A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs

Abstract

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal ''P'' site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNA i Met , eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed. Protein synthesis begins following assembly of an initiation complex in which the initiation codon of an mRNA and the anticodon of initiator tRNA are base-paired in the ribosomal ''P'' site. There are similarities and significant differences in the mechanisms of initiation complex formation in prokaryotes and eukaryotes. A universal characteristic is that initiation starts with separated ribosomal subunits.In prokaryotes, the small (30S) ribosomal subunit binds mRNA and initiator tRNA in random order to form a complex that then undergoes conformational rearrangement, promoting codon-anticodon base-pairing at the P site and joining of the large (50S) subunit (Gualerzi and Pon 1990). Ribosome binding results from interactions between the 30S subunit and multiple recognition elements in the mRNA, such as the Shine-Dalgarno sequence (McCarthy and Brimacombe 1994). It does not depend on initiation factors. Ribosome-binding sites can occur at any position within an mRNA and as a result many prokaryotic mRNAs are polycistronic.In contrast, the small (40S) ribosomal subunit in eukaryotes requires several eukaryotic initiation factors (eIFs), first to bind initiator tRNA (as a ternary complex with eIF2 and GTP) to form a 43S complex and then to bind mRNA to form a 48S complex (Merrick 1992). The most common mechanism for recruitment of an mRNA is mediated by its capped 5Ј end, which is bound by the eIF4E subunit of eIF4F and then by the 43S complex. Ribosomal binding to mRNA and scanning to the initiation codon require ATP hydrolysis and involve eIF4A, eIF4B, and eIF4F. Most mRNAs that use this mechanism of ribosomal binding are monocistronic because initiation is usually limited to the 5Ј-most AUG codon.

Structure of the full-length HCV IRES in solution

“…A common feature of all IRES is their inherent flexibility, which seems to increase in correlation with the need for more initiation factors. HCV IRES belongs to class II IRES and requires eIF3, eIF2 and initiator met-tRNA to assemble ribosomes 10 . We showed here that it is an articulated molecule in which structured elements undergo large reorientations relative to each other 16,18,28 .…”

“…Next, the IRES-40S complex interacts with the eukaryotic multi-subunit initiation factor 3 (eIF3) and the ternary Met-transfer RNA (tRNAi)-eIF2-GTP complex, forming a 48S IRES-preinitiation complex. GTP hydrolysis triggers the release of the initiation factors and the binding of the 60S ribosomal subunit, leading to the assembly of an elongation-competent IRES-80S initiation complex [9][10][11][12] .…”

Bunyavirus N: eIF4F surrogate and cap-guardian

“…[14][15][16] However, in the case of the flavivirus, hepatitis C virus (HCV), as well as some of the avian and porcine picornaviruses, the pre-initiation complex is recruited directly to the IRES by way of just the 40S ribosomal subunit and eIF3, and without eIF4F components. [17][18][19] Further, the dicistoviruses, such as cricket paralysis virus, recruit the 40S subunit its IRES without either eIF4F or eIF3. 20,21 These examples are indicative of various requirements for initiation factors during IRES-mediated translation.…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…2) and protein requirement (Table 1) (Fraser & Doudna, 2007). To assemble 48S initiation complexes in vitro, the IRES of HCV as well as classical swine fever virus (CSFV) requires the presence of the 40S ribosomal subunit, eIF3 and the eIF2/GTP/Met-tRNA(i) ternary complex (Ji et al, 2004;Pestova et al, 1998).…”