Figure 3

Rev induced disruption of the IN-LEDGF/p75 complex. (A) The presence of Rev, IN, LEDGF/p75 and Actin (control) was detected by Western blots in cell lysates obtained from HIV-1-infected H9 lymphocytes at different times after infection using the appropriate antibodies. Rev, IN, LEDGF/p75 and Actin also were precipitated from the cytoplasm, nuclei and PIC fractions obtained from HIV-1-infected H9 lymphocytes at different times PI by the indicated specific antibodies. (B) Co-IP of Rev-IN and Rev-LEDGF/p75, but not IN-LEDGF/p75, can be observed in Rev10⁺ cells. (C) Only IN-LEDGF/p75 co-IP can be observed in cells infected with ΔRev HIV-1. (D) Only Rev-IN co-IP can be observed in LEDGF/p75-knockdown cells infected with WT HIV-1. (E) In LEDGF/p75-knockdown cells infected with ΔRev HIV-1, only IN can be detected. (F) Co-IP of Rev-IN, IN-LEDGF/p75 and Rev-LEDGF/p75, which is observed in infected cell lysates, is interrupted specifically by pretreating the cells with 150 µM of specific peptides that contain the interaction-mediating sequences. Quantitative estimation of the bands, performed by Image Gauge V3.46 software, is presented in supplementary Table 1A-F, respectively. All other details are described in Materials and Methods.