The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus

Abstract

Internal ribosomal entry site (IRES) functions as a cis-acting RNA element, which drives an alternative and cap-independent translation initiation pathway. Currently, there are few studies on effects of nucleotide usages at key nucleotide positions +4 and-3 flanking start codon mediated by IRES of hepatitis C virus (HCV). Herein, we focus on the effect of nucleotide usages at-3 and +4 positions mediated by HCV IRES. The nucleotide contexts flanking AUG start codon employed by HCV IRES is firstly analyzed. We found that each position in the six nucleotide positions (-4 to +6) flanking start codon of HCV has a strong tendency to select the specific nucleotide. A set of bicistronic expression vectors containing CAT gene, HCV IRES and EGFP gene were constructed, including 16 different nucleotide combinations at position-3 and +4. Each set, in which nucleotide at the-3 and +4 position has been changed into different nucleotides, included 16 types of bicistronic expression vectors. It was found that the purine nucleotide at the position-3 or +4 obviously impacts on HCV IRES-related expression, and IRES-driven translation is potentially influenced by the Kozak rule. Our results suggest that optimization of nucleotides at positions-3 and +4 is a convenient and efficient way to enhance the level of IRES-mediated translation.

Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES

“…The occupancy of the mRNA-binding channel for 48S ICs prepared on both wt and DdII IRESs (Structures 12 wt/DdII ) is identical, with mRNA density present from the exit channel through the E, P, and A sites to the entry region. The observation that eIF2a interacts via Arg55 with the (À3) context nucleotide, as in the canonical initiation process, is consistent with its importance in HCV IRES function (Ma et al, 2018).…”