Both codon context and leader length contribute to efficient expression of two overlapping open reading frames of a cucumber necrosis virus bifunctional subgenomic mRNA.

The importance of codon context and leader length in the translational regulation of p20 and p21 from the bifunctional 0.9-kb subgenomic mRNA cucumber necrosis virus was investigated. Nucleotide substitutions introduced into the -3 and +4 positions of the p21 AUG codon (where the A of the AUG is +1) verified that purines in these positions are favored and demonstrated the similar contribution of the -3 and +4 positions to the efficiency of initiation codon selection in plants. The effect of nucleotide substitutions in the +5 position, most clearly demonstrated when pyrimidines occupy the -3 and +4 positions, also provided direct insight into the influence of the +5 position in plants. The codon context of the upstream p21 initiation codon affected expression from the downstream p20 AUG codon. In addition, an increase in the length of the subgenomic mRNA leader decreased expression from the downstream p20 initiation site. These latter observations are in accordance with the "Kozak rules" for accession of internal AUG codons by leaky ribosomal scanning and provide the first example of an effect of leader length on the efficiency of translation initiation in a plant (viral) mRNA.