Modulation of translational efficiency by contextual nucleotides flanking a Baculovirus initiator AUG codon

In a previous study of translational regulation of a baculovirus gene, we o bserved that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang a nd G. W. Blissard, 1997, J. Virol 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codo n in modulating translation initiation in lepidopteran insect cells. The ro les of nucleotides flanking the AcMNPV gp64 initiator codon were examined b y site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiato r context was cloned in-frame with a chloramphenicol acetyl transferase rep orter gene and under the control of a heterologous promoter. All possible s ingle-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated 1. Constructs were transfected into lepidopteran cells and translation prod ucts were quantified by an enzyme-linked immunosorbent assay procedure. Sub stitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, s pecific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitut ion of any nucleotide for T in the +5 position resulted in approximately 1. 9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6 ) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopt eran cells. Consensus translation initiation contexts were generated from b aculovirus genes and lepidopteran genes, then compared with the experimenta l results from the gp64 initiator context. (C) 1999 Academic Press.