Figure 5

Proteolytic processing of cellular proteins by active HIV PR. (a) HEK-293 cells transfected with HIV-1 Gag and HA-BCA3 (500 ng each) and various amounts of c-myc-HIV-1 PR (25–1000 ng) were analyzed 48 h post-transfection. (b-e) HEK-293 cells transfected with HIV-1 Gag and HA-BCA3 were lysed 48 h post-transfection as described in Materials and Methods and incubated with recombinant HIV-1 PR (1.25 μM), optionally in the presence of ritonavir (4 μM) (d) or calpain inhibitor I (100 μM) (e) at 37°C for the time indicated. The proteins were then separated on 12-15% SDS-PAGE and analyzed by Western blot. (f) Autoradiographic analysis of HIV-1 PR in vitro cleavage of full-length p21Bax and N-terminally truncated p18Bax proteins obtained by TNT transcription/translation system. Protein samples were treated with HIV-1 PR (1.25 μM), optionally in the presence of ritonavir (8 μM), at room temperature for the time indicated. The proteins were then separated on 15% SDS-PAGE and visualized by autoradiography. (g) In vitro cleavage of mitochondrial proteins by HIV-1 PR. Freshly isolated mitochondria from HEK-293 cells were incubated with various amounts of recombinant HIV-1 PR, optionally in the presence of ritonavir (4 μM). The proteins in the samples were separated on 12-15% SDS-PAGE and analyzed by Western blot. (h) Supernatant after cleavage of proteins from purified mitochondrial fraction. The samples from (g) were centrifuged, and supernatants were analyzed by Western blot.