The conserved acidic cluster domain of HIV-1 Nef is required to induces a proinflammatory state in primary macrophages: involvement of TNF alpha Receptor Associated Factor 2

Background: HIV-1 Nef is a virulence factor of primate immunodeficiency viruses. It acts as a molecular adaptor that interacts with and influence the activity of many cellular factors. It has been proposed that Nef intersects the CD40 signalling in macrophages, leading to the production of factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of signalling cascades involving the recruitment of specific Tumor Necrosis Factor Receptor Associated Factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF- ? B, MAPKs and IRF-3, that induce the synthesis and secretion of proinflammatory chemokines/cytokines in Nef-treated macrophages. Principal Findings: We have found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster of Nef. In pull-down experiments Nef interacts in vitro with TRAF2, but not TRAF6. We have expressed a 4E -> A recNef mutant that retains its overall structure measured by circular dichroism and its ability to interact with GST-HckSH3 Using this mutant we show that all the signalling effects we observed in macrophages treated with wt Nef require the integrity of the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that TRAF2 and TRAF6 are both required for the tyrosine phosphorylation of STAT1 and STAT2 induced by the production of proinflammatory cytokines in Nef-treated cells . Conclusions: The results reveals TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF- ? B and TRAF/IRF-3 signalling axes.