Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus 1b 5' untranslated region (5'UTR) lead to mutants severely defective in translation which cannot be complemented in trans by the wild-type 5'UTR sequence.
Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5' untranslated region (5'UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5'UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5'UTR sequence were identified at various locations throughout the 5'UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.
Tang
S
S
Institute of Virology, University of Glasgow, Glasgow G11 5JR, Scotland, United Kingdom.
Collier
A J
AJ
Elliott
R M
RM
D020121:5' Untranslated Regions / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D003673:Defective Viruses / Q000235:genetics; D005816:Genetic Complementation Test; D016174:Hepacivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry*; D013329:Structure-Activity Relationship