Modulation of Translational Efficiency by Contextual Nucleotides Flanking a Baculovirus Initiator AUG Codon

Abstract

In a previous study of translational regulation of a baculovirus gene, we observed 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 and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-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 products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific 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 substitution 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 lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.

Transfection of insect cell lines using polyethylenimine

“…For transfection were used the following ratios of DNA:ExGen500: wells 1, 2, 3-0.5 lg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 4, 5, 6-1.0 lg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 7, 8, 9-2.0 lg plasmid DNA and 6 eq (1), 8 eq (2), 10 eq (3) ExGen500; wells 10, 11, 12-mock, Sf9 cells transfected with plasmid DNA in the absence of ExGen500 (10), Sf9 cells transfected in the absence of plasmid DNA (11), mock Sf9 cells transfection for 5 days and revealed maximal GFP fluorescence occurred at 72 h post transfection. This is consistent with previous results obtained with the gp64 promoter (Slack and Lawrence 2003;Slack et al 2001;Chang et al 1999). Inoculation of cells with plasmid DNA in absence of transfection reagent (i.e., a negative control) resulted in no fluorescent cells.…”

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

“…This molecular mass had already been predicted from the amino acid sequence of hDPPIV (2,300 bp) (Tanaka et al 1992;Thoma et al 2003). The result was in consistence with previous studies (Chang et al 1999;Slack et al 2001;Slack and Lawrence 2002;Ogay et al 2006).…”

Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions

“…Gradient fractions were assayed by double-label scintillation spectrometry. To minimize the 35 S spillover into the 3 H window, about 10-fold more 3 H counts were loaded on each gradient. wt PaV particles sedimented as a single sharp peak near the middle of the gradient centered on fraction 15 ( Fig.…”