Figure 2: Intracellular complexes containing radiolabeled Gag and ABCE1 appear shortly after translation and disappear upon intracellular processing of radiolabeled p55Gag.
A) COS-1 cells expressing HIV-1 were pulsed for 15 min with 35S-methionine/cysteine and chased with unlabeled media. At chase times indicated above panels, cells were harvested and lysates were subjected to immunoprecipitation under native conditions with αABCE1. Dot indicates position of coimmunoprecipitated p55Gag. B) Collections of medium at chase times indicated were subjected to immunoprecipitation with αGag. Medium collections in the first 24 h were cumulative, starting with the beginning of the chase period, while medium collected at later times shows p24Gag release during intervals (24–48 h and 48–72 h). Marker lanes (M Gag, M ABCE) show the positions of p55Gag and ABCE1. All lanes are from the same autoradiograph, with lanes spliced from elsewhere on the same autoradiograph indicated by dividing line. C) The amount of radiolabeled p55Gag coimmunoprecipitated with αABCE1 in A was quantitated as percentage of maximum (squares, left-hand y-axis) and was plotted against the amount of radiolabeled Gag (p24Gag and p25Gag) present in medium in B as percentage of maximum radiolabeled p24Gag and p25Gag accumulated at 72-h chase (circles, right-hand y-axis). Inset shows data with chase time plotted on a logarithmic scale in order to highlight early time-points. D) Data shown in C are plotted against increase in processed Gag bands (p24Gag and p25Gag) from cell lysates (triangles, right-hand y-axis) and against the amount of radiolabeled Gag (p24Gag and p25Gag) present in medium as in C (circles, right-hand y-axis). Graph shows data with chase time plotted on a logarithmic scale in order to highlight early time-points (up to 9.25-h chase). Accumulation of processed p24Gag and p25Gag bands in lysate (triangles, right-hand y-axis) was quantitated as percentage p24Gag and p25Gag present at maximum intracellular levels (9.25 h). Processed Gag bands in the media were quantitated as percentage of maximum radiolabeled p24Gag and p25Gag accumulated at 72-h chase (circles, right-hand y-axis). C,D) When identical time-points were available in two experiments (filled squares, circles or triangles), data were averaged, with error bars showing SEM. Open squares, circles and triangles show time-points that were only examined in one of the two experiments. All data are representative of four independent experiments.