The 22-nt Rbm3 IRES directs downstream reporter gene expression from bicistronic and tricistronic HBV pgRNA mimics.

<p>(A) EGFP as reporter. <i>Left</i>: Schematic representation of the expression cassettes used. The upstream part is identical to the HBV vector pCH-9/3093 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060306#pone.0060306-Sun1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060306#pone.0060306-Liu1" target="_blank">[21]</a> in which the CMV-IE promoter drives transcription of authentic pgRNA starting at nt position 3100. In constructs I - III the sequence following the end of the core gene was replaced by the indicated control elements and a gene for EGFP; in construct IV, the EGFP gene was fused to the authentic Pol translation start. pC denotes the preC region; its start codon (nt positions 3096–3098) is not part of the pgRNA. BsdR, blasticidin resistance. <i>Right</i>: EGFP fluorescence in HepG2 cells transfected with constructs I - IV on day 4 post transfection. All micrographs were taken under identical conditions. Comparable results were obtained in Huh7 cells. (B) RLuc as reporter. The constructs are based on the HBV expression vector pHBV1.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060306#pone.0060306-Ren1" target="_blank">[46]</a> in which pgRNA transcription is driven by the authentic HBV core promoter which overlaps with coding information for the RNase H (RH) domain of Pol and HBx (X), and which also generates precore mRNA, thus giving rise to genuine HBeAg. No significant differences in HBeAg levels between constructs I, II, and III were detected by HBeAg ELISA (n = 3; <i>P</i>>0.05). Histograms on the right show relative RLuc activities in HepG2 and Huh7 cells compared to firefly luciferase activities from cotransfected pGL3 control vector, as determined by the dual luciferase assay. Values for the EMCV IRES construct were set as 100%. Mean values from three independent experiments are shown; error bars indicate standard deviation (SD). All differences between the groups were significant, with <i>P</i><0.01 for all pairs except III vs. IV (<i>P</i><0.05).