Fig 4.

Impact of amino acid substitutions on TPV's dimerization inhibition. (A) COS7 cells were exposed to each of the drugs (TPV, DRV, and APV) at various concentrations and subsequently cotransfected with plasmids encoding each full-length molecular infectious HIV-1 (HIV_NL4-3) clone producing CFP- or YFP-tagged wild-type protease. After 72 h, cultured cells were examined in the FRET-HIV-1 assay system and the CFP^A/B ratios 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. *, not significant; **, P < 0.05. (B) COS7 cells were cotransfected with a pair of clones, pHIV_NL4-3^CFP and pHIV_NL4-3^YFP, carrying I54V (cHIV_NL4-3^I54V), V82T (cHIV_NL4-3^V82T), or I54V/V82T (cHIV_{NL4-3^I54V/V82T}) or a pair of clones, pHIV_B^CFP and pHIV_B^YFP, carrying no additional substitution (cHIV_B), I54V (cHIV_B^I54V), or I54V/V82T (cHIV_B^I54V/V82T) in the absence or presence of 1 or 10 μM TPV or 0.01 or 0.1 μM DRV, and CFP^A/B ratios were determined. *, not significant; **, P < 0.05. The statistical differences (P values) between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{no drug}}) and the CFP^A/B ratios in the presence of 1 μM TPV (CFP^{A/B_{1 TPV}}) and those between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{no drug}}) and the CFP^A/B ratios in the presence of 10 μM TPV (CFP^{A/B_{10 TPV}}) were, respectively, <0.0001 and <0.0001 for cHIV_NL4-3^I54V, 0.0067 and 0.0005 for cHIV_NL4-3^V82T, 0.0009 and 0.0005 for cHIV_{NL4-3^I54V/V82T}, 0.359 and 0.2865 for cHIV_B, 0.132 and 0.0234 for cHIV_B^I54V, and 0.3689 and 0.1653 for cHIV_B^I54V/V82T. The statistical differences (P values) between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{no drug}}) and the CFP^A/B ratios in the presence of 0.01 μM DRV (CFP^{A/B_{0.01 DRV}}) and those between the CFP^A/B ratios in the absence of drug (CFP^{A/B_{no drug}}) and the CFP^A/B ratios in the presence of 0.1 μM DRV (CFP^{A/B_{0.1 DRV}}) were 0.5956 and 0.0021, respectively, for cHIV_B^I54V/V82T. (C) COS7 cells were cotransfected with a pair of clones, pHIV_NL4-3^CFP and pHIV_NL4-3^YFP, carrying L24I, L24M, L33I, or L33F or a pair of clones, pHIV_B^CFP and pHIV_B^YFP, with Leu-33 (wild-type) reverted from Ile-33 (cHIV_B^I33L) with an additional I54V substitution (cHIV_B^I33L/I54V) or with an additional I54V/V82T substitution (cHIV_B^{I33L/I54V/V82T}) in the absence or presence of 1 or 10 μM TPV, and CFP^A/B ratios were determined. *, not significant; **, P < 0.05.