Figure 5
Defects in gene expression of Gag VLPs containing a bicistronic expression cassette in target cells. (A) Schematical representation of expression plasmids used for trans-packaging and subsequent protein expression. HIV-1 Gag expression plasmid and PGAP-GagFL-IRES-EGFP-TGAP were used for production of Gag VLPs (as a helper plasmid) and synthesis of reporter RNA (as a vector plasmid), respectively. HIV GagFL-EGFP was used as a control for GagFL and EGFP expression in mammalian cells. (B) Expression of HIV-1 GagFL and EGFP in HeLa cells. Yeast was cotransformed with the helper and vector plasmids. After removal of the cell wall, yeast spheroplasts were cultured overnight for Gag VLP release as before. Produced Gag VLPs (equivalent to 1 μg RNA) were added to HeLa cells. Following incubation at 37°C for 2 days, HeLa cells were subjected to immunostaining using anti-FLAG (for detection of GagFL) antibody. Nuclei were stained with DAPI. Representative images were shown at the same magnification. Bar, 10 μm.