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

<p>Sf9 cells were infected with (-) AcMNPV-Pr55Gag alone, or (+) coinfected with AcMNPV-Pr55Gag and another recombinant baculovirus expressing (<b>a</b>) Vpr or (<b>b, c</b> )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. (<b>a</b>), 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>b</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). (<b>c</b>), 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).</p

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

<p>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.</p

Quantification of VLP assembly and egress using luciferase assay.

<p>(<b>a</b>), <b><i>Ultracentrifugation analysis of VLP.</i></b> 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>b)</b>, <b><i>Dose-response curve of PA-457 inhibitory effect on VLP production</i></b>. 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. <b><i>Inset</i></b> : VLP production (<b><i>top</i></b>) and intracellular expression of Pr55Gag (<b><i>bottom</i></b>) were evaluated in parallel by Western blot analysis using anti-Gag rabbit antibody and phosphatase-labeled conjugate.</p

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

<p>Sf9 cells infected with AcMNPV-Pr55Gag were untreated (<b>a</b>) or treated (<b>b, c</b>) 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>b</b>) and high (<b>c</b>) magnification, respectively. Control, membrane-enveloped VLP released from untreated cells (<b>a</b>) are shown at the same magnification as in (<b>c</b>). (<b>d</b>), 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.</p

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

<p>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.</p

Efficiency of inhibition of HIV-1 VLP assembly by betulinic acid derivatives ^(a).

(a)<p>The mean values (m) for the 50% inhibitory activity (IC₅₀) on VLP assembly were given as µg/ml (mean, m ± SEM ; n = 4), or as µM (m). The mean values for cytoxicity (CC₅₀) were only given as µM.</p>(b)<p>The selectivity index (SI) was given by the ratio CC₅₀∶IC₅₀.</p>(c)<p>ND, not determined.</p>(d)<p>NA, not applicable.</p

Structure of betulinic acid derivatives.

<p>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.</p

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

<p>Luminograms of SDS-PAGE and Western blot analysis of recombinant Gag polyproteins cross-linked <i>in situ</i> 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 (<b>a</b>) corresponds to underexposed control lanes 0 (without BS3 cross-linking) from the blot shown in panel (<b>b</b>). Panel (<b>c</b>) is an enlargement and overexposure of the top of panel (<b>b</b>).</p

Effect of EP-39 on sedimentation properties of VLP.

<p>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.</p

Electron microscopy of Pr55Gag-expressing cells.

<p>Samples of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-LucVpr at equal MOI, were (<b>a</b>) untreated, or (<b>b, c</b>) 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.</p