HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner

Abstract

27 kDa accessory HIV and simian immunodeficiency virus (SIV) protein necessary for efficient virus replication and high viral load, has for several years been considered as a progression factor to AIDS (Greene and Peterlin, 2002). According to several excellent reviews, a variety of diverse functions including downregulation of cell-surface molecules have been assigned to the protein (Das and Jameel, 2005;Piguet and Trono, 1999;Roeth and Collins, 2006). Moreover, HIV-associated dementia (HAD) correlates with infiltration of monocytes into the brain. The accessory HIV-1 negative factor (Nef) protein, which modulates several signaling pathways, is constitutively present in persistently infected astroctyes. We demonstrated that monocytes responded with chemotaxis when subjected to cell culture supernatants of nef-expressing astrocytic U251MG cells. Using a protein array, we identified CC chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) as a potential chemotactic factor mediating this phenomenon. CCL2/MCP-1 upregulation by Nef was further confirmed by ribonuclease protection assay, RT-PCR and ELISA. By applying neutralizing antibodies against CCL2/MCP-1 and using CCR2-deficient monocytes, we confirmed CCL2/MCP-1 as the exclusive factor secreted by nefexpressing astrocytes capable of attracting monocytes. Additionally, we showed that Nef-induced CCL2/MCP-1 expression depends on the myristoylation moiety of Nef and requires functional calmodulin. In summary, we suggest that Nef-induced CCL2/MCP-1 expression in astrocytes contributes to infiltration of monocytes into the brain, and thereby to progression of HAD.

HIV-1 Nef Inhibits Lipopolysaccharide-induced IL-12p40 Expression by Inhibiting JNK-activated NFκB in Human Monocytic Cells

“…Recombinant myristoylated HIV-nef enhanced the release of interferon-␤ in monocytes/macrophages through the activation of MAPKs, IB kinase and IRF-3 (52). HIV-nef was also shown to up-regulate CCL2/MCP-1 expression through calmodulin activation (53). In contrast, HIV-nef was shown to inhibit macrophagecolony stimulating factor receptor signaling through Hck activation in monocytes/macrophages (54).…”

Chemokine (C-C Motif) Receptor 1 Is Required for Efficient Recruitment of Neutrophils during Respiratory Infection with Modified Vaccinia Virus Ankara

“…Chemotaxis assay. Chemotaxis assays were performed as previously described (30). Briefly, freshly isolated neutrophils were resuspended at a concentration of 5 ϫ 10 6 cells/ml in HBSS and allowed to migrate for 40 min in a 96-well Multi-Screen-MIC plate (3-m pore size; Merck Milli- pore, Billerica, MA).…”

Modified Vaccinia Virus Ankara Triggers Chemotaxis of Monocytes and Early Respiratory Immigration of Leukocytes by Induction of CCL2 Expression

“…Monocytic THP-1 cells were assayed in 96-well MultiScreen-MIC plates equipped with hydrophilic polycarbonate filters (8.0-m pore size; Millipore Corp., Billerica, MA) as previously described (22). Cells that had migrated into the lower chambers were subsequently fixed with 1% paraformaldehyde, and the number of was cells determined by means of a BD LSR II flow cytometer and BD TruCount beads.…”

“…To begin with, supernatants of MVA-infected and mock-infected cells were incubated either with antibodies certified to exclusively neutralize the chemotactic activity of CCL2 or with isotype-matched control antibodies. Next, a chemotaxis assay with naive THP-1 cells, previously shown to express the CCR2 receptor (22), was undertaken. Cell migration induced by supernatants both of MVA-infected THP-1 cells and of primary human monocytes was significantly reduced by the antibody neutralizing the chemotactic activity of CCL2 and reached the level achieved with supernatants of mock-infected cells.…”