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Journal of Virology, October 2002, p. 9686-9694, Vol. 76, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.19.9686-9694.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of a cis-Acting Replication Element (cre) Adjacent to the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus

Peter W. Mason,* Svetlana V. Bezborodova, and Tina M. Henry

Plum Island Animal Disease Center, U.S. Department of Agriculture, Greenport, New York 11944

Received 19 March 2002/ Accepted 25 June 2002

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 3Dpol 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.

* Corresponding author. Mailing address: PIADC, USDA, ARS, NAA, P.O. Box 848, Greenport, NY 11944-0848. Phone: (631) 323-3177. Fax: (631) 323-2507. E-mail: pwmason{at}piadc.ars.usda.gov.

Journal of Virology, October 2002, p. 9686-9694, Vol. 76, No. 19
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.19.9686-9694.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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