Over the last few years, an essential RNA structure known as the cis-acting replicative element (cre) has been identified within the protein-coding region of several picornaviruses. The cre, a stem-loop structure containing a conserved AAACA motif, functions as a template for addition of U residues to the protein primer 3B. By surveying the genomes of representatives of several serotypes of foot-and-mouth disease virus (FMDV), we discovered a putative cre in the 5 untranslated region of the genome (contiguous with the internal ribosome entry site [IRES]). To confirm the role of this putative cre in replication, we tested the importance of the AAACA motif and base pairing in the stem in FMDV genome replication. To this end, cre mutations were cloned into an FMDV replicon and into synthetic viral genomes. Analyses of the properties of these replicons and genomes revealed the following. (i) Mutations in the AAACA motif severely reduced replication, and all viruses recovered from genomes containing mutated AAACA sequences had reverted to the wild-type sequence.(ii) Mutations in the stem region showed that the ability to form this base-paired structure was important for replication. Although the cre was contiguous with the IRES, the mutations we created did not significantly reduce IRES-mediated translation in vivo. Finally, the position of the cre at the 5 end of the genome was shown not to be critical for replication, since functional replicons and viruses lacking the 5 cre could be obtained if a wild-type cre was added to the genome following the 3D pol coding region. Taken together, these results support the importance of the cre in replication and demonstrate that the activity of this essential element does not require localization within the polyprotein-encoding region of the genome.Foot-and-mouth disease, one of the most important known pathogens of livestock, is caused by a small RNA virus of the family Picornaviridae. Foot-and-mouth disease virus (FMDV) is the prototype member of the Aphthovirus genus of this family, and although aspects of FMDV replication resemble those of many other picornaviruses, there are notable differences between FMDV and other viruses that include FMDV's broad host range and several unique genetic features.The FMDV genome is over 8,300 bases in length and is covalently bound at its 5Ј terminus to a 23-to 24-amino-acid genome-linked protein, 3B. The genome encodes three copies of 3B, all of which are apparently utilized (8,15). No natural isolates of FMDV with fewer than three 3B coding regions have been identified, suggesting that there is a strong selective pressure maintaining this redundancy, since homologous recombination within the FMDV genome (22) should readily eliminate redundant copies of 3B. Despite the observed retention of three 3Bs in all natural FMDVs, viruses lacking one of the 3Bs or with two nonfunctional 3Bs have been derived by genetic engineering (7). Recently, we have observed that viruses lacking two of the three 3Bs can be generated by similar methodology, an...