Internal ribosome entry site is not used to translate uENF2 or PP ORF from the uENF1-uENF2-PP tricistronic mRNA. A, shown are the effects of insertion of the Renilla luciferase ORF (0.9 kb), indicated by a dark gray box with a double diagonal line, in front of the uENF1 ORF on the translation of proteins from the uENF1, uENF2, and PP ORFs. B, shown are the effects of replacement of the uENF1 or uENF2 ORF with the Renilla luciferase ORF on the translation of proteins from the downstream ORFs. C, shown are the effects of insertion of a 22-bp stem loop at the 5′-end of the mRNA on the translation of proteins from the uENF1, uENF2, and PP ORFs. Schematic structures of mRNAs expressed in Sf9 cell lines were shown on the left side of each graph. Error bars represent S.E. (n = 3–4 for each experiment. Each replicate is an average of results from 3–4 wells). Experiments were repeated on two to four different days.