Abstract
BackgroundThe HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies.ResultsWe have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication.ConclusionsTogether, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.
Highlights
The human immunodeficiency virus (HIV)-1 Negative factor (Nef) protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors
Architecture of the Nef–Single domain antibody 19 (sdAb19)–SH3B6 complex The Nef–antibody complex was formed by mixing a purified recombinant form of HIV-1 NefSF2 (45–210) deleted of the first 44 N-terminal residues with sdAb19, and adding the SH3 domain of human Hck, termed SH3B6 and engineered for high affinity binding to Nef, to this complex [17,18]
Here we define the structural basis of HIV-1 Nef inhibition by the camelid-derived sdAb19 antibody fragment. sdAb19 binds to a C-terminal surface epitope on Nef that overlaps with multiple interaction sites of the viral protein
Summary
The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Of the fifteen proteins encoded by the HIV genome, the three viral enzymes, protease, integrase and reverse transcriptase are indispensable for the production of viral progeny These enzymes are core targets of highly active anti-retroviral therapy (HAART) together with proteins mediating virus entry [2,3]. Nef affects signal transduction through interaction with cellular kinases like Pak and Hck to modulate signaling pathways in infected cells [9,10]. To achieve this multitude of activities, Nef has evolved as a versatile adaptor for protein interactions that lacks intrinsic enzymatic activity. The structure of HIV-1 Nef is characterized by its flexible loop regions that contain several sequence motifs as an N-terminal myristoylation site, a central poly-proline PxxP motif for SH3 domain binding and C-terminal motifs for interaction with clathrinassociated endosomal adaptor protein complexes [11]
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