Picornavirus Internal Ribosome Entry Site Elements Can Stimulate Translation of Upstream Genes

Abstract

Certain viral and cellular mRNAs initiate translation capindependently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K ؉ concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K ؉ concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m 7 GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety. Internal ribosome entry site (IRES)3 elements are cis-acting RNA regions that confer the internal entry of ribosomes to the RNA translation start site independent of the process of capdependent scanning from the 5Ј-end of the RNA (1, 2). Such IRES elements were first discovered in picornaviruses like encephalomyocarditis virus (EMCV) (3), poliovirus (4), and foot-and-mouth disease virus (FMDV) (5). Also the translation of hepatitis C virus (HCV) (6) and of several cellular mRNAs is driven by IRES elements (2).The picornaviral IRES elements are classified in three groups according to their sequences and secondary structures, the type I elements of the entero-/rhinovirus group (including poliovirus), the type II elements of the cardio-/aphthovirus group (including EMCV and FMDV), and the type III element of hepatitis A virus (1). After infection of a susceptible cell, these IRES elements guide the small ribosomal subunit to an AUG triplet in a starting window at the 3Ј-border of the IRES (1, 2, 7). Ribosome binding to the picornavirus IRES elements is mediated by a number of cellular RNA-binding proteins (8) that fall into two groups. On one hand, all standard eukaryotic translation initiation factors (eIFs) are required (9), except the actual cap-binding protein eIF4E, a protein that associates with the large adaptor protein eIF4G (and the RNA-helicase eIF4A) i...

The 3′ cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation

“…Therefore, BLucB is eIF4F, and thus, BTE dependent. Finally, observations that some plant and mammalian virus IRESes can stimulate translation of upstream AUGs (Jaag et al 2003;Herbreteau et al 2005;Nicholson et al 2006;Junemann et al 2007) may be explained by mechanisms similar to that shown in Figure 7.…”

Ribosomal tethering and clustering as mechanisms for translation initiation

“…Recently, Junemann et al (32) showed that translation can initiate at a cistron located upstream of picornavirus IRESs. These results would require the ribosomal subunits to initiate translation up to Ϸ900 nt upstream of the IRES.…”

Activation of hepatitis c virus translation by a liver-specific microRNA

“…Our concerns came from our finding that picornavirus IRES elements can stimulate the translation not only of the gene downstream of the IRES but also that of an upstream gene. 23 A picornavirus IRES in a dicistronic mRNA system can bind translation initiation factors and ribosomes and hand them over to the upstream translation start site. From the above study it was clear to us that we must take care when interpreting results obtained in the carbohydrate and lipid metabolism of the liver.…”