Fig 9.

Gag binding to PI(4,5)P₂-containing liposomes requires the specific HBR amino acid sequence, whereas the overall positive charge of the HBR is sufficient for RNA binding and acidic lipid binding in the absence of RNA. (A) Liposome binding assays were performed to examine binding of wild-type Gag-YFP, HBR/RKswitch/Gag-YFP, and 6A2T/Gag-YFP to control liposomes that contain PC and PS in a 2:1 ratio [PC;PS (2:1)] or liposomes that contain 7.25 mol% PI(4,5)P₂ in addition to PC:PS (2:1). M, membrane-bound Gag; NM, non-membrane-bound Gag. (B) The amount of labeled Gag in each fraction was quantified using a phosphorimager, and the percentage of labeled Gag in the membrane-bound fraction versus the total amount of labeled Gag was calculated. The relative liposome-binding efficiency for each condition was calculated in comparison to the binding efficiency of wild-type Gag-YFP with PI(4,5)P₂-containing liposomes. Data from three different experiments are shown as means ± standard deviations. Liposome binding efficiency was quantified, with all conditions normalized to Gag-YFP binding to PI(4,5)P₂-containing liposomes. (C) Liposome binding assays were performed to examine binding of wild-type Gag-YFP, HBR/RKswitch/Gag-YFP, and 6A2T/Gag-YFP to PC:PS (2:1) liposomes with or without RNase treatment. M, membrane-bound Gag; NM, non-membrane-bound Gag. (D) Liposome binding efficiency was quantified as described for panel B, and the relative liposome-binding efficiency for each condition was calculated in comparison to the binding efficiency of RNase-treated Gag-YFP with PC:PS (2:1) liposomes. Data from three different experiments are shown as means ± standard deviations. Note that results shown in panels A and C were obtained in the same experiment, and therefore the same gel images are shown for the negative-control conditions (left side).