###### Abstract  
Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the _Picornaviridae_ family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed \(\mathrm{uridylylation}\), is catalyzed by the viral RNA-dependent RNA polymerase named \(\mathrm{3D^{pol}}\). 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg \(\mathrm{uridylylation}\). Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg \(\mathrm{uridylylation}\) by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with \(\mathrm{3D^{pol}}\). Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of \(\mathrm{3D^{pol}}\) in the presence of VPg1-FUMP showed an 8 A movement of the \(\mathrm{\SIUnitSymbolFace{9}}\)-\(\alpha\)11 loop of the polymerase towards the active site cavity relative to the complex of \(\mathrm{3D^{pol}}\) with VPg1-UMP. The conformational rearrangement of this loop preceding the \(\mathrm{3D^{pol}}\) B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the \(\mathrm{uridyylation}\) of their VPg proteins to initiate the viral RNA synthesis. foot-and-mouth disease virus; RNA-dependent RNA polymerase; 5-fluorouracilouracil; uridyylation inhibition; VPg 2019 24 2360 10.3390/molecules24132360  
1  
Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the _Picornaviridae_ family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed \(\mathrm{uridylylation}\), is catalyzed by the viral RNA-dependent RNA polymerase named \(\mathrm{3D^{pol}}\). 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg \(\mathrm{uridylylation}\). Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg \(\mathrm{uridylylation}\) by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with \(\mathrm{3D^{pol}}\). Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of \(\mathrm{3D^{pol}}\) in the presence of VPg1-FUMP showed an 8 A movement of the \(\mathrm{\SIUnitSymbolFace{9}}\)-\(\alpha\)11 loop of the polymerase towards the active site cavity relative to the complex of \(\mathrm{3D^{pol}}\) with VPg1-UMP. The conformational rearrangement of this loop preceding the \(\mathrm{3D^{pol}}\) B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the \(\mathrm{uridyylation}\) of their VPg proteins to initiate the viral RNA synthesis. foot-and-mouth disease virus; RNA-dependent RNA polymerase; 5-fluorouracilacil; uridyylation inhibition; VPg 2019 24 2360 10.3390/molecules24132360