In vitro treatment of human monocyte-derived macrophages with the HIV-1 regulatory protein Nef induces a rapid activation of the NF-kB pathway.

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phase of HIV replication. Nef expression in cellular model systems induces downregulation of CD4 and MHC-I, alteration of T cell receptor signaling, pro-apoptotic effects in uninfected bystander cells and anti-apoptotic effects in infected cells. In addition, Nef expression in human primary monocyte-derived macrophages (MDM) induces a remarkable modifications in the pattern of secreted factors that appear able to recruit and activate T lymphocytes rendering them susceptible to HIV infection. We have observed that Nef induces the activation of the signal transducers and activator of transcription STAT1 and STAT3 in hu MDM purified from PBMC of healthy donors via the release of factor(s). Treatment of MDM with recNef induces also the activation of the NF-kB pathway in a cycloheximide-independent way (Federico et al., Blood 2001, 98:2752; Olivetta et al., J. Immunology 2003, 170:1716; Percario et al., J. Leuk. Biology 2003, 74:21). To gain more insights into the activation of NF-kB, we have used as experimental system both MDM and the human monocytic cell line THP-1. In particular, a rapid NF-kB activation is detected treating those cells with recNef. NF-kB activation has been evaluated by EMSA analyzing the induction of specific NF-kB DNA binding complexes and has been shown Nef-specific as demonstrated treating MDM with Nef-immunodepleted medium. We also observed a transient phosphorylation of the alfa and beta subunits of the Ik-B kinase complex (IKKalfa and IKKbeta). Finally a specific IKKs inhibitor is able to inhibit the Nef-dependent STAT1and STAT3 activation in THP-1 cells, suggesting that the activation of the NF-kB pathway is required for the subsequent production of STATs activating factors.