Since À1 PRF studies are affected by a huge number of different parameters, it is not an easy task to determine the real ratio between the proteins produced via this mechanism in vivo. However, in viral systems, the efficiency of À1 PRF is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus. For example, frameshifting in retroviruses determines the ratio of structural (Gag) to enzymatic (Gag-Pol) proteins, and plays a critical role in viral particle assembly (5) . In this study, the capacity of CfMV frameshift signals to direct efficient À1 PRF was analysed in vivo by using dual reporter vectors. The length of the CfMV sequence clearly affected the actual efficiency percent in vivo. The PRF efficiency was elevated when longer viral sequences were directing the À1 PRF, but the deletion analysis did not identify any specific region as being solely responsible for the enhancement. Up-and downstream sequences nearby or far away from the cis-acting signals have been reported to enhance À1 PRF in other viruses, such as HIV, human T-cell leukaemia virus and BYDV (6, 19, 20) . Also out-of-frame stop codons have been shown to influence À1 PRF frequency in vitro in retroviruses (17) and in CfMV (18) . A study on the spacer sequences located between the cis-acting signals showed that high slippage frequencies were obtained when the first three nucleotides were G/U, G/A and G/A, the first two being the most important (37) . In CfMV, the spacer starts with UAC, which partially explains the capacity of the CfMV sequence to promote high slippage levels. In this study, the observed enhancement of À1 PRF was, however, caused by sequences that were not in the immediate vicinity of the Figure 5 . Co-expression of CfMV P27 or replicase simultaneously with the minimal frameshift signal construct pAC-A or the corresponding inframe control pAC-Am in yeast. Yeast total protein samples were separated in 12% SDS-PAGE gels, transferred onto PVDF membranes, and immunocomplexes detected by ECL chemiluminescent system. CfMV P27 expression was verified by western blotting with antisera raised against ORF2A (A), and CfMV replicase expression was detected with antisera raised against ORF2B (B). Abbreviations: À, repressed; +, induced; C1, pMAL-VPg $53 kDa; and C2, baculovirus expressed CfMV replicase. slippery sequence thus indicating that CfMV sequences further away also have an influence on the level of frameshifting in vivo. We conclude that the most reliable estimates for À1 PRF and consequently for the amount of replicase versus the 0-frame translation product P27 can be obtained only by using the full-length viral sequences. In reality, such a study would however be hampered by the non-quantitative nature of the western blot analysis, the presence of different polyprotein processing intermediates, and the differences in the stabilities of the end products in the infected cells.