Digoxin blocks cytoplasmic accumulation of HIV-1 US RNA and its effect is partially reversed by expression of Rev <i>in trans</i>.

<p>(A) To assess the effect of digoxin treatment on viral RNA transport, in situ hybridization of US RNA was performed on HeLa rtTA-HIV-Δ<i>Mls</i> cells treated with digoxin, 3TC, or DMSO solvent. After 4 h, viral expression was left uninduced (−) or induced by addition of Dox (+). After 24 h, cells were fixed and incubated with labeled oligonucleotides specific to HIV-1 US RNA (US RNA). After washing to remove unbound probe, cells were stained with DAPI to allow detection of nuclei and images captured at 630× magnification. Shown is a representative sample of the results observed from ≥4 independent experiments. (B) To determine whether Rev expression <i>in trans</i> could alleviate the inhibition of Gag protein synthesis by digoxin, cells were transfected with vectors expressing dsRed or a dsRed-Rev fusion protein. At 24 h post-transfection, cells were treated with digoxin for 4 h then Dox was added to induce provirus expression. At 24 h post-doxycycline addition, cells were fixed, DAPI stained, and viewed for co-expression of dsRed (Cy3) and Gag signal (FITC). Shown is a representative sample of the results obtained. Note: intense foci in the nuclei seen in the FITC channel in cells transfected with dsRed-Rev is due to bleed through from the Cy3 channel. To assess the extent of reversing the effects of digoxin by expressing Rev in trans (C), >100 dsRed+ cells were examined in each experiment and the frequency of dsRed+ cells also having Gag+ expression was determined for each condition and displayed as “% of cells rescued”. Error bars of the data are SEM and significant changes from dsRed indicated by asterisk as detailed in “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#s4" target="_blank">Materials & Methods</a>”.