Yellow Fever 5′ Noncoding Region as a Potential Element to Improve Hepatitis C Virus Production through Modification of Translational Control

Abstract

The lengthy 5′ noncoding region (5′ NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5′ NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was comparedin vitroin rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5′ NCR. Surprisingly, the related distanced YFV 5′ NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effectivein vivoafter transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5′ NCR among theFlaviviridaefamily?