Internal Ribosomal Entry Site-Mediated Translation Initiation in Equine Rhinitis A Virus: Similarities to and Differences from That of Foot-and-Mouth Disease Virus

Abstract

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinasesLab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.Equine rhinitis A virus (ERAV), formerly known as Equine rhinovirus 1, is a member of the Picornaviridae family and has been recently reclassified as the only non-foot-and-mouth disease virus (non-FMDV) member of the genus Aphthovirus (26). This reclassification was based largely on nucleotide sequence determination of the ERAV genome (14, 32), although it is also consistent with many of the known physicochemical and biological properties of the virus (20). ERAV infection of horses results in an acute febrile respiratory disease that is accompanied by viremia and persistent virus shedding in the urine and feces (see reference 30 for a review). It is also pathogenic for a broad range of other animal species, including humans (24, 25). The recent finding that strains of ERAV which are noncytopathic in in vitro-cultured cells are responsible for outbreaks of febrile respiratory disease in horses suggests that the virus has been underdiagnosed and that its relative significance as an equine pathogen may have been underestimated (15). Further investigation into the epidemiology and pathogenesis of this virus is clearly required. In this paper we characterize the role of the ERAV 5' nontranslated region (5'-NTR) in translation initiation, an important pathogenic determinant in picornaviruses.Picornaviruses initiate translation in a cap-inde...

Comparative Genomics of Foot-and-Mouth Disease Virus

“…FMDV L pro is expressed as long (Lab) and short (Lb) isoforms that result from alternative usage of either the first or second start codon, respectively, with the sequence between the two AUGs predicted to form a stable hairpin structure crucial to IRES activity (39,66). L pro isoforms have indistinguishable activities and specificities and have been proposed to play a role in virulence that may involve the regulation of host interferon responses (89).…”

The Capsid Protein of Satellite Panicum Mosaic Virus Contributes to Systemic Invasion and Interacts with Its Helper Virus

“…Initiation of translation in eukaryotes is regulated by several factors, including mRNA length, the 5Ј-UTR, secondary structure, and the nucleotide context surrounding AUG start codons (7,11,13,17,32). Eukaryotic mRNAs generally adhere to the first AUG rule-in most cases, the AUG codon nearest the 5Ј end is the sole site of initiation of translation (13).…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…These elements are cis-acting RNA regulatory sequences endowed with the capacity to govern cap-independent translation initiation in mRNAs that are translated when cap-dependent translation is compromised. Subsequent studies demonstrated that IRES elements drive internal initiation of translation in the RNA of all members of the Picornaviridae family (Bakhshesh et al, 2008;Borman and Jackson, 1992;Brown et al, 1991;Hinton and Crabb, 2001;Hinton et al, 2000;Kaku et al, 2002;Kuhn et al, 1990;Nateri et al, 2000;Sweeney et al, 2012;Willcocks et al, 2011;Yu et al, 2011b). The genome of other RNA viruses also contains IRES elements, as illustrated by hepacivirus, pestivirus, dicistrovirus, retrovirus Tsukiyama-Kohara et al, 1992;Vallejos et al, 2010;Wilson et al, 2000), as well as some RNA viruses infecting plants and protozoa (reviewed in Martinez-Salas et al, 2012).…”