Pseudoknot mutants were cloned in KpnI/BamHI digested pDUAL-HIV(0) (4). The GGGAAAC slippery sequence was changed to the more efficient UUUAAAC (28) to obtain a better read-out. In these constructs the stopcodon of the first open-reading frame, Renilla luciferase, is located downstream of the pseudoknot. A non-frameshifting control was constructed by changing the slippery sequence to UUUAAGC, while in the in-frame control a C-residue was added directly downstream of this heptamer. In both constructs the pseudoknot was the NMR-pseudoknot. A list of oligonucleotide sequences is available on request. All constructs were confirmed by DNA sequencing. HeLa cells (24-well plate) were transfected with 250 ng of plasmid using lipofectamine-2000 (Invitrogen). Cells were lysed 20 h after transfection and luciferase activities were measured in a Glomax-multidetector (Promega) according to manufacturer's protocol.