## 2 Results and Discussion
### Crystal Structures of the UMP-VPg1 and FUMP-VPg1 Bound to the 3D Polymerase  
Previous studies with FMDV 3 DPol and the pyrimidine analogue 5-fluorouracilite triphosphate (FUTP) revealed that this nucleotide analogue acts in two different ways during FMDV replication: (i) as a mutagenic agent when it is incorporated into the nascent RNA by increasing misincorporation of GMP in place of UMP, and (ii) as a strong inhibitor of VPg uridyyllation [18]. Despite the fact that FUMP-VPg1 was observed, this fluororuidylylated form of VPg prevented the incorporation of the second UMP molecule [18]. To gain insight into the role of FUMP-VPg1 in inhibition of further uridyylation, we co-crystallized 3 DPol in the presence of the modified 15-mer peptides FUMP-VPg1 and UMP-VPg1. Tetragonal crystals of the 3 DPol complexes were obtained in both cases. X-ray diffraction data from both crystals were collected at 100 K using synchrotron radiation. The structures were solved by direct molecular replacement, using the tetragonal structure of the unbound polymerase (PDB id. 1U09) as a model. Direct rigid body refinement was sufficient to position the 3 DPol model in the new unit cells (Table 1). Analyses of the electron density maps showed that the VPg1 peptides could not be traced in any of the complexes, most likely due to disorder. However, best-fit structural superpositioning showed that 3 DPol crystallized in the presence of FUMP-VPg1 exhibited a large conformational change in the \(\beta\)9-\(\alpha\)11 loop, located at the N-terminal motif B. On the contrary, the structure of 3 DPol in complex with UMP-VPg1 was practically identical to that of the apo form (Figure 3A). Motif B is formed by a \"loop-\(\alpha\)-helix\" structure located at the base of the template channel and is involved in ribonucleoside triphosphate (rNTP) recognition, template binding and translocation of the nascent dsRNA [29; 30; 31; 32; 33]. In the apo form of 3 Dpol, the \(\beta\)9-\(\alpha\)11 loop is packed against the fingers domain, forming part of the template channel and participating in template binding. But in the presence of FUMP-VPg1, loop residues from Ser298 to Thr303 in this motif B appear displaced by 7.8 A towards the active site, thereby closing the template channel and preventing the correct positioning of the nucleotide template in the active site pocket (Figure 3B). Different studies have shown that the motif B loop is one of the most flexible elements in the active sites of RdRps (reviewed in [31]). Large movements of this loop have been observed ranging from an open conformation, in which the loop is packed against the fingers domain, leaving the active site cavity accessible for template entry, to a closed conformation where the loop is found inside the catalytic pocket, resulting in a conformation that sterically clashes with the template nucleotide (Figure 3B). The structural data shown in this work, together with previous evidence of VPg fluorouridylylation [18], prompt us to speculate that the inhibitory effect of FU on VPg uridyyllation might be explained by the stabilization of the \(\beta\)9-\(\alpha\)11 loop in the closed conformation, thus preventing the correct positioning of the template nucleotide in the active site cavity of 3 Dpol. Because FUMP-VPg1 is detected experimentally [18] this inhibition would occur following attachment of the first FU unit to VPg.