Characterization of a Novel Type of HIV-1 Particle Assembly Inhibitor Using a Quantitative Luciferase-Vpr Packaging-Based Assay

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.

Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site

“…Moreover, for the first time, a direct interaction between compound 16 and the peptide CA-SP1eNC was proved by NMR. More recently, we showed that compound 16 was able to generate an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100 nm in diameter [37]. All together, these data constitute a first step in the understanding of the mechanism by which bevirimat inhibits HIV-1 assembly and maturation as proposed in a previous model [27,38].…”

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

“…AcMNPV gag expresses the full-length, wild-type, N-myristoylated HIV-1 Gag polyprotein in Sf9 cells, resulting in the assembly and extracellular release of membrane-enveloped VLPs at high yields. The high number of particles produced per cell in this heterologous model system of VLP assembly and egress, allowed quantitative analyses of morphologically normal VLPs compared to aberrant particles 31 , 33 , 48 – 51 .…”

“…S1). The aim of the N-myristoylation of αRep4E3-GFP and αRep9A8-GFP was to compensate for the difference in cell content between the N-myristoylated Pr55Gag polyprotein, expressed at high levels under the control of the strong polyhedrin promoter 31 , 33 , 48 – 51 , and αRep proteins, expressed at lower levels under the control of the weaker OpIE2 promoter. Addressing (Myr+)αRep proteins to the plasma membrane was meant to promote the interaction between Pr55Gag and αRep proteins at the assembly sites of membrane-enveloped VLPs.…”

“…Control, nonmodified Sf9 cells, Sf/(Myr+)αRep4E3-GFP and Sf/(Myr+)αRep9A8-GFP cells were infected with AcMNPV gag , and the amounts of VLPs assembled and released into the culture medium were estimated by SDS-PAGE and Western blot analysis of pelletable, extracellular Pr55Gag 31 – 33 . The intensity of the Pr55Gag band on immunoblots did not radically differ between the three samples (data not shown).…”

A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

“…However, chemiluminescent signals obtained from this protein were very low, consistent with our own experience that HIV-1 Gag-Luciferase fusion proteins are poorly incorporated into VLPs in mammalian cells (unpublished observations). Recently, a Vpr-firefly luciferase fusion has been used to monitor HIV-1 particle release in a baculovirus-based Gag and Vpr expression system (Gonzalez et al, 2011). However this is obviously a non-native context, with highly overexpressed Gag and Vpr proteins.…”