For the in vitro analysis, the lacZ-gene of pAC-A/Am, -B/Bm and -C/Cm vectors was replaced with PCR-amplified Renilla luciferase (Rluc) gene from pRLnull vector (Promega). The resulting pACRF plasmids were used as templates for PCR in order to add T7 promoter upstream of the Rluc gene. These PCR products were used for RNA synthesis with RiboMax kit (Promega). Transcripts were treated with RQ1-DNase (Promega), purified with Qiagen RNeasy columns, and quantified spectrophotometrically. The integrity of the transcripts was checked in agarose gels. In vitro translations were carried CfMV À1 PRF test and control sequences were cloned between the b-galactosidase (LacZ) and firefly luciferase (Luc) genes into a dual reporter vector pAC74 (26) . Inframe control constructs had one extra nucleotide inserted in front of the slippery heptamer, which fused the reporters into the same reading frame. Thus, translation of the inframe control results in the production of a b-galactosidase-CfMV-firefly luciferase fusion. Translation of the test constructs in the incoming 0-frame yields a b-galactosidase-CfMV fusion, whereas À1 PRF produces a b-galactosidase-CfMV-firefly luciferase fusions identical to those produced from the inframe controls. À1 PRF efficiencies were calculated from the firefly luciferase activities after b-galactosidase normalization with the given formula. (B) CfMV polyprotein is encoded by two overlapping ORFs, 2A and 2B via À1 PRF. Sequence regions tested in the dual reporter vectors for their activity to promote À1 PRF are indicated. The numbering refers to the CfMV RNA sequence as published in (23) . out in wheat germ extract (WGE) according to the manufacturer's protocols (Promega). Reactions were incubated in room temperature for 60 min, and stopped on ice prior to enzymatic measurements.