Differential Effect of CLK Overexpression on HIV-1 RNA Accumulation and Splicing. Cells were transfected with CMVmyc 3xTerm (-) or CMVtTa (+, to induce provirus expression) along with control plasmid (CMVmyc 3xterm) or vectors expressing GFP-CLK1, GFP-CLK2, GFP-CLK3 or GFP-CLK4. Forty-eight hours post-transfection, cells were harvested and total RNA extracted. (A) Abundance of unspliced (US), singly spliced (SS), and multiply spliced (MS) viral RNAs was determined by qRT-PCR as outlined in "Materials & Methods". Shown are the average of >7 independent analyses. (B) To examine the effect of overexpression of individual CLK proteins on viral RNA splicing, radioactive RT-PCR was performed on MS viral RNAs. Products were fractionated on 8M urea-PAGE gels and gels exposed to phosphor screens to detect the different splice products. For explanation of the products generated, please refer to additional file 2, Figure S2. On the left is a representative RT-PCR gel of the pattern observed and on the right, a summary of the relative abundance of each splice product (fraction of total viral MS RNA) for >3 independent assays. Asterisks denote values determined to be significantly different from control at a p value < 0.05.