Digoxin alters HIV-1 pre-mRNA splice site usage to suppress Rev expression.

<p>Effects of digoxin on HIV-1 splice site use were assayed by RT-PCR of the 2 kb, MS class of HIV-1 mRNA. (A) Diagram of the HIV-1 provirus indicating the splice sites (SD1–4, SA1–7) and position of the primers (arrow points) used to amplify MS mRNA species (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#s4" target="_blank">Materials and Methods</a>). Also shown below are the forms of viral RNA used to express the two isoforms of Tat: p14 generated from SS RNA and p16 from MS RNA. Open boxes represent exons, closed boxes are alternative exons, and dashed lines are excised introns. The SS RNA generates a truncated form of Tat (p14) because of the presence of a termination codon immediately 3′ of SD4. (B) A representative RT-PCR gel of the levels of each MS mRNA species (arrows) from cells treated with digoxin (Dig) or control (C) using cDNAs described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#ppat-1003241-g003" target="_blank">Fig. 3</a>. (C) Graph summarizing the effects of digoxin (white) and control treatments (gray) on the level of each MS mRNA species (x-axis) relative to the total HIV-1 MS mRNA (y-axis) and displayed as a fraction of the total MS HIV-1 RNA. Data was averaged from ≥6 experiments, error bars are SEM, and significant changes of treatments from DMSO (+) control are indicated by asterisks. (D) Western blot analysis of HIV-1 regulatory factors Rev (top) and Tat (bottom) from cells treated with digoxin or DMSO as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#ppat-1003241-g001" target="_blank">Fig. 1</a>. Anti-α-tubulin blots served as an internal loading control for the relative amount of protein lysate in each lane. In each experiment, Dox induced (+) compared to uninduced (−) shows successful activation of HIV-1 protein expression. Protein products (right), specific antibodies detecting them (left), and MW standards (top left) are as indicated. Each immunoblot is a representative of the effects observed from ≥7 independent experiments.