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.
Highlights
The majority of antivirals against HIV-1 target the following steps of virus-cell interaction : (i) fusion between virus envelope and cell plasma membrane, (ii) reverse transcription of the viral genomic RNA, (iii) provirus integration into the host genome, and (iv) viral protease (PR)-mediated processing of HIV-1 polyprotein precursors
This suggested that (i) the luciferase-Vpr fusion protein (LucVpr) fusion protein was competent for packaging into virus-like particles (VLP), and (ii) that the luciferase moiety of LucVpr retained its enzymatic activity after fusion with Vpr
We have shown that the p6 domain is dispensable for VLP budding and egress from recombinant baculovirus-infected insect cells [12,32,33,53], and, interestingly, that VLP constituted of p6deleted Gag precursor molecules (GagDp6 of 47 kDa) showed a more regular shape and higher sphericity than VLP constituted of wild type (WT) Pr55Gag [28]
Summary
The majority of antivirals against HIV-1 target the following steps of virus-cell interaction : (i) fusion between virus envelope and cell plasma membrane, (ii) reverse transcription of the viral genomic RNA, (iii) provirus integration into the host genome, and (iv) viral protease (PR)-mediated processing of HIV-1 polyprotein precursors (reviewed in [1,2]). The accumulation of immature, noninfectious virus particles due to unprocessed or partially processed polyprotein precursors could be provoked by anti-PR inhibitors, or by a novel class of antivirals derived from betulinic acid [3]. The prototype of this family of antiviral drugs is the 3-O(3’,3’-dimethylsuccinyl)-betulinic acid, abbreviated DSB, known as YK-FH312 [4] or PA-457 [5], and used in the clinics as BevirimatTM [5,6,7,8,9,10]. Further confirmation of the structural effects of PA-457 was provided in a recent study, showing that PA-457 used at micromolar concentrations freezes the lattice of protein shell underlying the HIV-1 envelope in its immature configuration [13]
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