Gonzalez, G., DaFonseca, S., Errazuriz, E., Coric, P., Souquet, F., Turcaud, S., Boulanger, P., Bouaziz, S. and Hong, S. S. (2011) Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative Luciferase-Vpr packaging-based assay. PLoS ONE, 6(11), e27234. (doi: 10.1371/journal.pone.0027234) (PMID:22073298) (PMCID:PMC3207847)
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Abstract
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 ε-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.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Gonzalez, Ms Gaelle |
| Authors: | Gonzalez, G., DaFonseca, S., Errazuriz, E., Coric, P., Souquet, F., Turcaud, S., Boulanger, P., Bouaziz, S., and Hong, S. S. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
| Journal Name: | PLoS ONE |
| Publisher: | Public Library of Science |
| ISSN: | 1932-6203 |
| ISSN (Online): | 1932-6203 |
| Copyright Holders: | Copyright © 2011 The Authors |
| First Published: | First published in PLoS One 6(3):e27234 |
| Publisher Policy: | Reproduced under a Creative Commons License |
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