Natural variation in translational activities of the 5 ' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: Evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site

The 5' nontranslated RNA (5'NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about tw ofold less efficiency than the 5'NTR of a genotype la virus under physiolog ic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222: 31-42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (p resent in HCV-H) restores the translational activity to that of the HCV-H 5 'NTR both in vitro and in vivo. These nucleotides are located upstream of t he minimal essential internal ribosome entry site (IRES), as a 6-nt deletio n spanning nt 32 to 37 also increased the translational activity of the HCV -N 5'NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, t his inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analy sis of RNA transcripts containing frameshift mutations demonstrated that th e nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation ef ficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in ret iculocyte lysates containing high K+ concentrations, with a sevenfold diffe rence evident at 130 to 150 mM K+. These results suggest that there is an R NA-RNA interaction involving 5'NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the gen otype 1b virus, HCV-N.