Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-0

crystals and analysis by SDS-PAGE and Coomassie blue staining. Lane 1 : solution of purified EED3-H(10 mg/mL) used for crystallogenesis (protein load : 50 μg). Lane 2 : protein content of solubilized single crystal. MW : markers of protein molecular mass, indicated in kilodaltons (kDa) on the right side of the panel.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></p

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-1

. Shown is a ribbon representation of the polypeptide backbone atoms of EED3 isoform (amino acid residues 84–441), with secondary and tertiary structures of the different β-blades. , 3D-model of the EED3 seven-bladed β-propeller, deduced from crystallographic data (modified, from [30]). The black arrow indicates the major difference between our putative model (A) and the crystal model (B), consisting of the α1 helical region facing the β-strand β17 in β-blade IV. , Position of immunogenic epitopes (depicted in green) on the 3D-model of EED polypeptide backbone (represented in blue). , Primary and secondary structures of EED3, deduced from crystallographic data [30]. The amino acid sequence was numbered according to the accepted nomenclature [12] : Met95 in EED1 isoform represented Met1 in EED3 ; thus, the C-terminal residue L440 in EED3 corresponded to L535 in EED1. Regions in β-strand structure are represented by horizontal arrows, with reference to the blade number and β-strand letter a, b, c or d ; α-helices are represented by spirals, and turns by TT. Helical regions marked α1 and η1, and the β-strand region marked β17, were structurized domains of EED which were unique among representatives of WD-40 proteins. The relative accessibility of each residue () in the 3D structure was extracted from the dictionary of protein structure [45], and indicated as coloured bars under the sequence with the following colour code : dark blue, highly accessible ; light blue, accessible ; white, buried. Discrete regions recognized by anti-EED IgG are indicated by green boxes. The binding sites of HIV-1 matrix protein (MA) and integrase (IN) are underlined by solid black lines.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></p

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-2

crystals and analysis by SDS-PAGE and Coomassie blue staining. Lane 1 : solution of purified EED3-H(10 mg/mL) used for crystallogenesis (protein load : 50 μg). Lane 2 : protein content of solubilized single crystal. MW : markers of protein molecular mass, indicated in kilodaltons (kDa) on the right side of the panel.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></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

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

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

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