Among VLP constituents, Vpx is necessary and sufficient to rescue HIV-1 from type I IFN. (A) MDDCs were treated with LPS for 24 hrs, then treated for 3 hrs with media or the indicated VSV-G-pseudotyped HIV-2ROD or SIVMAC-251 VLPs, and finally challenged with a VSV-G-pseudotyped HIV-1NL4-3 GFP reporter virus. Infectivity was measured by flow cytometry. (B) As indicated, 293T cells were co-transfected with a codon optimized SIVMAC251 vpx expression plasmid and HIV-1 GFP reporter vectors bearing either wild-type Gag or Gag with an engineered Vpx binding motif (DPAVDLL). Proteins from the cell lysate and from virion preparations were separated by SDS-PAGE and then immunoblotted with anti-Vpx or anti-p24 antibodies. (C) MDDCs treated with IFN-β for 24 h and were then challenged with VSV-G-pseudotyped HIV-1 GFP reporter vectors with wild-type HIV-1 Gag or HIV-1 Gag bearing the engineered Vpx binding motif (DPAVDLL). Both HIV-1 reporter vectors were produced in the presence of empty pcDNA3.1 plasmid or pcDNA3.1 containing a codon-optimized SIVMAC-251 vpx cDNA. Data are representative of one of at least three independent experiments. Error bars represent ± SD (n = 3).