Figure 4
Preferential incorporation of HIV-1 genomic RNA into Gag VLPs in yeast. (A) Efficiency of incorporation of HIV-1 genomic RNA into Gag VLPs in yeast. Yeast was cotransformed with the Gag expression plasmid (helper) and PGAP-HIV-TGAP plasmid (vector). After spheroplast formation, cells were cultured overnight for Gag VLP release from yeast spheroplasts were carried out as before. RNA was isolated from yeast spheroplasts and Gag VLPs, serially diluted, and analyzed by Northern blotting using minus-strand biotinylated RNA probe for the HIV-1 pol gene. Representative blots were shown. HeLa cells transfected with HIV-1 molecular clone pNL43 were used as control. Arrow indicates gag mRNA. A 0.24-9.5 kb RNA ladder (GIBCO BRL) were used as molecular weight markers. (B) Semi-quantification of incorporation efficiency of HIV-1 genomic RNA into Gag VLPs. RNAs of the yeast spheroplasts and Gag VLPs were serially diluted, slot-blotted, and detected by with hybridization with the minus-strand RNA probe for the HIV-1 pol gene. Representative blots were shown. (C) Semi-quantification of incorporation efficiency of yeast actin mRNA into Gag VLPs. RNAs of the yeast spheroplasts and Gag VLPs were serially diluted, slot-blotted, and detected by with hybridization with the minus-strand RNA probe for yeast actin mRNA. Representative blots were shown. (D) Quantification of incorporation efficiency of HIV-1 genomic RNA into Gag VLPs by real-time RT-PCR. RNA was isolated from yeast spheroplasts or Gag VLP and was reverse-transcribed. Aliquots of the reaction samples were used for amplification up to 40 PCR cycles with specific primers for unspliced HIV-1 mRNA. Data were shown as means with standard deviations from 3 independent experiments.