The effect of didemnin B on RT-eEF1A interaction <i>in vitro</i> and viral RTCs in cells.

<p>(<b>A</b>) Cell lysates from HEK293T cells transiently expressing HIV RTp51-FLAG were incubated for 30 min at 37°C with concentrations of Did B, CHX or DMSO as indicated. Immunoprecipitation was performed using a rabbit anti-eEF1A antibody and visualized by western blot using anti-FLAG (upper panel) or mouse anti-eEF1A antibodies (lower panel). The experiment was performed three times with similar results. (<b>B</b>) Western blots in (<b>A</b>) were analyzed by densitometry. Normalized density values for RTp51 were calculated by dividing the relative density of RTp51 in each sample lane by the relative density of the eEF1A in the same lane [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005289#ppat.1005289.ref045" target="_blank">45</a>]. The columns represent the mean normalized densities and standard deviations of three independent experiments relative to the DMSO treated value. Student’s two-tailed T test was used to calculate statistical significance (<b>C</b>—<b>F</b>) HEK293T cells were treated with Did B at 0.8 nM or with DMSO carrier. The cells were infected with VSV-G psuedotyped wild type (<b>C</b> and <b>E</b>) or W252A RT mutant (<b>D</b> and <b>F</b>) HIV-1. A lysate was prepared and viral RTCs were purified by isopycnography using a 20% to 70% linear sucrose gradient. Fractions collected from the tube bottom were assayed for (<b>C</b> and <b>D,</b> left y-axis) CA by ELISA and (<b>E</b> and <b>F,</b> left y-axis) viral strong stop DNA by qPCR. The density of the sucrose in each fraction (<b>C</b>–<b>F</b>, right y-axis) is indicated by the blue line. The box in each graph indicates a fraction with a density of 1.33 g/ml. The experiment was repeated twice with similar results and a representative outcome is shown.