Specific Interaction of Eukaryotic Translation Initiation Factor 3 with the 5′ Nontranslated Regions of Hepatitis C Virus and Classical Swine Fever Virus RNAs

Abstract

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the ∼350-nucleotide internal ribosomal entry segment (IRES) at the 5′ end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES 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 III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V1. eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This 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 [32P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling 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 ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

“…Previously, a IIIb position much closer to the 40S subunit and overlapping with the IIIabc four-way junction in our present model was suggested (Boehringer et al, 2005;Siridechadilok et al, 2005). Because IIIb interacts with the eIF3 initiation factor complex (Sizova et al, 1998;Ji et al, 2004;Otto & Puglisi, 2004), our model places this major eIF3 binding site further away from the ribosome in excellent agreement with the direct visualization of eIF3 on the related 40S-bound CSFV IRES with a deletion of domain II (Hashem et al, 2013).…”

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

“…The class III IRES in HCV and pestivirus RNAs uses a prokaryote-like mode of translation initiation by positioning ribosomes on the mRNA through interactions with both eIF3 and the ribosome itself, thereby circumventing any need for eIF4F Sizova et al 1998;Ji et al 2004;Otto and Puglisi 2004;Pisarev et al 2004;Fraser and Doudna 2007;Locker et al 2007;Babaylova et al 2009;Berry et al 2010). Translation of HCV RNA is also enhanced by the binding of host miR-122 to two target sites in the 5 0 UTR, thereby promoting ribosome recruitment (Jopling et al 2005(Jopling et al , 2008Henke et al 2008;Jangra et al 2010).…”

Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-α correlates with a reduced La protein

“…[15][16][17] The IRES activity is highly dependent on both the primary sequence of this segment and its ability to form complex secondary and tertiary RNA structures. [18][19][20][21][22] A number of in vitro studies have suggested that several cellular proteins, including both conventional translation initiation factors such as eukaryotic initiation factor 3 (eIF3) 23,24 and noncanonical translation initiation factors such as the nuclear La protein [25][26][27][28][29] or polypyrimidine tract binding protein (PTB), [30][31][32] may stimulate HCV translation. We previously reported that HCV translation is regulated in a cell-cycle-dependent manner and that cellular proteins that vary in abundance during the cell cycle may be involved in this process, 33 but to date, the mechanism by which HCV translation is regulated in vivo is not well understood.…”