Human immunodeficiency virus type 1 (HIV-1) Nef activates STAT3 in primary human monocyte/macrophages through the release of soluble factors: involvement of Nef domains interacting with the cell endocytotic machinery

Abstract

Increasing evidence indicates that the expression of the human immunodeficiency virus-1 (HIV-1) Nef protein significantly influences the activation state of the host cell. Here we report that Nef specifically activates STAT3 in primary human monocyte-derived macrophages (MDM). This was demonstrated by both single-cycle infection experiments driven by Vesicular Stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 and treatment with exogenous recombinant Nef. The analysis of the effects of Nef mutants revealed that domains of the C-terminal flexible loop interacting with the cell endocytotic machinery are involved in the STAT3 activation. In particular, our data suggest that the Nef-dependent STAT3 activation relies on the targeting of Nef to the late endosome/lysosome compartment. In addition, we found that Nef activates STAT3 through a mechanism mediated by the release of soluble factor(s), including MIP-1alpha, that requires de novo protein synthesis but appears independent from the activation of src tyrosine kinases. The results presented here support the idea that the first intervention of Nef in the intracellular signaling of monocyte-macrophages could generate, by means of the release of soluble factor(s), a secondary wave of activation that could be of a potential pathogenetic significance.

HIV-1 Nef Transfer and Intracellular Signalling in Uninfected Cells

“…Overall the experimental results indicated that myristoylation of the protein was required for the activation of the signaling cascades, because G 2 →A and ΔN-term Nef were the only two Nef mutants unable to induce signalling. These data were in agreement with lack of induction of STAT1, STAT2 and STAT3 tyrosine phosphorylation in MDMs 8 to 16 h after infection with VSV-G Δenv HIV-1 pseudotypes expressing the G 2 →A Nef mutant (Mangino et al, 2007;Percario et al, 2003). As previously mentioned myristoylation of Nef is a weak membrane-targeting signal and N-terminal basic residues, especially an arginine-rich cluster (R 17 to R 22 ), are needed for the stable association of the viral protein with cellular membranes.…”

“…Nef treatment of primary human MDMs down-regulated CD4 surface expression, thus reproducing an effect widely observed in cells endogenously expressing the viral protein. In addition, myr+ Nef treatment of MDMs induces the rapid (15-30') activation of IKK/NF-B, MAPKs (i.e., ERK1/2, JNK and p38) and IRF-3, the main transcriptional regulator of the IFN gene expression, thereafter regulating the expression of many cellular transcripts (Federico et al, 2001;Mangino et al, 2007;Olivetta et al, 2003;Percario et al, 2003). The prompt transcriptional reprogramming leads in 2 hours to the synthesis and release of a set of proinflammatory cytokines/chemokines, including TNF, IL-1, IL-6, CCL2/MIP-1 and CCL4/MIP-1, and of IFN that, in turn, immediately activate the signal transducers and activators of transcription STAT1, -2 and -3 in autocrine and paracrine manner.…”

Macrophages and HIV-1: dangerous liaisons

“…The secretion of inflammatory chemokines and cytokines is associated with the activation of the transcription factors NF-B and AP-1 Varin et al, 2003). The release of soluble factors, including CCL3, induced by exogenous Nef is also involved in the activation of STAT elements 1 and 3, which may contribute to the deregulation of signalling pathways associated with cell survival or the IFN response (Federico et al, 2001;Percario et al, 2003). (Chougnet, 2003).…”

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

“…In any case, the cell reprogramming induced by Nef is not only limited to manipulation of apoptosis. It also includes the activation or suppression of cell signaling pathways (Mahlknecht et al, 2000;Mangino et al, 2007;Percario et al, 2003;Varin et al, 2003). Although the effects of Nef sometimes seem contradictory it must be stressed out that its functions are the product of many protein-protein interactions that can take place during different steps of the viral replication cycle in the infected cell and are subjected to Nef cellular trafficking and conformational status.…”

“…This effect can take place in many cell types as promonocytic cells, Monocyte-Derived Macrophages (MDMs), and DCs Quaranta et al, 2006;Varin et al, 2003). The soluble mediators synthesized as the result of the activation of these pathways, as IL-6 and MIP-1alpha, activate JAK, leading to dimmerization of STAT1 and 3 (Mangino et al, 2007;Percario et al, 2003). Also, as Nef activates IRF3, leading to the synthesis of IFNbeta, it also leads to STAT2 activation (Mangino et al, 2007).…”