeficiency Virus Type 1 Leads to the Activation of Mitogen-Activated Protein Kinases, IκB Kinases, and Interferon Regulatory Factor 3 and to the Release of Beta Interferon

Abstract

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-B and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the ␣ and ␤ subunits of the IB kinase complex and of JNK, ERK1/2, and p38 mitogenactivated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.The 27-to 34-kDa Nef protein is an important virulence factor of primate lentiviruses; it is the regulatory protein expressed earliest and most abundantly in the infection cycle. Studies of animal models and seropositive patients showed that Nef-defective viruses led to an attenuated clinical phenotype with a reduced viral load (29,30,50,59,60). In addition, nef transgenic mice develop an AIDS-like disease (51) characterized by failure to thrive/weight loss, diarrhea, wasting, premature death, thymus atrophy, loss of CD4 ϩ T cells, interstitial pneumonitis, and tubulointerstitial nephritis. Inside the cell, Nef induces effects that are genetically distinguishable yet highly conserved and that appear to be mediated via specific protein-protein interaction domains (7,33,44). Nef is cotranslationally modified by an N-terminal myristoylation site whose lipidation is required for membrane association. However, cellular-fractionation assays from transient transfections showed that less than 50% of the protein was localized at membranes, while the remaining portion was found to be cytosolic (25,34,58,68,98). The protein adopts a two-domain structure that is characterized by a flexible N-terminal arm of about 60 amino acids, followed by a well-conserved and folded core...

HIV-1 Proteins Preferentially Activate Anti-Inflammatory M2-Type Macrophages

“…Using the widely used method to prepare M2-MF (M-CSF derived) and M1-MF (GM-CSF derived) (13-16), we examined whether soluble Tat, gp120, or Nef differentially activated these MF by comparing the activation of MAPK, such as p38, JNK, and ERK. As reported (18)(19)(20)(21)(22)(23)(24)(25)(26)(27), all soluble HIV-1 proteins clearly activated p38 (Supplemental Fig. 1B).…”

“…It was shown that soluble Nef enters MF or dendritic cells and accumulates in perinuclear regions (41,42) through endocytosis, pinocytosis, or as-yet-unknown mechanisms (26). Of importance, we found that FITC-labeled Nef entered M2-MF more rapidly than it entered M1-MF (Fig.…”

“…Soluble Nef was shown to activate MAPK and NF-kB pathways in MF (25)(26)(27). However, the in vitro preparations of differentiated MF used in these studies varied; for instance, monocytes were cultured with GM-CSF and then FCS alone (26) or were cultured with a high concentration of FCS alone (27). To our knowledge, the current study is the first report in which the response to exogenous Nef was compared between two major MF populations.…”

“…We also analyzed the activation of the NF-kB pathway. In NF-kB pathway activation, IKKa/b are activated through serine phosphorylation, which phosphorylates IkB, leading to its degradation and the nuclear translocation of NF-kB (26). Thus, we…”

“…1A), but such sustained ERK activation has been found in several stimuli such as TPA-induced megakaryocytic differentiation of K562 cells (57)(58)(59). Soluble Nef was shown to activate MAPK and NF-kB pathways in MF (25)(26)(27). However, the in vitro preparations of differentiated MF used in these studies varied; for instance, monocytes were cultured with GM-CSF and then FCS alone (26) or were cultured with a high concentration of FCS alone (27).…”