Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes

Abstract

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem-loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA(Phe) or its anticodon stem- loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

“…1D). Previous reports have implicated domains 1 and 2 as the parts of the IGR IRES primarily responsible for ribosome recruitment (14,17,18); our assays indicate that domain 3 by itself can bind to ribosomes, but at a much lower affinity than does the full-length IRES (∼2-10 nM) (18). Binding of the domain 3 RNA to 70S ribosomes was reduced by competitor tRNA, suggesting that the IRES domain occupies a tRNA binding site (Fig.…”

“…Mutational analysis of IGR IRES RNAs has shown that the primary determinants for binding to the ribosome reside in domains 1 and 2; deletion of domain 3 has only a minor effect on ribosome binding affinity (17,42). In contrast, certain localized changes in domain 3 have profound effects on gene expression.…”

“…1A) (5,6). IGR IRES RNAs bind directly to host cell ribosomes, initiate translation of the downstream message using a non-AUG codon, and induce translocation before a peptide bond is made (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Thus, the IGR IRESs are direct manipulators of the translation machinery, and they show how an RNA can drive its own translation using direct ribosome recruitment without protein intermediaries, reminiscent of an RNA world.…”

“…The IGR IRES RNAs fold before encountering the ribosome into a conformation with three structural domains: Domains 1 and 2 fold together and are important for initial recruitment of the ribosome (14,17,18), whereas domain 3 is implicated in correct positioning of the IRES-containing viral RNA with respect to the ribosomal reading frame (Fig. 1B) (14).…”

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

“…On the other hand, structural studies performed on the HCV IRES and the IGR of members of the family Dicistroviridae have shown the capacity of these IRES elements to be accommodated in the interface of the ribosomal subunits (Spahn et al, 2001;Boehringer et al, 2005;Schuler et al, 2006;Pfingsten et al, 2006). Although the IGR and the HCV IRES elements exhibit different structural organization (Kieft et al, 2002;Rijnbrand et al, 2004;Jan & Sarnow, 2002;Nishiyama et al, 2003) and their binding sites in the ribosomal subunit are different, similar conformational changes are induced in the 40S ribosomal subunit (Spahn et al, 2004). This finding opens the possibility that IRES elements could share the property of having a universal structural IRES motif (USIM) that could mediate its direct interaction with the 40S subunit.…”