Protein analysis of DIII-pseudotyped VLPs. (A-C), VLPs were purified from the culture medium of Sf9 cells infected with AcMNPV-Pr55GagHIV or coinfected with AcMNPV-Pr55GagHIV and AcMNPV-DIII-DENV1-RIgE or AcMNPV-DIII-WNVKun-RIgE. (A), VLPs from Pr55GagHIV-expressing cells (lane 1), from Pr55GagHIV + DIII-DENV1-RIgE coexpressing cells (lane 2), or from Pr55GagHIV + DIII-WNVKun-RIgE coexpressing cells (lane 3) were isolated by centrifugation through a sucrose cushion. VLP samples were analyzed by SDS-PAGE and Western blotting. Dual color was obtained by reaction of the blot with anti-Pr55Gag rabbit antibody and peroxidase-labeled anti-rabbit IgG antibody, which revealed the Pr55GagHIV polyprotein and its major cleavage products (Pr47GagHIV and Pr41GagHIV) in sepia red; this was followed by a second reaction with monoclonal anti-His6 tag antibody and phosphatase-labeled anti-mouse IgG, which revealed the DIII-RIgE fusion proteins in purple blue. Lane m, molecular weight markers. (B, C), VLP pellets in (A) from cells coexpressing DIII-RIgE and Pr55GagHIV were further purified by density ultracentrifugation in sucrose-D2O isopycnic gradient, and analyzed by SDS-PAGE and Western blotting, as above. (B), DIII-DENV1-RIgE; (C), DIII-WNVKun-RIgE. (D), VLPs were isolated from cells coexpressing DIII-WNVKun-RIgE and (lane 1) human virus Pr55GagHIV, (lane 2) simian virus Pr57GagSIV, (lane 3) chimeric murine-human virus Pr72GagMLV-HIV, or (lane 4) murine virus Pr65GagMLV. VLPs were analyzed by SDS-PAGE and Western blotting. Blots were reacted with rabbit anti-Gag antibodies and mouse anti-His6 tag monoclonal antibody, followed by peroxidase-labeled anti-rabbit IgG and anti-mouse IgG antibodies, then revealed by chemiluminescence substrate. Due to differences in the intensity of immunoreactivity of Gag proteins with polyclonal antibodies, compared to that of DIII-WNVKun-RIgE with monoclonal antibody, the blot was subjected to different exposure times: the luminogram showing the DIII-WNVKun-RIgE protein (lower panel) corresponds to a longer exposure.