title: Single-domain antibody-SH3 fusions for efficient neutralization of HIV-1 Nef functions.
abstract: HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.
authors.affiliation: Institut Cochin, CNRS (UMR8104), Université Paris Descartes, Paris, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Bouchet
authors.forename: Cécile
authors.initials: C
authors.lastname: Hérate
authors.forename: Carolin A
authors.initials: CA
authors.lastname: Guenzel
authors.forename: Christel
authors.initials: C
authors.lastname: Vérollet
authors.forename: Annika
authors.initials: A
authors.lastname: Järviluoma
authors.forename: Julie
authors.initials: J
authors.lastname: Mazzolini
authors.forename: Salomeh
authors.initials: S
authors.lastname: Rafie
authors.forename: Patrick
authors.initials: P
authors.lastname: Chames
authors.forename: Daniel
authors.initials: D
authors.lastname: Baty
authors.forename: Kalle
authors.initials: K
authors.lastname: Saksela
authors.forename: Florence
authors.initials: F
authors.lastname: Niedergang
authors.forename: Isabelle
authors.initials: I
authors.lastname: Maridonneau-Parini
authors.forename: Serge
authors.initials: S
authors.lastname: Benichou
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation / Q000276:immunology; D023061:Gene Order; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008969:Molecular Sequence Data; D010587:Phagocytosis / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D021381:Protein Transport; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics / Q000276:immunology
chemical_list: D000998:Antiviral Agents; D015704:CD4 Antigens; D015395:Histocompatibility Antigens Class I; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D018808:Transcription Factor AP-1; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051572:Proto-Oncogene Proteins c-hck