A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation.

The 5'-untranslated region (5'-UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) that directs translation of the viral open reading frame (ORF). The 5'-UTR consists of 341 nucleotides (nt) in most strains, and multiple segments within this region are important for its IRES activity. Sequencing analysis of a full-length HCV cDNA clone derived from a Japanese HCV1b-positive patient showed the 5'-UTR was 342 nt long due to a nucleotide T insertion at position 207. The influence of this T insertion on the IRES activity in directing cap-independent translation was investigated. The IRES of the 5'-UTR342 was approximately five- and two- to sevenfold more active in directing luciferase expression in monocistronic and bicistronic expression systems, respectively, when compared with the IRES of the 5'-UTR341 of a previously reported HCV1b strain. In addition to the T insertion, another point mutation involving an A to C transition at position 119 was also present in the 5'-UTR342. Simultaneous comparison of the IRES activities in engineered constructs that contained each of the two mutations indicated that the insertion at position 207 is responsible for the enhanced IRES activity of the 5'-UTR342. Further determination of the abilities of the engineered 5'-UTRs harbouring A, G, or C insertions at the same position to initiate translation indicated that both T and non-T nucleotide insertions lead to enhanced cap-independent translation.