Figure 3
Trans -packaging of HIV-1 genomic RNA into Gag VLPs in yeast. (A) Schematical representation of HIV-1 expression plasmids used for trans-packaging. HIV-1 Gag expression plasmid was used for production of Gag VLPs (as a helper plasmid). PGAP-HIV-TGAP and PGAP-ΔHIV-TGAP were used for synthesis of HIV-1 genomic RNA (as vector plasmids). (B) Production of HIV-1 Gag VLPs and packaging of HIV-1 RNA in yeast. Yeast was cotransformed with the helper and vector plasmids. After removal of the cell wall, yeast spheroplasts were cultured overnight for Gag VLP release. Cells and purified Gag VLPs were analyzed by Western blotting using anti-HIV-1 p24 antibody. RNA was isolated from cells and purified Gag VLPs and was analyzed by Northern blotting using minus-strand biotinylated RNA probe for the HIV-1 pol gene. All blots are representative from 3–4 independent experiments. (C) Electron microscopy of Gag VLPs. Purified Gag VLPs were analyzed by electron microscopy. Representative micrographs were shown at the same magnification. Bar =100 nm.