SPECIFIC INTERACTION OF EUKARYOTIC TRANSLATION INITIATION-FACTOR-3 WITH THE 5'-NONTRANSLATED REGIONS OF HEPATITIS-C VIRUS AND CLASSICAL SWINE FEVER VIRUS RNAS

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap independent attachment (internal entr y) of ribosomes to the similar to 350-nucleotide internal ribosomal en try segment (IRES) at the 5' end of both RNAs, Eukaryotic initiation f actor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an e ssential role in the initiation process on them. Here we report the re sults of chemical and enzymatic footprinting analyses of binary eIF3-I RES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain LII of the CSFV IRES from chemical modification a nd protected bonds in and adjacent to this bulge from cleavage by RNas es ONE and V-1. eIF3 protected an analagous region in domain III of th e HCV IRES from cleavage by these enzymes. These results are consisten t with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpi n IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. Th is is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [P-32]UTP-labelled HCV and CSFV IRES elements resulted in strong label ling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S rib osomal subunits. The results presented here are consistent with a mode l in which binding of these two translation components to separate, sp ecific sites on both HCV and CSFV IRESs enhances the efficiency and ac curacy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.