The 39 end of the foot-and-mouth disease virus genome establishes two distinct long-range RNA–RNA interactions with the 59 end region Paula Serrano, 1 Miguel Rodriguez Pulido, 2 Margarita Sa ´iz 1,2 and Encarnacion Martı ´nez-Salas 1 Correspondence Encarnacion Martı ´nez-Salas emartinez@cbm.uam.es 1 Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, 28049 Madrid, Spain 2 CISA-INIA, Valdeolmos, 28130 Madrid, Spain Received 22 March 2006 Accepted 16 June 2006 The untranslated regions (UTRs) of the foot-and-mouth disease virus (FMDV) genome contain multiple functional elements. In the 59 UTR, the internal ribosome entry site (IRES) element governs cap-independent translation initiation, whereas the S region is presumably involved in RNA replication. The 39 UTR, composed of two stem–loops and a poly(A) tract, is required for viral infectivity and stimulates IRES activity. Here, it was found that the 39 end established two distinct strand-specific, long-range RNA–RNA interactions, one with the S region and another with the IRES element. These interactions were not observed with the 39 UTR of a different picornavirus. Several results indicated that different 39 UTR motifs participated in IRES or S region interactions. Firstly, a high-order structure adopted by both the entire IRES and the 39 UTR was essential for RNA interaction. In contrast, the S region interacted with each of the stem–loops. Secondly, S–39 UTR interaction but not IRES–39 UTR interaction was dependent on a poly(A)-dependent conformation. However, no other complexes were observed in mixtures containing the three transcripts, suggesting that these regions did not interact simultaneously with the 39 UTR probe. Cellular proteins have been found to bind the S region and one of these also binds to the 39 UTR in a competitive manner. Our data suggest that 59–39-end bridging through both direct RNA–RNA contacts and RNA–protein interactions may play an essential role in the FMDV replication cycle. INTRODUCTION The foot-and-mouth disease virus (FMDV) genome consists of a positive-sense RNA of about 8500 nt encoding a polyprotein, which is processed into the mature viral products. In common with all picornaviruses, the 59 end of the viral RNA is linked covalently to the viral protein VPg and the 39 end is polyadenylated. 59 and 39 untranslated regions (UTRs) of approximately 1300 and 100 nt, respec- tively, flank the single open reading frame (Belsham & Martı´nez-Salas, 2004). The FMDV 59 UTR displays the most complex organization among picornavirus, comprising the S region, a poly(C) tract, several pseudoknots, the cis-acting replication element (cre) and the internal ribosome entry site (IRES). The S region, spanning about 360 nt at the 59 terminus of the viral RNA, is predicted to form a long hairpin structure (Escarmis et al., 1992; Witwer et al., 2001). An important role in replication is assumed for this region, although direct evidence for protein interactions or the molecular mechanisms involved is still lacking. In poliovirus (PV), the 59 end of the genome adopts a cloverleaf structure that has been shown to interact with the cellular and viral proteins poly(rC)-binding protein 2 (PCBP2) and 3CD proteinase, respectively (Gamarnik & Andino, 1997; Parsley et al., 1997). A role for these interactions has been proposed in regulating the switch from translation to replication (Gamarnik & Andino, 1998). A second role in RNA circularization has been also suggested, as PCBP2 binds to the poly(A)-binding protein (PABP), which is known to interact with the viral poly(A) tail (Herold & Andino, 2001). The FMDV IRES (about 450 nt), mediating cap-indepen- dent translation of the viral RNA, has been studied extensively (Martı´nez-Salas & Ferna ´ndez-Miragall, 2004). The essential domains, RNA secondary structure and interaction with cellular proteins have been described (Ferna ´ndez-Miragall & Martı´nez-Salas, 2003; Ferna ´ndez- Miragall et al., 2006; Lo ´pez de Quinto & Martı´nez-Salas, 1997, 2000; Pilipenko et al., 2000; Stassinopoulos & Belsham, 2001; Walter et al., 1999). Secondary structure analysis of the FMDV 39 UTR sequence predicts the presence of two stable stem–loops. This region is strictly required for replication and infectivity (Sa ´iz et al., 2001). In other picornaviruses, the 39 UTR is organized into two stem–loops that adopt a quasi-globular organization (Melchers et al., 2000) and constitute essential determinants of virus replication (Brown et al., 2005; Dobrikova et al., 2003; Melchers et al., 1997). 0008-2059 G 2006 SGM Printed in Great Britain 3013 Journal of General Virology (2006), 87, 3013–3022 DOI 10.1099/vir.0.82059-0