Fig 1.
Structure of the FMDV 3Dpol-3B1 complex.
(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.
Fig 2.
The FMDV 3Dpol-3B1 complex forms long fibres in the crystal packing.
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.
Fig 3.
Conservation of the 3B contact surface at the base of the palm of 3Dpol among picornaviruses.
(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).
Fig 4.
3B1 binds the bottom of the 3Dpol palm also in solution.
(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.
Fig 5.
Polymerase recruitment to E. coli membranes containing the 3AB1 precursor.
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.
Fig 6.
The binding of 3AB1 at the bottom of the palm subdomain of 3Dpol increases localization of polymerase to intracellular membranes.
(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).