FIGURE 5.
The NC domain is required for inhibition of Gag binding to PC + PS liposomes mediated by psi, yeast tRNA, and tRNAPro. Gag constructs containing MA or NC changes were examined for susceptibility of their liposome binding ability to various RNA species. (A) Schematic representation of Gag derivatives used in these experiments. (B) [35S]-labeled WT Gag, HBR switch Gag, or delNC Gag, synthesized using rabbit reticulocyte lysates, were treated (or left untreated) with RNase A and examined for susceptibility of their liposome binding ability to yeast tRNA or psi in the add-back experiments performed as described in Figure 1. In these experiments, liposome binding efficiency normalized to the binding efficiency observed upon RNase treatment is shown as 100% for each Gag construct. The actual mean liposome binding efficiencies under this condition for WT Gag, HBR switch Gag, and delNC Gag are 47%, 42%, and 46%, respectively. Data from five different experiments are shown as mean ± standard deviation. P values were determined in comparison to the no RNA add-back condition using Student's t-test. (***) P < 0.001. (C,D) Dependence of the inhibition of Gag–liposome binding on the MA HBR sequence (C) and NC (D) was examined for yeast tRNA, tRNALys3, and tRNAPro as in panel B. The actual mean liposome binding efficiencies upon RNase treatment are 33% (C) and 31% (D) for WT Gag, 35% for HBR switch Gag, and 72% for delNC Gag. Data from three different experiments are shown as mean ± standard deviation. P-values were determined in comparison to the no RNA add-back condition using Student's t-test. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.