HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

Abstract

BackgroundHIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted 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 different signalling cascades that require 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-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.Methodology/Principal FindingsSearching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2.ConclusionsResults reported here revealed 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-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.

Exosomes from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells License Quiescent CD4 + T Lymphocytes To Replicate HIV-1 through a Nef- and ADAM17-Dependent Mechanism

“…The results we obtained allowed us to exclude the possibility that the lack of effects on resting lymphocytes of exosomes from Nef 4EA -expressing cells depended on the intrinsic instability of the Nef mutant or a defect in its incorporation in exosomes. Of note, and as previously reported (12), in SDS-PAGE analysis, Nef 4EA migrates faster than the wt counterpart.…”

“…To produce HIV-1 pseudotyped with the G protein from vesicular stomatitis virus (VSV-G), a cytomegalovirus (CMV) immediate early-promoted VSV-G-expressing vector was cotransfected in a molar ratio of 1:5 with vectors expressing either wild-type (wt), ⌬nef (11), or Nef 4EA HIV-1. The latter molecular clone was obtained by amplifying the pcDNA3/Nef 4EA vector (12) with primers carrying the MluI (forward) and ClaI (reverse) restriction sites. The amplification product was then inserted in the respective restriction sites of a pNL4-3 clone where MluI and ClaI sites were created at the 5= and 3= ends of the nef gene (13).…”

“…Infections with HIV-1 were carried out by spinoculation at 400 ϫ g for 30 min at room temperature (RT). For the production of exosomes from 293T-transfected cells, IE-CMVpromoted expression vectors expressing either ADAM17 (8), wt Nef (15), or Nef 4EA (12) were used. Azidothymidine (AZT) was obtained from the NIH AIDS Research and Reference Reagent Program.…”

HIV-1 Nef Interacts with HCV Core, Recruits TRAF2, TRAF5 and TRAF6, and Stimulates HIV-1 Replication in Macrophages

“…Additionally, HCV Core, TRAF2, TRAF5 and TRAF6 can be recruited to lipid rafts and have been reported to activate NF-κB [31,44]. Recently, HIV-1 Nef has been reported to induce a proinflammatory state in macrophages through its acidic cluster domain and the involvement of TRAF2 [45]. Our results expand these previous observations by indicating that both HIV-1 Nef and HCV Core bind to several members of the TRAF family, namely TRAF2, TRAF5 and TRAF6.…”

A variant in IRF3 impacts on the clinical outcome of AML patients submitted to Allo-SCT

“…11 Thus, after infectious or non-infectious injury stimulation, TLR3 and TLR4 initiate TLR adaptor TICAM-1 (TRIF)-dependent stimulus to activate intracellular signaling cascades that converge on NF-kappaB and IFN regulatory factor-3 (IRF3) activation, inducing production of pro-inflammatory cytokines. [12][13][14][15] Besides, IRF3 activation through TRIF signaling in BM-derived DCs results in natural killer (NK) cell activation by direct cell-cell contact inducing an antitumor NK response. 16,17 We and others have previously analyzed the clinical impact of gene polymorphisms of the innate immune system on the outcome of allo-SCT.…”