Figure 2.

The S40F mediated ubiquitination of Gag occurs irrespective of Gag processing or other viral proteins. HeLa cells were co-transfected with HA-ubiquitin and either p∆R PR⁻ plasmids (A) or syngag plasmids encoding for either wt Gag, or the p6 mutant S40F or ∆PTAP, respectively (B). Gag was recovered by immunoprecipitation from denatured whole cell lysates with anti-HIV antibodies, and ubiquitinated Gag molecules were subsequently visualized by Western blot using antibodies specific for HA. The amount of Gag recovered by immune precipitation, as well as Gag recovered from whole cell lysates was detected with CA specific antibodies; (C) Evaluation of three or five independently performed experiments for the p∆R or the syngag system, respectively. Values were matched to the ubiquitin signal of the ∆PTAP mutant (100%) and represent the arithmetic mean ± SD.