Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: Similarities to and differences from that of foot-and-mouthdisease virus

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovir us, a genus otherwise comprised of the different serotypes of Foot-and-mout h disease virus (FMDV). FMDV initiates translation via a type II internal r ibosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5 ' nontransl ated region also possesses the core structures of a type II IRES. The funct ional activity of this region was characterized by transfection of bicistro nic plasmids into BHK-21 cells. In this system the core type Il 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 secon d 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 al so has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Usin g the bicistronic expression system, we detected initiation from both AUG p airs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is t he major initiation site, although AUG1 can be accessed, albeit inefficient ly, in the absence of AUG2. Further mutational analysis indicated that codo ns 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 reveal s important differences in IRES function between aphthoviruses.