Different fusion precursors respond differentially to darunavir inhibition. All fusion precursors were derived from the GST-p6*-PR-HA construct. Differences in the p6* region are highlighted on the top (A). The p6*-PRNL(B) contains the wild type NL4-3 protease sequence with two native cleavage sites. The P site was mutated in the MG-PRNLconstruct (C); the D site was deleted in the M1- PRNLprecursor (D) as previously described . The p6*-PRpseprecursor (E) is similar to the p6*-PRNLexcept that it contains the pseudo wild type protease sequence. The p6*-PRH69D (F) precursor carries H69D point mutation in the p6*-PRNLbackbone. Precursor autoprocessing in transfected HEK293T cells was examined in the presence of increasing concentrations of darunavir. Post-nuclear cell lysates were prepared at 30 h post transfection and subjected to western blot analysis. Each blot was simultaneous probed using polyclonal rabbit anti-GST (left) and mouse anti-HA (middle) primary antibodies, respectively, for dual visualization through two separate infrared channels. Open triangles indicate the apparent IC50 (left) for the cleavage reaction mediated by the embedded protease. Asterisks denote the IC50 for self degradation of the released PR-HA and p6*-PR-HA (middle). The band intensity of each PR-containing fragment was quantified, normalized to the highest pixel value (1000), and plotted against darunavir concentrations (right). Thick lines with solid symbols represent PR-HA produced by P site cleavage reaction; thin lines with open symbols represent p6*-PR-HA produced by D site cleavage reaction.