Differential factor requirement to assemble translation initiation complexes at the alternative start codons of foot-and-mouth disease virus RNA

Abstract

The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that eIF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem-loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic" toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap-dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.

FIGURE 1.

Dependence of the 48S complex formation at (A) AUG1 and (B) AUG2 on the initiation factors eIF1 and eIF1A. Positions of toeprint signals (denoted on the left of the gels) were determined using dideoxynucleotide sequences generated with the same primers (not shown in this figure; see Fig. 4A,B).

FIGURE 2.

(A) Schematic representation of the FMDV constructs used for translation assays in transfected cells to analyze simultaneously initiation at AUG1 and AUG2. (Empty rectangle) The FMDV IRES; (black rectangle) the luciferase gene fused to AUG2. Initiation at triplets AUG1 and AUG2, underlined on the sequence, gives rise to the synthesis of 20- or 13-kDa polypeptides, respectively, in constructs with a deleted version of the luciferase-coding region. (B) The sequence encompassing the first 84 nt of the FMDV polyprotein including the two functional initiation codons, AUG1 and AUG2. The nucleotide sequence of in-frame mutants (C) FMDV–LH and (D) FMDV-SH is also shown. (B–D) Secondary structures of the spacer region for the wild-type FMDV RNA and its mutants FMDV–LH and FMDV–SH, respectively. A transcript encompassing the entire IRES sequence extended to the second AUG was incubated in vitro with DMS or RNase A or T2, and then analyzed by primer extension using a 5′-end-labeled primer. (Arrows) Attacks were used in conjunction with (gray solid rectangles) the RNA phylogenetic conservation to represent the secondary RNA structure of (B) wild-type sequence, and (C) FMDV–LH. The secondary structure of FMDV–SH was built using computer modeling. The functional FMDV start codons AUG1 and AUG2 are boxed. Nucleotide numbering is used according to previous FMDV IRES literature (Fernandez-Miragall and Martinez-Salas 2003). The genetic variability in the region between the AUGs of 31 FMDV sequences deposited in databanks, belonging to serotypes C (12), O (nine), A (seven), and SAT (three), were used to derive the phylogenetic conservation of RNA structure. Compensatory nucleotide substitutions, consistent with base-pair formation, affected 16 bases (gray boxes in B); natural variation tolerated changes that converted G:C pairs to G.U or to A:U (in five positions), and U:A pairs to C:G or to U.G (in two positions). A strong tendency to covariate G492 and C500 to form a G.U pair was noticed. Independent nucleotide substitutions were also noticed in 15 positions, all located in loops in the secondary structure depicted in B.

FIGURE 3.

Effect of the secondary structure of the spacer region on the translation initiation at AUG1 and AUG2. (A) The gel showing toeprint assays in the reconstitution system from purified components. (Lane RT) No initiation factors added; (FMDV) control wild-type RNA. (B) Western blot assay of the luciferase truncated proteins schematically shown in Figure 2 synthesized in BHK-21 cells, 20 h post-transfection with the indicated constructs. (Three upper arrows) Point to the peptides (p) initiated at the AUG1 in FMDV wt, FMDV–LH, and FMDV–SH, respectively. (Solid arrow) The protein initiated at AUG2.

FIGURE 4.

Effect of PTB and ITAF45 on the reconstitution of 48S complexes at (A) AUG1 and (B) AUG2. Positions of the toeprint bands were determined using dideoxynucleotide sequences generated with the same respective primers (see Materials and Methods). These sequences are shown on the left and right of the gels, respectively.

FIGURE 5.

Kinetic toeprint analysis in RRL of the 48S complex formation on AUG1 and AUG2 of FMDV-Luc and β-globin mRNA. (A) Gel showing toeprint bands corresponding to aliquots withdrawn from the same reconstitution mixture at different time points for the wild-type FMDV-Luc and the FMDV-Luc with the mutated AUG1 (AUG1→AUA). (B) Similar to A but for the FMDV-Luc constructs with either AUG1 or AUG2 mutated. (C) Similar to A and B but for β-globin mRNA. (D) Time course of formation of the 48S complex at AUG 2 of FMDV-Luc mRNAs (filled diamonds) with or (open circles) without AUG1. The plot was generated on the basis of the data presented in A. (E) Comparison of the time courses of the 48S complex accumulation at AUG1 and AUG2 of FMDV-Luc mRNA with that for the initiation codon of β-globin mRNA. The plot was created on the basis of the data presented in B and C. The points in the plots represent the average of several independent experiments. The standard deviation was within 10%.