Identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus

Abstract

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.

FIG. 1.

Two-dimensional representation of a portion of the 5′ UTR of FMDV serotype A12. The structures of domains 2 to 5 are based on the model of Pilipenko et al. (30). The cre (cis-acting replicative element) and its conserved AAACA sequence (boxed) are described in the text.

FIG. 2.

Schematic diagram of replicon and infectious FMDV genomes encoded by plasmids pA12-βgal and pCRM4H, respectively. Open boxes indicate protein-coding regions, and lines indicate RNA structures. The hatched box shows the position of the E. coli β-galactosidase-coding region in the replicon plasmid pA12-βgal. βgal, β-galactosidase; S, short fragment of the genome; poly(C), polycytosine tract; PKs, pseudoknots; cre, cis-acting replicative element; IRES, internal ribosome entry site. Hairpin loop structures below the genome contain the wild-type and mutant versions of the cre, with altered nucleotides circled.

FIG. 3.

Time course of β-galactosidase (βgal) production by cells transfected with β-galactosidase-expressing replicons from pA12-βgal (wild-type cre; ♦), pA12-C2-βgal (wild-type cre; ▴), and pA12-βgal in the presence of 5 mM guanidine HCl (▪).

FIG. 4.

Replication and in vivo translation activity of replicon RNAs electroporated into BHK cells and truncated replicon-derived cDNAs transfected into BHK cells, respectively. The structures of the wild-type (WT) and mutant replicons in the pA12-βgal-derived RNAs shown along the bottom are illustrated in Fig. 2. Open bars show “in vivo” β-galactosidase (βgal) translation activity from the average of duplicate transfections containing equimolar amounts of plasmid DNA encoding replication-incompetent (truncated) genomes and the bacteriophage T7 polymerase (harvested 22 h posttransfection; see the text). Hatched bars show β-galactosidase activity recovered from a single representative experiment in which cells were transfected by electroporation with 10 μg of LiCl-purified, in vitro-transcribed RNA (harvested 12 h postelectroporation; see the text).

FIG. 5.

Predicted structures of the cres of five different plaque isolates recovered from cells transfected with a viral genome encoding cre mutant 2a. Substitutions relative to the transfected RNA are shown in lowercase letters and identified with arrowheads (wild-type and mutant 2a structures are shown at the left for comparison).

FIG. 6.

Photographs of plaques of the indicated viruses on BHK cell monolayers. Plaques from the wild-type virus (derived from pCRM4H) were produced by the second passage from the electroporated stock. Plaques from the plaque-picked isolates were produced by the BHK-amplified plaques picked from the first passage postelectroporation (this is the same passage used for the sequence analyses; see text for details).

FIG. 7.

Schematic diagram of replicon and infectious FMDV genomes showing the positions of the cre deletion (mutant 7) and the insertion of the cre at the 3′ end of the genome. For this portion of the figure, lowercase letters indicate specific mutations to the genome to facilitate construction, and underlined lowercase letters indicate bases added to the genome to facilitate construction. Other abbreviations and nomenclature are explained in the legend to Fig. 2.

FIG. 8.

Photographs of plaques produced by the indicated viruses on BHK cell monolayers. Plaques for all three viruses were produced by virus recovered from the second passage of lysates of cells transfected with synthetic genome-length RNAs by using Lipofectin (see the text).