FIG 4 .

DRV’s protease dimerization inhibition activity also increases against HIV^V32I compared to HIV^WT. (A, top) Plasmids encoding full-length molecular infectious HIV_NL4-3 clones, which produce CFP- or YFP-tagged protease (PR), were generated using the PCR-mediated recombination method as described in Materials and Methods. A linker consisting of five alanines was inserted between PR and each fluorescent protein. The phenylalanine-proline site (F/P) that PR cleaves was introduced between the fluorescent protein and reverse transcriptase (RT). AA, amino acids. (Bottom) Structural representations of PR monomers and dimer in association with the linker atoms and fluorescent proteins are shown below the schematic representation of the plasmids. FRET occurs when the two fluorescent proteins come close in the proximity of 1 to 10 nm. If an agent that inhibits the dimerization of 2-PR monomer subunits is present when the CFP- and YFP-tagged PR monomers are produced within the cell upon cotransfection, FRET does not occur. (B) COS-7 cells were cotransfected with plasmids encoding either of full-length molecular infectious HIV_NL4-3 clones producing CFP- or YFP-tagged wild-type protease (HIV^WT) and cultured in the presence or absence of various concentrations of DRV. COS-7 cells were also cotransfected with plasmids encoding either of HIV_NL4-3 clones producing CFP- or YFP-tagged V32I-carrying protease (HIV^V32I). After 72 h, cultured cells were examined in the FRET-HIV-1 assay system and the CFP^A/B ratios (y axis) were determined. The mean values of the ratios obtained are shown as bars. A CFP^A/B ratio that is greater than 1 signifies that protease dimerization occurred, whereas a ratio that is less than 1 signifies the disruption of protease dimerization. All the experiments were conducted in a blind fashion. For rHIV^WT, the P values were 0.3816 for the CFP^A/B ratio in the absence of drug (CFP^A/B_{No Drug}) versus the CFP^A/B ratio in the presence of 10 nM DRV (CFP^A/B_10-DRV), 0.004 for CFP^A/B_{No Drug} versus CFP^A/B_100-DRV. For rHIV^V32I, the P values were 0.1111 for CFP^A/B_{No Drug} versus CFP^A/B_0.1-DRV, 0.001 for CFP^A/B_{No Drug} versus CFP^A/B_1-DRV, and 0.0086 for CFP^A/B_{No Drug} versus CFP^A/B_10-DRV.