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Caliciviruses have positive-sense RNA genomes, typically with short 5′-untranslated regions (5′UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5′UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200–300 codon-long ORF (designated ORF1*) that overlaps the 5′-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3′-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES's 3′-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF45, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1). [ABSTRACT FROM AUTHOR]

Research has been conducted on ribosomes assembled on the Cricket paralysis virus without initiation factor or initiator tRNA involvement. Results demonstrate that ribosomes can begin elongation on natural mRNA after initiation without initiator tRNA or initiation factors.

The assembly of 80S ribosomes in the cricket paralysis virus involves 40S and 60S ribosomal subunits with a CCU and GCU triplets at the P and the A site respectively. Data indicate that although the A site lacks translation initiation factors, initiation of protein synthesis occurs at this site suggesting involvement of unknown factors.

Author(s): Tatyana V. Pestova (corresponding author) [1, 2]; Ivan B. Lomakin [1]; Joon H. Lee [3]; Sang Ki Choi [3]; Thomas E. Dever [3]; Christopher U. T. Hellen [1] Initiation [...]

INTRODUCTION AND BACKGROUND Poliovirus, discovered in 1909 by Landsteiner & Popper, is possibly the best known of all viruses. During the first half of this century poliovirus caused devastating epidemics [...]

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (elFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex elF4F and the factors elF4A and elF4B are required for binding of 43S complexes (comprising a 40S subunit, elF2/GTP/Met-tRNAi and elF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon, elFIA enhances the ability of elF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires elF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of elF2-bound GTP induced by elF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The elF4A and elF4G subunits of elF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require elF1, elFIA, and the elF4E subunit of elF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of elF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only elF2 and elF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap–eukaryotic initiation factor 4E (eIF4E)–eIF4G–eIF3–40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5′-terminal AUGs was stimulated by the eIF4E–cap interaction and followed “the first AUG” rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5′ end of mRNA, implying that Met-tRNAiMet inspects mRNA from the first nucleotide and that initiation does not have a “blind spot.” In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E–eIF4G–eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m7G cap specifically interacts with eIF3l. [ABSTRACT FROM AUTHOR]