Exploring IRES Region Accessibility by Interference of Foot-and-Mouth Disease Virus Infectivity

Abstract

Translation initiation of picornavirus RNA is driven by an internal ribosome entry site (IRES) element located upstream of the initiator codon. RNA structure organization as well as RNA-protein interaction plays a fundamental role in internal initiation. IRES activity has been mainly analyzed in the context of reporter genes, lacking regions of the viral genome potentially affecting translation efficiency. With the aim to understand the vulnerability of the IRES and translation start region to small molecules in the context of the viral genome, we designed a set of customized RNase-resistant 2′O-methyl antisense oligoribonucleotides (2′OMe AONs) based on RNA structure data. These AONs were then used to monitor their capacity to interfere viral RNA translation, and thus, to inhibit virus yield. Foot-and-mouth disease virus (FMDV) RNA translation can be initiated at two in-frame AUG codons. We show here that a 2′OMe AON complementary to AUG2 inhibited viral multiplication more efficiently than the one that targeted AUG1. Furthermore, the response of the viral RNA to AONs targeting the IRES region denoted important differences between tissue culture cells and cell-free systems, reinforcing the need to analyze viral RNA response in living cells. Importantly, we have identified four specific motifs within the IRES element that are targets for viral inhibitors both in tissue culture cells and in cell-free systems. The identified targets define accessible regions to small molecules, which disturb either the RNA structural organization or the RNA-protein interactions needed to initiate translation in FMDV RNA.

The effects of nucleotide usage in key nucleotide positions +4 and -3 flanking start codon on translation levels mediated by IRES of hepatitis C virus

“…The usage of IRES in bicistronic expression plasmids enables the expression of two genes controlled by one promoter in the same cells, which are important tools in today's cell biology (Fajardo et al, 2012;Ma et al, 2016). In order to obtain ratio-controlled or improved IRES-mediated protein expression, many methods were developed, for example, removal of unnecessary AUG start codon, using multiple IRES elements (Bouabe et al, 2008;Koh et al, 2013).…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…A peculiarity of aphthovirus RNA is that the second AUG triplet (AUG2) is preferentially used to initiate translation (Cao et al, 1995;Lopez de Quinto and Martinez-Salas, 1999). Further information in favor of AUG2 as the functional initiator codon was derived from interference studies using modified oligonucleotides targeting the initiator codons in RNA-transfected cells (Fajardo et al, 2012). A conserved polypyrimidine tract is located 15-25 nts upstream of AUG1, within a single-stranded region that accumulates mutations in viral RNAs isolated from persistently infected cells (MartinezSalas et al, 1993).…”

Biological function of Foot-and-mouth disease virus non-structural proteins and non-coding elements

“…There are evidences to support that these domains are involved in translational control. Some highly conserved sequences exist in these domains, like Domain II containing a polypyrimidine tract (UCUUU) that provides a polypyrimidine tract-binding protein (PTB) binding site [ 26 ]. Domain III includes two conserved essential motifs, GNRA and RAAA (N is any nucleotide and R stands for purine) at the apical region, and a conserved C-rich loop at the middle region [ 27 , 28 ].…”