FIG. 3.

iVLP assay with VP24-deficient particles. (A) VeroE6 cells were infected with WT or VP24-deficient iVLPs. The reporter gene activity was determined 2 days p.i. As a negative control, the plasmid encoding VP40 was omitted in p0. Error bars indicate standard deviations. (B) Recovery of infectivity of VP24-deficient iVLPs by pretransfection of p1. VeroE6 cells were pretransfected by electroporation with 1.25 μg of plasmid encoding the indicated protein. One day posttransfection, the cells were infected with WT or VP24-deficient iVLPs, and 2 days p.i., the reporter activity was measured. Error bars indicate standard deviations. (C) Electron microscopy analysis of iVLPs. WT and VP24-deficient iVLPs were produced and purified by ultracentrifugation over a 20% sucrose cushion. Particles were visualized using electron microscopy after negative staining. Arrows show nucleocapsid-like structures. (D) Analysis of iVLP composition. WT (lanes 2, 5, and 8) or VP24-deficient (lanes 3, 6, and 9) iVLPs were produced and purified over a 20% sucrose cushion. As a control, the plasmid encoding VP40 was omitted in p0 (lanes 1, 4, and 7). The purified iVLPs were either mock treated (lanes 1 to 3), treated with proteinase K (lane 4 to 6), or treated with proteinase K and Triton X-100 (lanes 7 to 9). The samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by Western blotting using specific antibodies. Proteins inside the iVLPs are visible in lanes 4 to 6.