The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus

Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus , a new genus in the Picornaviridae family. ERBV is distantly related to me mbers of the Cardiovirus and Aphthovirus genera which utilize a type II int ernal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem-loop structures (H-L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro t ranscription and translation in rabbit reticulocyte lysates. In both system s, a region encompassed by nucleotides (nt) 189-920 downstream of the poly( C) tract was required for maximal translation. This sequence includes stem- loops H-L as well as four additional upstream stem-loops. Nt 904 correspond s to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869-880). Site-directed mutagenes is demonstrated that AUG2 is the major initiation codon despite the appropr iate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In d irect IRES competition experiments, the ERBV IRES was able to compete stron gly for translation factors with the IRES of Encephalomyocarditis virus (EM CV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.