Downloaded from www.microbiologyresearch.org by IP: 54.157.13.203 On: Sat, 13 Feb 2016 02:07:50 Journal of General Virology (2001), 82, 2257–2269. Printed in Great Britain ................................................................................................................................................................................................................................................................................... The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus Tracey M. Hinton 1 and Brendan S. Crabb 1, 2 1 Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology, The University of Melbourne, Australia 2 The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, VIC 3050, Australia Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal 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 transcription and translation in rabbit reticulocyte lysates. In both systems, 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 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869–880). Site- directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). 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. Introduction Equine rhinitis B virus (ERBV), formerly known as equine rhinovirus type 2, is the only member of the newly recognized Erbovirus genus in the family Picornaviridae (King et al., 2000 ; Pringle, 1999). The closest known relatives are the members of the Cardiovirus and Aphthovirus genera. Although the nucleotide sequence of the ERBV genome is known (Wutz et al., 1996) the virus has not been extensively studied. ERBV infection of horses can result in an acute febrile respiratory tract disease characterized by coughing and lymphadenitis (Mumford & Thomson, 1978) and horses can carry ERBV for Author for correspondence : Brendan Crabb (at The Walter and Eliza Hall Institute of Medical Research). Fax 61 3 9347 0852. e-mail crabbwehi.edu.au long periods of time (Burrows, 1978 ; Thorsen, 1991). Recent studies have suggested that ERBV may be an important cause of respiratory disease in horse populations (Carman et al., 1997 ; McCollum & Timoney, 1992). Further investigation into the epidemiology and pathogenesis of ERBV infections is clearly required. The positive-sense RNA genome of picornaviruses is not capped but instead contains a long 5-nontranslated region (NTR) which allows cap-independent, internal translation initiation. This process is facilitated by an internal ribosomal entry site (IRES) which forms an extensive and highly stable secondary structure that interacts with both canonical and non- canonical translation factors (Andino et al., 1999; Belsham & Sonenberg, 1996). Some picornaviral IRESs are recognized as virulence determinants, perhaps through the involvement of these non-canonical factors. The predicted IRES structure and 0001-7653  2001 SGM CCFH