Figure 4.

Dominant-negative inhibition of RSV budding in 293/E cells by the LDI-1 WW domains. (A) Cells were transfected with the wild-type RSV Gag expression plasmid along with increasing amounts of either an expression plasmid for the WW domains of LDI-1 (lanes 3–7), or the LDI-1 C2 domain (lanes 8–12). Transfected cells were labeled with [³⁵S]methionine, and Gag was immunoprecipitated from cell lysates and media, separated by SDS/PAGE, and quantified by phosphorimage analysis. Untransfected cells (lane 1), cells transfected with wild-type Gag (lane 2). Cells transfected with Gag and varying amounts of LDI-1 WW as follows: 5 μg (lane 3), 7.5 μg (lane 4), 10 μg (lane 5), 12.5 μg (lane 6), and 15 μg (lane 7). Cells transfected with Gag and varying amounts of LDI-1 C2 as follows: 5 μg (lane 8), 7.5 μg (lane 9), 10 μg (lane 10, 12.5 μg (lane 11), and 15 μg (lane 12). (B) The effect of cotransfection of plasmids expressing the WW domains of LDI-1 (□) or LDI-1 C2 (▴) on budding of RSV Gag in 293 cells. The data are presented as the ratio of Pr76^Gag in the media/lysate as a function of varying concentration of cotransfected expression plasmid. The LDI-1 WW and C2 data are derived from A. The extent of Gag budding in the absence of competing coexpressed proteins was set to 1.0. (C) Western blot analysis of expression of LDI-1 C2 and WW domains. Human 293/E cells were untransfected (lane 1) or transfected with 100 ng of AGP5 and 5 μg of LDI-1 C2 (lane 2) or LDI-1 WW (lane 3). Proteins were identified by Western blotting with a rabbit anti-HA antibody.