HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

Abstract

Macrophages are a major target of HIV-1 infection. HIV-1–infected macrophages form multinucleated giant cells (MGCs) using poorly elucidated mechanisms. In this study, we show that MGC formation was reduced when human macrophages were infected with nef-deleted HIV-1. Moreover, expression of Nef, an HIV-1 protein required in several aspects of AIDS, was sufficient to trigger the formation of MGCs in RAW264.7 macrophages. Among Nef molecular determinants, myristoylation was dispensable, whereas the polyproline motif was instrumental for this phenomenon. Nef has been shown to activate hematopoietic cell kinase (Hck), a Src tyrosine kinase specifically expressed in phagocytes, through a well-described polyproline–SH3 interaction. Knockdown approaches showed that Hck is involved in Nef-induced MGC formation. Hck is expressed as two isoforms located in distinct subcellular compartments. Although both isoforms were activated by Nef, only p61Hck mediated the effect of Nef on macrophage fusion. This process was abolished in the presence of a p61Hck kinase-dead mutant or when p61Hck was redirected from the lysosome membrane to the cytosol. Finally, lysosomal proteins including vacuolar adenosine triphosphatase and proteases participated in Nef-induced giant macrophage formation. We conclude that Nef participates in HIV-1–induced MGC formation via a p61Hck- and lysosomal enzyme-dependent pathway. This work identifies for the first time actors of HIV-1–induced macrophage fusion, leading to the formation of MGCs commonly found in several organs of AIDS patients.

Placental Macrophages Are Impaired in Chorioamnionitis, an Infectious Pathology of the Placenta

“…Because myeloid MGCs are characterized by specific concentrations of F-actin and the organization of their microtubules (17,30), we examined the cytoskeleton in placental MGCs. In pMw, F-actin was concentrated in dotted structures with a characteristic vinculin ring called podosomes (Fig.…”

“…The cytoskeletal organization of placental cells was studied as described previously (30). Briefly, pMw and MGCs were permeabilized with 0.3% Triton X-100, incubated with mouse monoclonal anti-vinculin or anti-tubulin Abs (Sigma-Aldrich) for 1 h, and then incubated with Alexa 488-conjugated secondary Abs, phalloidin-Texas Red (Invitrogen), which specifically binds F-actin, and DAPI (Invitrogen) for 30 min.…”

HIV-1–Mediated BAFF Secretion in Macrophages Does Not Require Endosomal TLRs, Type-I IFN, and Nef, but Depends on the Cellular Phenotype Status

“…Virus particles were produced by calcium-phosphate transfection of 293T cells, as described previously (37), using a plasmid coding for R5-tropic Bal Env glycoproteins inserted within the NL4-3 backbone (NL4-3Balenv) (38), a plasmid coding for wild type R5-tropic ADA or the same molecular clone of HIV-1 disrupted for the Nef open reading frame (ADADnef) (39,40). Cell-free supernatants were ultracentrifugated to eliminate free p24, resuspended in endotoxin-free PBS (Sigma-Aldrich), and quantified by an in-house double-Ab sandwich ELISA specific for the major core p24 protein (41).…”

Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions

“…Nef is able to activate both Hck isoforms, leading to the formation of actin-rich protrusions and podosome rosettes (43). By activating p61Hck, Nef also triggers the fusion of individual infected macrophages, leading to the formation of giant multinucleated cells that could contribute to the establishment of latent virus reservoirs in some tissues (40). In addition, we recently showed that Nef expression in HIV-1-infected macrophages also resulted in a significant diminution of their phagocytosis ability (28).…”

“…Infected macrophages also participate in the propagation of virus in nonlymphoid tissues, such as lungs or brain. Furthermore, we have recently shown that Nef expression during HIV-1 infection disturbs specialized functions of macrophages: (i) Nef induces the fusion of infected macrophages, leading to the formation of multinucleated giant cells through the activation of the Hck tyrosine kinase (40), and (ii) Nef also inhibits phagocytosis through the alteration of the polarized exocytosis of recycling endosomes regulated by AP-1 (28).…”