HIV-1 Counteracts GCN2 Inhibitory Effect Figure 2. HIV-1 protease directly cleaves GCN2. (A) BHK-21 cells were transfected by electroporation with in vitro transcribed capped Sindbis virus RNAs from plasmids pT7SV-HIV-1PR (C+HIV-1Pro), pT7SV-2Apro (C+2APro), pT7SVwt (C) for the expression of HIV-1 and PV-2A proteases. After 4 h cells were metabolically labeled with [35S]-Met-Cys. Equivalent amounts of total protein were subjected to 12% SDS-PAGE, transferred to PDVF membranes and subjected to autoradiography (lower panel) to visualize protease expression. The membranes were then probed with specific antisera for detection of endogenous GCN2 and eIF4GI as indicated. (B) COS-7 cells were subjected to coupled infection/DNA transfection with recombinant vaccinia virus (vvT7), pTM1 (2) or pTM1-derived plasmid encoding HIV-1Pro and pcDNA3.1/Myc-His plasmid, empty (2) or encoding human or mouse GCN2. At 18 h post-transfection cells were lysed and equivalent amounts of total protein were analyzed by western blot using a specific antiserum for detection of GCN2. (C) Affinity purified mouse or human GCN2 was incubated for 3 h at 30uC in the absence or the presence of HIV-1 or HIV-2 proteases. Incubation was stopped by addition of SDS-PAGE sample buffer and proteins were analyzed by western blot using a specific antiserum for detection of GCN2. (D) HeLa cell-free extracts were incubated with recombinant HIV-1Pro in the absence or the presence of 2.5 mM SQ for the indicated times. Proteins were analyzed by western blot using specific antisera for detection of GCN2, eIF4GI, PABP or actin as indicated. (E) Purified mouse and human GCN2 GST-fusion proteins were incubated in the presence of recombinant HIV-1Pro for 3 h at 30uC. Proteins were resolved PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47272