VLP release by Gag and HIV are differently sensitive to PTAP and YP inactivation.
(A) Gag versus HIVR9 and HIVR8.2 expressions in 293T cells and corresponding virion/VLP release. 200 ng of each construct was used for transfection, and samples were collected 24 hours post-transfection. Mutations in primary binding sites of Tsg101 and ALIX (PTAP and YP, respectively) and the subsequent retention of Tsg101 and ALIX in the released VLPs are shown. (B) Effect of Gag mutations on the yield of VLP release in 293T cells. 200 ng of each humanized Gag construct were used for transfection, and samples were collected 24 hours post-transfection. Tsg101 and ALIX retention in VLPs is also shown. (C) Effect of Gag mutations on the yield of VLP release in 293T cells by non-humanized Gag. 1 μg of each Gag construct plus 300 ng HIV Rev encoding vector were used, and samples were collected 24 hours post-transfection. (D) Effect of Gag mutations on the yield of VLP release in 293T cells by non-humanized Gag in the context of HIV. The ribosomal slippage site on Gag cDNA was inactivated by mutagenesis without changing the corresponding translated amino acids. 250 ng of each GagΔFrameshift construct were used and samples were collected 24 hours post-transfection. Densitometry values correspond to the ratio of p24 in VLPs/Cells relative to WT values. All experiments were performed at least 3 times with similar results; specifically, the variability in the final densitometry values is <0.05).
