Genetic mechanisms underlying the structural elaboration and dissemination of viral internal ribosomal entry sites

Viral internal ribosomal entry sites (IRESs) are highly structured cis-acting RNAs that mediate end-independent initiation of translation. Their origin remains obscure. The simplest IRESs (type 6) occur in the intergenic region of Dicistroviridae genomes (order Picornavirales), consist of two pseudoknots, and initiate translation by factor-independent binding to ribosomes. Larger variants contain a third pseudoknot that modifies the mechanism of IRES function by engaging with the ribosomal head and promoting binding to the ribosomal aminoacyl site. Metagenomic analyses undertaken to identify structurally distinct type 6 IRESs identified subsets ranging from ∼120-260 nt in length. They differ by the cumulative addition of structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication, and form additional subdomains. Biochemical analysis showed that these novel classes of type 6 IRES all bound directly to the ribosomal peptidyl site. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of type 6 IRESs at the 5'-end of dicistrovirus-like genomes and in families other than Dicistroviridae, including Marnaviridae (order Picornavirales) and Tombusviridae (order Tolivirales).