FIG. 8.

Comparison of buoyant densities of Gag-GFP constructs and HIV IIIB. Particles were prepared and labeled as described in Materials and Methods. Labeled supernatants from cells expressing GAGB/GFP, GAG391/GFP, MACA/GFP, and HIV-1 IIIB were separately centrifuged through a 20% cushion. The resuspended pellets were loaded onto the top of a single 20 to 60% sucrose gradient and centrifuged to equilibrium. Thirty fractions were collected; data for the bottom 20 fractions are presented. Gag proteins and Gag-GFP proteins in each fraction were immunoprecipitated with HIV patient sera and further analyzed by SDS-PAGE and autoradiography. Relative absorbance values were obtained by scanning the resulting autoradiogram on a flatbed scanner equipped for transparencies, and quantitation was performed with NIH Image software. (A) Plot of absorbance (y axis, relative values) for immunoprecipitated bands on the autoradiogram shown below. The x axis indicates fraction numbers, with the bottom of the gradient to the left. (B) Autoradiogram corresponding to the graphed results. Arrows point to the individual Gag-GFP fusion proteins. IIIB indicates the position of HIV-1 IIIB virions as demonstrated by radiolabeled and immunoprecipitated p24 (CA). The positions of molecular mass markers are indicated at the left in kilodaltons.