Mutational analysis of the different bulge regions of hepatitis C virus domain II and their influence on internal ribosome entry site translational ability.
The hepatitis C virus (HCV) 5'-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.
Odreman-Macchioli
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International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy.
Baralle
F E
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Buratti
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E
D001483:Base Sequence; D016174:Hepacivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism*
D012367:RNA, Viral