Mechanism of ribosome recruitment by hepatitis C IRES RNA

Abstract

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 59 untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA•40S•eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Structure of the full-length HCV IRES in solution

“…As domains II and IV are required for efficient translation in vitro and in vivo 7,26,36 but provide no significant stabilization to the IRES-preinitation complex 7,9,12 and that domain III has a slightly higher affinity for the ribosomal 40S subunit than for eIF3 7,12 , it is tempting to speculate that domain III binds first onto the back of the ribosomal 40S subunit, and subsequently to eIF3, and that conformational changes occur in the complex (Fig. 7a).…”

“…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] .…”

“…In these low-resolution structures, the HCV IRES adopts similar elongated conformations [26][27][28] . Upon formation of the 48S IRESpreinitiation complex, the basal part of domain III binds to the solvent-exposed side of the ribosomal 40S subunit, whereas the apical part binds to eIF3 9,12,26 ( Supplementary Fig. S1).…”

Evolution of naturally occurring 5′ non-translated region variants of hepatitis C virus genotype 1b in selectable replicons

“…This shows that although the combination of the two mutants had the highest effect on IRES activity, the C168G mutation contributed most to the reduction in translation. When considering that stem-loop IIIa is a major determinant of eIF3 and 40S ribosomal subunit binding (Hellen & Sarnow, 2001;Kieft et al, 2001), this decrease of~20 % in translation activity seems small for a mutation that is predicted to disrupt the stem-loop (see also Laporte et al, 2000Laporte et al, , 2003. …”

Biography of Jennifer A. Doudna

“…''I think this is the project in the lab that has the greatest potential to lead to something that might have an impact on human health,'' Doudna says. In a series of experiments, Doudna and Jeff Kieft determined that HCV IRES RNA actually has a threedimensional structure responsible for its activity (12,13).…”