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

Jerome Bouchet, Cecile Herate, Carolin A. Guenzei, Christel Verollet, Annika Jarviluoma, Julie Mazzolini, Salomeh Rafie, Patrick Chames, Daniel Baty, Kalle Saksela, Florence Niedergang, Isabelle Maridonneau-Parini, Serge Benichou

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)4856-4867
Number of pages12
JournalJournal of Virology
Volume86
Issue number9
DOIs
Publication statusPublished - May 2012

Keywords

  • I DOWN-REGULATION
  • VIRUS TYPE-1 NEF
  • SH3 DOMAINS
  • 3-DIMENSIONAL MIGRATION
  • CYTOPLASMIC TAIL
  • HIGH-AFFINITY
  • INDUCED CD4
  • COMPLEX
  • BINDING
  • PROTEIN

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