DDX3 facilitates translation of reporter mRNAs containing the 5’ UTR of HIV-1 mRNAs.

<p><b>A</b>. Schematic representation of the 5’ UTR of HIV-1 mRNAs. The 5’ UTR of all HIV-1 transcripts shares the same 289 nt noncoding region, which contains numerous cis-acting elements, including the transactivation response (TAR) hairpin, the polyadenylation (poly(A)) hairpin, the primer binding site (PBS), the dimerization initiation site (DIS), and the major splice donor site (SD). RNA secondary structures in the 5’ UTR of HIV-1 mRNA were adapted from Wilkinson et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068665#B24" target="_blank">24</a>]. The functional motifs are indicated above each stem-loop domain. <b>B</b>. Schematic representation of the firefly luciferase (Fluc) reporters and details on the 5’ UTR of the pFL-SV40 derived reporters. HIV-5’ UTR represents the complete 5’ UTR of HIV-1 mRNAs (nucleotides 1-289). HIV-TAR contains the TAR hairpin (nucleotides 1-57), while HIV-5’ UTRΔTAR encompasses the 5’ UTR devoid of the TAR hairpin (nucleotides 58-289). HIV-TP contains the TAR-poly(A) region (nucleotides 1-104), while HIV-5’ UTRΔTP encompasses the 5’ UTR devoid of the TAR-poly(A) region (nucleotides 105-289). <b>C</b>. HeLa cells were co-transfected with a pFL-SV40 derived reporter and the control pRL-SV40 vector encoding the <i>Renilla</i> luciferase (Rluc) together with empty pSilencer 1.0-U6 vector (mock) or the pSilencer 1.0-U6 vector expressing sh-DDX3#2 (DDX3-KD) for 48 h. For each transfectant, the Fluc activity was first normalized to that of the Rluc control. Normalized Fluc activity of sh-DDX3#2-transfectants (DDX3-KD) was then compared to that of the mock. The bar graph shows the relative Fluc/Rluc activities in DDX3-KD cells relative to mock cells (upper panel). The pFL-SV40 derived reporter containing the 5’ UTR of <i>cyclin E1</i> (CCNE1) served as a positive control. Fluc and Rluc mRNAs were analyzed by quantitative RT-PCR (lower panel). All data are shown as mean (± SEM) from at least three independent experiments (<b>*</b><i>p</i> < 0.05, <b>**</b><i>p</i> < 0.01, <b>***</b><i>p</i> < 0.001).</p