Exploring IRES region accessibility by interference of foot-and-mouth disease virus infectivity.

Teodoro Fajardo,Encarnacion Martinez-Salas,Francisco Sobrino,Maria Flora Rosas

doi:10.1371/journal.pone.0041382

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.

Highlights

Foot-and-mouth disease virus (FMDV) is the prototype of the aphthovirus genus of the Picornaviridae family
FMDV RNA translation initiation is peculiar in that protein synthesis can be initiated at two start codons, AUG1 and AUG2 (Fig. 1A)
We have made use of 29OMe AONs complementary to the FMDV internal ribosome entry site (IRES)-AUG translation start region to explore the capacity of these molecules to interfere viral RNA translation, and viral multiplication

Summary

Introduction

Foot-and-mouth disease virus (FMDV) is the prototype of the aphthovirus genus of the Picornaviridae family. The viral genome consists of a positive sense, single-stranded RNA of about 8500 nts, encoding a single polyprotein, flanked by a heavily structured untranslated region (UTR) at the 59end (Fig. 1A). Upon entry into the cell, the viral open reading frame is translated into a polyprotein that is proteolytically processed by virus-encoded proteases [3]. The 59UTR comprises a long hairpin (termed S), a poly(C) tract of variable length, a variable region folding as two to four pseudoknots, the cisreplication element (cre), and the internal ribosome entry site (IRES) element that mediates cap-independent translation of the viral RNA [5,6,7]. The 39UTR consists of a region of about 90 nt and a poly(A) tail, which stimulate IRES activity and participates in viral RNA replication [8,9]. Both AUGs are used as start codons for viral protein synthesis, the second one is preferentially utilized in a variety of assays [11,12]

Methods

Results

Conclusion