Regulation of -1 ribosomal frameshifting directed by cocksfoot mottle sobemovirus genome

The polyprotein of Cocksfoot mottle virus (CfMV) is encoded by two overlapping open reading frames (ORFs). The putative replicase of CfMV is produced as a part of the polyprotein from ORF2b by the -1 ribosomal frameshifting mechanism. The signals leading to -1 ribosomal frameshifting directed by CfMV RNA are the slippery heptamer UUUAAAC and a stem-loop structure starting seven nucleotides downstream from the heptamer. We studied the effect of different parts of the CfMV genome on the -1 ribosomal frameshifting efficiency using a wheat germ extract transcription/translation system. A point mutation in the slippery heptamer and a mutation deleting the stem-loop structure prevented frameshifting. Seventy nucleotides of CfMV sequence, including the slippery sequence and the stem-loop structure, was found to act as a minimal region for frameshifting. Interestingly, a termination codon introduced into the -1-frame 27 nucleotides downstream of the stem-loop structure increased frameshift efficiency threefold, while a similarly located termination codon in the 0-frame had no effect. Even fourfold to fivefold efficiencies were observed when the polyprotein encoding ORFs were fused together, which led simultaneously to the formation of a termination codon downstream of the frameshift signal. Possible reasons underlying these observations are discussed.