Figure 4

DUb-Alix interferes with Alix mediated virus release. (A) Co-expression of DUb-Alix inhibits EIAV release. 293T cells were transfected with EIAV proviral DNA alone (500 ng throughout the study unless otherwise specified) (lane 1), with Flag-Alix, Flag-DUb-Alix, Flag-DUb-AlixF676D or Flag-DUb*-Alix (lanes 2, 3, 4, 5; respectively). (B) The active DUb-Alix fusion protein failed to replace cellular Alix to promote EIAV release. 293T cells were transfected twice with Alix RNAi oligonucleotides at 36-h intervals. At the second transfection, cells were transfected with EIAV provirus alone (lanes 1 and 2) or with 100 ng of RR versions of Flag-Alix, Flag-DUb-Alix or Flag-DUb*-Alix (lanes 3, 4, 5; respectively). (C) DUb-Alix fails to rescue HIV-1 PTAP- budding. 293T cells were transfected with expression plasmids of HIV-1 PTAP- alone (1 μg) (lane 1), or in combination with Flag-Alix (500 ng), Flag-DUb-Alix (100 ng) or Flag-DUb*-Alix (100 ng) (lanes 2, 3, 4; respectively). (D) Co-expression of DUb-Alix has no effect on MoMLV release. 293T cells were transfected with MoMLV provirus alone (1 μg) (lane 1), with Flag-DUb-Alix (100 ng) (lane 2) or Flag-DUb*-Alix (100 ng) (lane 3). Protein content of pelleted virions and cell lysates was analyzed 24 h later by WB blotting with the indicated antibodies. Release ratio (%) was calculated using the formula described in supplemental material. Error bars represent standard deviations (SD).