Figure 5
SIV MAC Vpx association with DCAF1 (VPRBP) is dispensable for Vpx-mediated rescue of HIV-1 from the antiviral state. (A) 293T cells were transfected with FLAG-tagged DCAF1 and either wild type SIVMAC-251 Vpx or SIVMAC-251 Vpx containing the indicated alanine-substitution mutations that disrupt associated with DCAF1. Immune complexes were isolated from clarified, 0.5% CHAPSO detergent lysates using anti-FLAG antibody conjugated to Protein G magnetic beads. Panels show immunoblots (IB) of the immunoprecipiated (IP) proteins (top panels) and immunoblots of the inputs (bottom panels). (B) Immunoblots of wild-type Vpx and the indicated mutants incorporated into SIVMAC-251 VLPs (top panels) and expression in the 293T producer cells (bottom panels). (C) MDDCs were treated with LPS, then treated with SIVMAC-251 VLPs containing wild-type Vpx or the indicated mutants, and challenged with an HIV-1NL4-3 GFP reporter virus. Data represent one of at least three independent experiments. Error bars represent ± SD (n = 3).