BUSCADOR RECOLECTA

(A) Scheme representing the fluorescence microscopy experiments comparing the distribution of 3Dpol in the presence of 3AB1, wild type and mutant. The left panel shows 3Dpol interacting with the 3AB1 precursor bound to the membrane. The right panel mimics the scenario in the presence of the 3AB1 mutant 3AB1(P6S/R9A/R16A/L19S), unable to bind 3Dpol. (B) Fluorescence images of HeLa cells showing the different distribution of 3Dpol bound to 3AB1 wild type or the 3AB1(P6A/R9A/R16A/L19S) mutant. Upper panels show the control cells transfected with the polymerase only (green), which appears distributed throughout the cell. Middle panels show cells transfected with 3AB1 wild type (red) and 3Dpol (green), where 3Dpol mostly co-localizes with the 3AB1 protein in a continuous compartment in the cytoplasm. The lower panels show cells transfected with the 3Dpol and the 3AB1 mutant (P6A/R9A/R16A/L19S), where 3Dpol recovers its localization throughout the cell. The images shown are representative of the total number of images obtained. (C) Fluorescence Intensity plots comparing the relative distribution of 3Dpol (green) in presence of wild type and mutant 3AB1 proteins (red), left and right panels, respectively. The nucleus is labelled in blue (DAPI)., Peer reviewed

E.coli membranes containing, wild-type 3AB1 or 3AB1(P6S/R9A), 3AB1(R16A/L19S) or 3AB1(P6S/R9A/R16A/L19S) mutants, disrupting the 3B1-3Dpol binding site were used in the analysis. (A) Western blot experiment that shows the 3Dpol protein recruited to the membrane. (B) Bars diagram indicating the percentage of 3Dpol bound by the membranes. To calculate the fraction of 3Dpol bound to 3AB, the volume of the 3Dpol band from a control membrane was subtracted from each of the other 3Dpol bands, and these were then normalized to the 3Dpol band pulled down by wild-type 3AB. Assays were performed in triplicate, and the mean value and the standard deviation are reported., Peer reviewed

(A) Enzyme-Linked Immunosorbent Assay (ELISA) to compare the binding affinities of 3Dpol I and 3Dpol II binding sites of 3B1, determined in the X-ray structure. A multi-well plate was coated with 10 μg/ml of 3Dpol and its interaction to GST-3B1 WT was measured at increasing concentrations (from 0.5μg/ml to 20 μg/ml) and compared with mutants: GST-3B1(P6S/R9A), GST-3B1(R16A/L19S), GST-3B1(P6S/R9A/R16A/L19S) and GST. (B) ELISA assay to compare the affinities of 3B1, 3B2 and 3B3 for 3Dpol. Data were obtained from three independent experiments and standard deviations are reported., Peer reviewed

(A) Structure-based sequence alignment of the picornavirus 3Dpol residues located the base of the palm that would participate in interactions with 3B, (B) Structural superimposition of the two quasi-equivalent 3B1 binding sites in FMDV 3Dpol. The polymerase residues and the bound 3B1 regions are shown in sticks, coloured as in Fig 1, but with molecule I shown in semi-transparent. (C) The 3B binding site in EV71 3Dpol, as seen in the X-ray structure of the EV71 3Dpol -3B complex [11] (PDB:4IKA). (D-G) Structural comparisons of the putative 3B binding region in 3Dpol of other representative picornaviruses whose structure is known: the enteroviruses PV [5] (PDB: 1RA7; light blue) (D) and HRV1B [7] (PDB: 1XR6; salmon) (E), the cardiovirus EMCV [14] (PDB: 4NYZ; cyan) (F), and the kobusvirus porcine aichi virus [13](PDB: 6R1I; green)., Peer reviewed

The two 3Dpol molecules (green and yellow cartoons) linked by 3B1 (grey ribbon) also interact with each other in the crystal packing through contacts between the finger subdomains, forming long fibres along the a-b diagonal. (B) Close up of the interactions involving the direct 3Dpol-3Dpol contacts that facilitate fibre formation., Peer reviewed

(A) Ribbon representation of the two 3Dpol molecules present in the asymmetric unit of the P212121 crystals (yellow and green ribbons for the 3Dpol I and 3Dpol II molecules, respectively). The 3B1 molecule bound to the bottom of the palm of the two 3Dpol molecules is shown as sticks in atom-type colour (carbons white). The ordered triphosphate moieties of the UTP molecules bound at the nucleotide-binding site of the polymerases are also shown in atom-type sticks (phosphates in orange). (B) Omit map around the 3B1 molecule, displayed at a contour of 1,0 σ (light blue mesh). 3B1 is shown in sticks as in A with the amino acids explicitly labelled. (C) Details of the intermolecular contacts stablished between 3Dpol I and the 3B1 N-terminus (Interface I). (D) Intermolecular interactions between 3Dpol II and the 3B1 C-terminus (Interface II). The interacting residues are shown in sticks representation, coloured as in A. Hydrogen bonds and salt bridges are shown as dashed lines in black., Peer reviewed

† Rwork = ∑hkl ||Fobs(hkl)|—|Fcalc(hkl)|| / ∑hkl |Fobs(hkl)|, where Fobs and Fcalc are the structure factors, deduced from measured intensities and calculated from the model, respectively. ‡ Rfree = as for Rwork but for 5% of the total reflections chosen at random and omitted from refinement., Peer reviewed

Summary of FMDV 3Dpol-3B complexes prepared and the number of crystals analysed that allowed obtaining X-ray diffraction data of sufficient quality to solve the structures., Peer reviewed

Picornavirus genome replication takes place in specialized intracellular membrane compartments that concentrate viral RNA and proteins as well as a number of host factors that also participate in the process. The core enzyme in the replication machinery is the viral RNA-dependent RNA polymerase (RdRP) 3Dpol. Replication requires the primer protein 3B (or VPg) attached to two uridine molecules. 3B uridylylation is also catalysed by 3Dpol. Another critical interaction in picornavirus replication is that between 3Dpol and the precursor 3AB, a membrane-binding protein responsible for the localization of 3Dpol to the membranous compartments at which replication occurs. Unlike other picornaviruses, the animal pathogen foot-and-mouth disease virus (FMDV), encodes three non-identical copies of the 3B (3B1, 3B2, and 3B3) that could be specialized in different functions within the replication complex. Here, we have used a combination of biophysics, molecular and structural biology approaches to characterize the functional binding of FMDV 3B1 to the base of the palm of 3Dpol. The 1.7 Å resolution crystal structure of the FMDV 3Dpol -3B1 complex shows that 3B1 simultaneously links two 3Dpol molecules by binding at the bottom of their palm subdomains in an almost symmetric way. The two 3B1 contact surfaces involve a combination of hydrophobic and basic residues at the N- (G5-P6, R9; Region I) and C-terminus (R16, L19-P20; Region II) of this small protein. Enzyme-Linked Immunosorbent Assays (ELISA) show that the two 3B1 binding sites play a role in 3Dpol binding, with region II presenting the highest affinity. ELISA assays show that 3Dpol has higher binding affinity for 3B1 than for 3B2 or 3B3. Membrane-based pull-down assays show that 3B1 region II, and to a lesser extent also region I play essential roles in mediating the interaction of 3AB with the polymerase and its recruitment to intracellular membranes., Peer reviewed

The strictly conserved residues are in red blocks and similar residues in red characters. The FMDV 3B1 residues interacting with FMDV 3Dpol are marked by green asterisks. Residues of EV71 3B previously shown to contact the bottom of the palm of EV71 3Dpol in the X-ray structure of the complex [11](PDB:4IKA) are highlighted in yellow boxes., Peer reviewed