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.

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. Keywords: Cap-independent translation; internal ribosomal entry site; hepatitis C virus; bicistronic expression vector; translation efficiency.