Figure 2

Inhibition of HIV-1 Gag protein expression by the R-U5 and SL regions in yeast. (A) Schematical representation of HIV-1 expression plasmids with deletions in the R-U5 and SL regions. (B) Semi-quantitative RT-PCR for HIV-1 RNA in yeast. Total cellular RNA was isolated from yeast transformed with HIV-1 expression plasmids with deletions in the R-U5 and SL and was subjected to semi-quantitative RT-PCR for HIV-1 gag mRNA and yeast actin mRNA. Using 300 ng of total cellular RNA, RT-PCR for HIV-1 gag mRNA and yeast actin mRNA was performed up to 26 PCR cycles (upper). Using a series of dilutions of the cellular RNA (10 to 300 ng), RT-PCR for HIV-1 gag mRNA and yeast actin mRNA was performed at 20 PCR cycles (lower). Representative blots were shown. The PCR product for HIV-1 gag mRNA corresponds to 670 bases and that for yeast actin mRNA 840 bases. (C) Semi-quantification of HIV-1 RNA in yeast. Total cellular RNA of the yeast transformants was serially diluted (3 to 1000 ng), slot-blotted, and detected by hybridization with the minus-strand RNA probe for the HIV-1 pol gene. Representative blots were shown. (D) Real-time RT-PCR for HIV-1 RNA in yeast. Total cellular RNA isolated from yeast transformants was reverse-transcribed and aliquots of the reaction samples were used for amplification up to 40 PCR cycles with specific primers for HIV-1 gag mRNA. Empty indicates total cellular RNA isolated form yeast transformant with an empty pKT10 plasmid (negative control). Data were shown as means with standard deviations from 3 independent experiments. (E) HIV-1 Gag protein expression in yeast. Whole cell lysates of yeast transformed with HIV-1 expression plasmids with deletions in the R-U5 and SL regions were subjected to Western blotting using and anti-HIV-1 p24 antibody. Representative blots were shown.