A3G Is Incorporated into Virion Cores but A3G Overexpression in Cells Results in Additional A3G Packaging Outside of the Core

<div><p>(A) HA-A3G–containing ΔVif virions were generated from 293T cells transfected with a fixed amount of proviral plasmid (60 μg) and increasing doses of HA-A3G (0 to 20 μg). Empty HA vector (0 to 20 μg) was used as balance DNA in the transfections. Sample number 1 = 0 (μg of HA-A3G):60 (μg of pNL4-3ΔVif), 2 = 1:60, 3 = 2:60, 4 = 5:60, 5 = 10:60, 6 = 20:60. ΔVif virions were also derived from the H9 T cell line and primary CD4 T cells, which endogenously express A3G. The virion lysates were subjected to immunoblotting with antibodies specific for p24-CA and A3G. The immunoblot is representative of several independent analyses used to generate the graph in (B).</p><p>(B) Graphical representation of quantification from immunoblots in (A) and unpublished data. Data are averaged from three independent transfections of 293T cells, five independent spinoculations of activated primary CD4 T cells, and three independent spinoculations of H9 T cells. The error bars represent standard deviation. The relative ratio of packaged A3G to p24-CA is plotted, with virions derived from CD4 T cells assigned a value of 1.</p><p>(C) Virions containing increasing amounts of HA-A3G relative to p24-CA were solubilized by brief Triton X-100 treatment to generate virion cores containing p24-CA, IN, RT, and NC and supernatants containing gp41 and p24-CA. The triangles represent the increasing dose of A3G and correspond exactly to the numbered samples presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030015#ppat-0030015-g001" target="_blank">Figure 1</a>A. The immunoblots (IB) were also probed for A3G to determine the amount packaged into virion cores.</p>