Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay
2011
GONZALEZ , GAELLE (INRA , Lyon (France). UMR 0754 Rétrovirus et Pathologie Comparée) | DaFonseca , Sandrina (Institut National de la Recherche Agronomique, Lyon(France). UMR0754 : Rétrovirus et Pathologie Comparée (RPC)) | Errazuriz , Elisabeth (Université de Lyon 1, Lyon(France). Faculté de Medicine, Centre Commun d’Imagerie Laennec) | Coric , Pascale (Centre National de la Recherche ScientifiqueUniversité Paris Descartes (Paris 5), ParisParis(France). UMR8015 Laboratoire de cristallographie et RMN biologiques UFR des Sciences Pharmaceutiques et Biologiques) | Souquet , Florence (Centre National de la Recherche ScientifiqueUniversité Paris Descartes (Paris 5), ParisParis(France). UMR8638 Synthèse et Structure de Molécules d'intérêt Pharmacologique UFR des Sciences Pharmaceutiques et Biologiques) | Turcaud , Serge (Centre National de la Recherche ScientifiqueUniversité Paris Descartes (Paris 5), ParisParis(France). UMR8638 Synthèse et Structure de Molécules d'intérêt Pharmacologique UFR des Sciences Pharmaceutiques et Biologiques) | BOULANGER , Pierre (INRA , Lyon (France). UMR 0754 Rétrovirus et Pathologie Comparée) | Bouaziz , Serge (auteur de correspondance) (Centre National de la Recherche ScientifiqueUniversité Paris Descartes (Paris 5), ParisParis(France). UMR8015 Laboratoire de cristallographie et RMN biologiques UFR des Sciences Pharmaceutiques et Biologiques) | HONG , Saw-See(auteur de correspondance) (INRA , Lyon (France). UMR 0754 Rétrovirus et Pathologie Comparée)
The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and epsilon-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.
Show more [+] Less [-]Bibliographic information
This bibliographic record has been provided by Institut national de la recherche agronomique