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.

Figure 1. Packaging of Vpr and LucVpr into HIV-1 VLP produced in Sf9 cells.

Sf9 cells were infected with (-) AcMNPV-Pr55Gag alone, or (+) coinfected with AcMNPV-Pr55Gag and another recombinant baculovirus expressing (a) Vpr or (b, c )Luciferase-Vpr fusion protein (LucVpr). Both Vpr and LucVpr were tagged with the His₆ epitope. VLP were isolated from the culture medium at 48 h pi, using ultracentrifugation in sucrose-D₂0 density gradient, and each gradient fraction analysed by SDS-PAGE and standard immunoblotting. (a), Western blot reacted with anti-Gag polyclonal antibody and peroxidase-labeled anti-rabbit IgG antibody, followed by monoclonal anti-His₆ tag and phosphatase-labeled anti-mouse IgG antibody. Pr55Gag polyprotein is revealed in brown, Vpr protein (14 kDa) in blue (lanes 1, 2). (b), Western blot reacted with anti-Gag polyclonal antibody and phosphatase-labeled anti-rabbit IgG antibody, followed by monoclonal anti-His₆ tag and peroxidase-labeled anti-mouse IgG antibody. Pr55Gag polyprotein is in blue, LucVpr protein (72 kDa) is in brown (lanes 3, 4). (c), Autoradiogram of dried SDS-gel of ³⁵S-labeled VLP released from control AcMNPV-Pr55Gag-infected cell cultures (lane 5), or from AcMNPV-Pr55Gag+AcMNPV-LucVpr-coinfected cell cultures, both labeled with ³⁵S-methionine and ³⁵S-cysteine. Lane m, PageRuler™ pretained protein ladder (Fermentas Inc.). Lane m', Dual Color™ molecular markers (BioRad). Molecular masses are indicated in kiloDaltons (kDa). (*), Asterisk indicates the position of the mono-ubiquitinated Gag polyprotein of 62 kDa, detected by its positive reaction with anti-ubiquitin antibody (not shown).

Figure 2. Gag-p6 domain-dependence of LucVpr packaging.

Isopycnic ultracentrifugation analysis in sucrose-D₂0 density gradient of extracellular VLP isolated from Sf9 cell culture medium. (a), Western blot of the gradient fractions analyzed by SDS-PAGE. Blot was reacted with anti-Gag polyclonal antibody and phosphatase-labeled anti-rabbit IgG antibody. Lane m, prestained molecular mass markers (PageRuler™; Fermentas Inc.). The position of VLP (fractions 10–13; 1.12–1.15 in density) is indicated at the top of the panel. (b), Luciferase activity was assayed on each gradient fraction, and expressed as relative light units (RLU). Open symbol, Sf9 cells expressing LucVpr alone; filled symbols, Sf9 cells coexpressing LucVpr and full-length Pr55Gag or p6-deleted Gag (GagΔp6). Each gradient fraction was assayed for luciferase activity. Density values are indicated on the right scale.

Figure 3. Quantification of VLP assembly and egress using luciferase assay.

(a), Ultracentrifugation analysis of VLP. Cells coexpressing Pr55Gag and LucVpr were untreated (control 0) or treated with PA-457 in DMSO for 24 h at 24 h pi, at increasing concentrations as indicated. VLP were isolated from the culture medium at 48 h pi by isopycnic ultracentrifugation in sucrose-D₂0 density gradient, and assayed for luciferase activity, expressed as relative light units (RLU). (b), Dose-response curve of PA-457 inhibitory effect on VLP production. The ratio of VLP-associated to intracellular luciferase activity was plotted versus PA-457 concentrations. The IC₅₀ value obtained was 2.2–2.4 µg/ml. Inset : VLP production (top) and intracellular expression of Pr55Gag (bottom) were evaluated in parallel by Western blot analysis using anti-Gag rabbit antibody and phosphatase-labeled conjugate.

Figure 4. Efficiency of packaging of LucVpr into VLP.

(a), Autoradiogram of ³⁵S-labeled VLP . VLP were isolated from the culture medium of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-Vpr (leftmost half of the panel), or AcMNPV-Pr55Gag and AcMNPV-LucVpr (rightmost half of the panel). After purification by ultracentrifugation in sucrose-D₂0 density gradient, VLP were analyzed by SDS-PAGE, and autoradiography of the dried gel. The position of Pr55Gag and its major cleavage products Pr41Gag, CAp24 and MAp17 are indicated, as well as Vpr, LucVpr and the monobiquitinated form of Pr55Gag (Ub-Gag). (b) Western blot analysis. VLP were analyzed by SDS-PAGE as above, followed by Western blot using anti-Histidine tag antibody and phosphatase-labeled conjugate. Only the portion of the blot showing the Vpr protein of 14 kDa is presented. (c) Graphic representation of the Gag:LucVpr ratios as a function of the inhibitor concentrations. Quantification of the VLP content of Pr55Gag, Pr41Gag, CAp24, MAp17, and LucVpr proteins was performed by excision of their corresponding ³⁵S-labeled band from SDS-gel as shown in (a), and counting radioactivity in scintillation spectrometer. After correction for the respective number of methionine and cysteine residues in proteins, the values of the Gag:LucVpr ratio were plotted versus the PA-457 concentrations.

Figure 5. Structure of betulinic acid derivatives.

Note that only carbon-3 and carbon-28 are numbered on the PA-457 formula. Compounds ST-327, EP-48, EP-39, EP-47 and EP-62 are schematically represented by their only difference with the leader compound PA-457, i.e. the substituant which amidifies the acidic function carried by carbon-28.

Figure 6. Evaluation of the inhibitory activity of BA, PA-457, ST-327, EP-47 and EP39 on VLP assembly, using luciferase-Vpr packaging-based assay.

Aliquots of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-LucVpr at equal MOI were treated at 24 h pi with increasing doses of each inhibitor for 24 h. Cells and culture medium were harvested at 48 h pi, and VLP isolated from the culture medium. Cell pellets and VLP were then processed for luciferase assay, and the values of the ratio of VLP-incorporated to cell-associated luciferase activity, in percentage of the control (0 inhibitor), were plotted versus the PA-457 concentrations. In control samples, the fraction of VLP-incorporated luciferase was usually 5 to 7% of the total activity recovered. E.g., in the experiment illustrated here, the activity recovered was 35.5×10⁶ RLU in cell pellets, versus 2.5×10⁶ in extracellular VLP. Note that the inhibition curve of EP-62, which resembled that of ST-327, was not represented for reason of clarity.

Figure 7. Effect of EP-39 on sedimentation properties of VLP.

Cells coexpressing Pr55Gag and LucVpr were untreated (control) or treated with EP-39 (10 mg/ml in DMSO) for 24 h at 24 h pi. VLP were recovered from the culture medium at 48 h pi by ultracentrifugation through a sucrose cushion, and the VLP pellet further analyzed by isopycnic ultracentrifugation in sucrose-D₂0 density gradient. Gradient fractions were assayed for luciferase activity, expressed as relative light units (RLU). Open symbols, control VLP ; solid symbols, VLP produced in the presence of EP-39.

Figure 8. Electron microscopy of Pr55Gag-expressing cells.

Samples of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-LucVpr at equal MOI, were (a) untreated, or (b, c) treated at 24 h pi with 10 µg/ml of EP-39 inhibitor for 24 h, harvested at 48 h pi, and processed for observation under the electron microscope.

Figure 9. Electron microscopy of EP-39-treated, Pr55Gag-expressing cells.

Samples of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-LucVpr at equal MOI, were treated at 24 h pi with 10 µg/ml of EP-39 inhibitor for 24 h, harvested at 48 h pi, and processed for observation under the EM. Note the morula-like shape of electron-dense particles of ca. 100 nm in diameter, some of which in the process of egressing into the extracellular milieu.

Figure 10. Structural analysis of extracellular EP-39-induced morula-like particles.

Sf9 cells infected with AcMNPV-Pr55Gag were untreated (a) or treated (b, c) at 24 h pi with EP-39 (10 µg/ml) for 24 h. Cell culture medium was harvested at 48 h pi, VLP pelleted by ultracentrifugation, and processed for EM analysis. Electron-dense 100-nm particles are viewed at low (b) and high (c) magnification, respectively. Control, membrane-enveloped VLP released from untreated cells (a) are shown at the same magnification as in (c). (d), Hypothetical model of an EP-39-induced nanoparticle of ca. 20 nm in diameter, composed of 12 to 16 copies of Pr55Gag assembled with a dodecahedral or octahedral symmetry.

Figure 11. Immunoelectron microscopy (IEM) of EP-39-treated, Pr55Gag-expressing cells.

Samples of AcMNPV-Gag-infected Sf9 cells were treated at 24 h pi with EP39 (10 µg/ml) for 24 h, harvested at 48 h pi, fixed and processed for IEM analysis. Ultrathin sections were incubated with rabbit anti-Gag antibody followed by 10-nm colloidal gold-tagged goat anti-rabbit IgG antibody. Panel (a) shows a festooned aspect of the cell surface, suggesting an abortive budding of particles. Panels (b) and (c) show immunogold-labeling associated with cytoplasmic 20-nm nanoparticles and with aberrant particles protruding from the plasma membrane. Note that the fainter staining of the specimens, compared to conventional electron microscopy (refer to Fig. 8-10), was destined to enhance the contrast between the colloidal gold grains and the background.

Figure 12. BS3 cross-linking of intracellular Pr55Gag in untreated and EP-39-treated cells.

Luminograms of SDS-PAGE and Western blot analysis of recombinant Gag polyproteins cross-linked in situ in untreated and EP-39 treated (10 µg/ml) Sf9 cells at 48 h pi. The spacer gel (delineated with dotted lines) was kept intact during the transfer of proteins to the membrane, as it potentially contained high order oligomers of Gag or/and aggregates of high molecular mass, too large to enter the resolving gel. Panel (a) corresponds to underexposed control lanes 0 (without BS3 cross-linking) from the blot shown in panel (b). Panel (c) is an enlargement and overexposure of the top of panel (b).