Figure 2
Pseudotyping of VLPs with CD16-RIgE protein. (A), Western blot analysis of CD16-RIgE-expressing cells and CD16-RIgE-pseudotyped VLPs. Left panel: whole cell lysates. Sf9 cells were mock-infected (lane 1), infected with AcMNPV-Pr55GagHIV (lane 2), coinfected with AcMNPV-Pr55GagHIV and AcMPV-CD16-RIgE at equal MOI each (10 pfu/cell; lane 3), or infected with AcMPV-CD16-RIgE (lane 4). Lysates of cells harvested at 48 hpi were analysed by SDS-PAGE and immunoblotting, using a dual color detection: blots were reacted with anti-Gag rabbit polyclonal antibody (Ab) and peroxidase-labeled anti-rabbit IgG Ab, followed by mouse monoclonal anti-CD16 Ab and phosphatase-labeled anti-mouse IgG Ab. The Pr55GagHIV polyprotein precursor shows as a sepia red-colored band at 55 kDa (Pr55Gag), and the chimeric CD16-RIgE protein as a sharp purple blue band at 37 kDa. Lane m, molecular weight markers. Right panel: extracellular VLPs. VLPs isolated from the culture medium of cells expressing Pr55GagHIV alone (lane 1), coexpressing Pr55GagHIV and CD16-RIgE (lane 2), and the corresponding samples from CD16-RIgE-expressing cells (no VLP control; lane 3) were analysed by SDS-PAGE and immunoblotting, as above. The Pr55Gag cleavage products (Pr47, Pr41 and CAp24 [9–15]) are indicated by dotted lines, and the chimeric CD16-RIgE protein, migrating as a blurred band at 72-75 kDa, by a blue arrow. (B), Electron microscopy (EM) of ultrathin sections of VLP-producing cells. (i), Double infection. Sf9 cells were coinfected with two recombinant baculoviruses AcMNPV-Pr55GagHIV and AcMNPV-CD16-RIgE, and processed for EM at 48 hpi. (ii, inset), Control single infection. Sf9 cells infected with AcMNPV-Pr55GagHIV alone produced nonpseudotyped VLP. Note the difference between the fuzzy (i) and smooth (ii) envelopes of VLPs.