Rare codons in uORFs of baculovirus p13 gene modulates downstream gene expression

The p13 gene is a group II nucleopolyhedroviruses (NPVs) specific gene and featured by containing upstream mini ORFs (uORF) in its 5' UTR region. However, there are almost no reports published on the functions of the uORFs of p13 gene. In this study, the Luciferase Reporter Assay System was employed to investigate how the mini ORFs of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) p13 gene (Ha-p13) and its rare codons regulated the downstream gene expression. After the coding sequence of uORFs in the Ha-p13 gene was fused to the luciferase reporter gene in the expression vector pGL3 and the plasmid DNA was then transfected into the Hz-AM1 cells, the translation of the fusion protein could be initiated from the start codon of the uORFs. The uAUG and its context in uORF2 seemed to be more efficient for translation initiation than that in uORF1. Mutation of the start codons in one or both of uORFs (uORF1 or uORF2) could significantly increase the expression of the downstream reporter gene. The start codon mutation in uORF1 produced a higher reporter gene expression than that in uORF2, indicating that the uORF1 could be a stronger inhibitor than the uORF2, and the length of uORFs seemed not to be crucial for down-regulating translation. The expression of both uORFs could co-regulate the associated gene expression. Substituting the rare codons in uORF1, uORF2 or both with less rare codons dramatically increased the expression of the downstream reporter gene. Rare codon mutations in both uORFs were much more efficient in up-regulating the associate gene expression than mutations in either of the two uORFs alone.