FIG. 4.

Expansion of the pseudoknot loop of the HCV IRES has no effect on IRES activity. (A) Structure and primary sequence of the HCV IRES pseudoknot, showing the position of insertion of five A residues in pXLHCV 40-372.NS′ ³²⁴insA₅. (B) Capped dicistronic transcripts of the parent pXLHCV 40-372.NS′ and a low activity deletion mutant pXLHCV 40-339.NS′ (29), the negative control pXLJ′OS, and the insertion mutant pXLHCV 40-372.NS′ ³²⁴insA₅ were translated at final concentrations of 50 (a), 25 (b), 12.5 (c), and 6.25 (d) μg/ml. The translation products were resolved on a 20% acrylamide gel, and the resulting autoradiograph is shown. XL, the cyclin B2 cistron translation product; NS′, the product derived from the downstream cistron. The smaller size of the downstream cistron product encoded by pXLHCV 40-339.NS′ is due to the fact that this construct has no viral coding sequences, whereas in pXLHCV 40-372.NS′ and its derivatives, the NS′ coding sequence is fused to the first 30 nt of viral coding sequences, and thus the product is NS′ with an N-terminal extension.