## 2 Results and Discussion
### Molecular Modeling Studies  
The x-ray crystallographic work was completed with some additional model building because, although a second VPg-binding site on picornavirus 3 Dpol enzymes has been demonstrated [7], one would expect each of the three distinct VPgs of FMDV (i.e., 3B1, 3B2 and 3B3) to bind into the active site of the enzyme in such a way as to make uridyylation possible. This reaction takes place by in-line attack of the Tyr3 hydroxyl of VPg onto the \(\alpha\)-phosphorus atom of UTP with the aid of the conserved triad of catalytic aspartates and the bound Mg2+ ions in 3 Dpol [34]. The 3B-uridyylation site (\"bus\"), located in a hairpin within the 5'-untranslated region of the FMDV genome [35], was predicted by the Mfold [36] and RNAcomoser [37] servers to consist of a base-paired stem and a loop region of sequence **CAAA**CACGAUCUA in which the underlined AAACA motif is strictly conserved. The two contiguous adenusers shown in bold (**A\({}_{2}\)** and **A\({}_{3}\)**) can be considered the putative templates for the addition to VPg peptides of the first and second UMP moieties (Figure 4A), in accordance with similar findings reported for poliovirus [7]. Nonetheless, the proposed \"slide-back\" mechanism [38], which is similar to that originally proposed for the initiation of protein-primed DNA synthesis by the DNA polymerase of phage \(\phi\)29 [6], may imply that only A\({}_{2}\) fulfils this role whereas the presence of both A\({}_{2}\) and A\({}_{3}\) ensures that the only dinucleotide produced will be VPgUpU. From a structural standpoint, VPg1-3 are predicted to be highly disordered peptides but, in order to place the Tyr3 hydroxyl close to the \(\alpha\)-phosphorus atom of the UTP molecule and the Mg2+-bound Asp240, Asp245 and Asp338, the only entry site possible is between motif A and motif F (Figure 4A). Once Tyr3 becomes uridyylylated, a second UTP nucleotide is recruited and a second uridyylylation takes place. Since further elongation of VPg1 pUpU by 3 Dpol using bus as the template is aborted [38], there must be a conformational change leading to the release of bus. Thereafter, the resulting 3 Dpol:VPg1 pUpU complex readily accepts the 3'-end of the poly(A) tail to be used as the primer for minus strand RNA synthesis. A model of this complex in the presence of the incoming UTP and Mg2+ ions is shown in Figure 4B. The intrinsically disordered character of VPg1 is in consonance with the  
Figure 3: **(A)** The structures of FMDV 3 Dpol (green) crystallized in presence of the fluororidylylated VPg that was disordered in the electron density. The structure of the 3 DpolVPg1 uridyylated previously has been superimposed to illustrate the conformational diferences (3 Dpol is in blue and the UMP-VPg1 molecule in white; PDB id 2F8E) The conformational rearrangement of the \(\beta\)9-\(\alpha\)11 loop has been highlighted with a dotted line. **(B)** Close-up of the polymerase catalytic cavity in the 3 Dpol FUMP-VPg1 crystals. The active site motifs A, B (where the \(\beta\)9-\(\alpha\)11 loop is located) and C are shown as a green cartoon. FUMP-VPg1 (white C atoms) has been modeled within the cavity with the side chain of Tyr3 covalently attached to FUMP depicted as sticks (the fluorine atom is colored in light cyan). A dinucleotide ApA template (cyan sticks) and an incoming UTP molecule (sticks with C atoms in white) have also been modeled to show the steric clashes that would take place between the template nucleotides and the \(\beta\)9-\(\alpha\)11 loop. fact that this peptide chain has to slide down the polymerase channel during RNA strand elongation in the 5\({}^{\prime}\)\(\rightarrow\) 3\({}^{\prime}\) direction (Figure 4C). Based on our models, we believe that the FUMP moiety is closer to Cys300 in the \(\beta\)9-\(\alpha\)11 loop when the active site is occupied by FUMP-VPg in the second uridylylation step than when it is occupied, in the first uridylylation step, by VPg and UTP in the presence of the template RNA hairpin (cre or bus).