Recombinant WT assemblies were produced following the protocol described previously33. Briefly, HIV-1 WT Gag was expressed in E. coli and purified by ion-exchange chromatography. In vitro assembly was done by mixing 4 mg/ml of protein with yeast transfer RNA overnight. PR digestion experiments with recombinant WT and T8I assemblies were performed by the addition of various concentrations of PR to the Gag WT assembly mixture and T8I cell lysate which was pre-treated and diluted to 2 mg/ml, and incubated at 37 °C for 2 h. For kinetic analysis of Gag WT and T8I cleavage, 3.3 μM of HIV-1 PR was added to the Gag assembly mixture and incubated at 37 °C, at different time points, 4ul of the digestion reaction mixture was taken out and mixed with NuPAGE® LDS Sample Buffer (Invitrogen) to stop the reaction, and then subjected to NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen) for cleavage products analysis and visualized by Coomassie blue staining.

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