Table 1.
Cytolytic passaging of the SAT1 and SAT2 viruses in BHK-21 cells: the plaque morphologies and their titres in PFU/ml on BHK-21, CHO-K1, CHO-677, CHO-745 and CHO-Lec2 cells are indicated.
Table 2.
Summary of the amino acid substitutions causing a change in the charge in the outer capsid proteins of serially passaged SAT1 and SAT2 viruses.
Fig 1.
A RIVEM representation [40] of the SAT1 and SAT2 pentamers.
(A) The SAT1 and SAT2 pentamers are based on the protein data bank co-ordinates 2WZR and 5ACA, respectively. Amino acid substitutions observed during the adaptation of SAT1 viruses in BHK-21 cells are indicated in yellow. The surface-exposed, positively charged mutations that occurred more than once in different SAT1 viruses, are highlighted in red. The five copies of VP1 show the positively charged residues cluster at the 5-fold axis. (B) Positively charged mutations are color-coded based on the frequency of occurrence in different viruses within the SAT1 serotype from orange (n > 1) to red (n > 5). (C) The SAT2 pentamer is modelled using the SAT1 co-ordinates as a template and the surface-exposed, positively charged mutations are shown in red. In SAT2, a Lys residue appeared twice in VP1 position 83 in two different viruses; however, in the current model VP1 83 is not surface exposed. Nonetheless, VP1 85R (seen in SAT2/KNP/2/89) is surface-exposed.
Fig 2.
GRID [41] was used to find the energetically favorable binding site for HSPG on the SAT1 modelled mutant capsid.
(A) The GRID calculation was performed for a 20 Å radius around the 5-fold axis using pyramidal sulfur as a probe. VP1 residue 112 is the most likely site of interaction with molecular interaction energy of -8.2 kcal/mole. The interaction energy increased to -10 kcal/mole when the grid was centered at VP1 residue 112. (B) Five linked heparin disaccharide molecules were docked using the default parameters of GOLD onto the SAT1 modeled mutant pentamer structures. A 30Å3 region from VP1 residue 112 was defined for docking and the GOLD fitness score function was used to rank the docking poses. The best docking pose is shown (GOLD score = 127). The equivalent process for the wild-type virus produced a less satisfactory docking (GOLD score = 102, docking not shown).
Fig 3.
Charge distribution and clustering of mutations on the capsid surface.
(A) Electrostatic potential of wild-type SAT1 capsid showing uniform charge distribution on the particle surface. (B) Electrostatic potential of the mutant SAT1 capsid showing clustering of positive charge at the 5-fold axis. The electrostatic potential was calculated using the APBS plugin embedded within Pymol. The colouring represents positive charge (blue), negative charge (red) and neutral (white). (C) The projection of the capsid on a 5-fold axis shows the clustering of the positively charged mutations from the top view. (D) The projection showing the side view and the surface exposure of the mutant side chains.
Table 3.
Titres (PFU/ml) of inter-serotype or intra-serotype FMDV chimera viruses with positive charged residue substitutions in the VP1 or VP3 capsid proteins.
Plaque morphology on BHK-21 cells are indicated.
Fig 4.
Infection of CHO-K1 cells by the recombinant mutants rvSAUVP3158K, rvSAUVP183K and rvSAUVP1158K is inhibited by heparin.
(A) Viruses with titres ranging from 5 × 107 to 7 × 107 PFU/ml were mock-treated or treated with different concentrations of soluble heparin, where i, ii, iii, iv, v and vi refers to 0mg/ml, 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml and 10mg/ml heparin respectively, before infecting CHO-K1 cell monolayers. Each treatment was performed in triplicate. Virus that had not been internalized was removed by washing with MES buffer (pH 5.5). The number of plaques formed on CHO-K1 cells was counted and expressed as the percentage of infectivity in relation to the non-heparin treated chimeric viruses. Statistical analyses were performed using a one-way ANOVA (followed by a Bonferroni's Multiple Comparison test). The confidence interval was 95%. Statistical analyses were carried out using GraphPad Prism v5.0 (GraphPad Software). Error bars represent the standard deviation. The a, b, c, d, or e denotations refer to statistically significant groupings where a is statistically significant to b, c, d and e; b is statistically significant to c, d and e; c is statistically significant to d and e and d is statistically significant to e. (B) Heparinase treatment of BHK-21 cells. BHK-21 cell monolayers were incubated with heparinase I or III enzymes or were mock-treated for 30 min at 37°C before virus infection (100 PFU/well). The number of plaques were determined at 24 hpi on BHK-21 cells and the percentage reduction in plaques calculated. The data represent means ± SD from three independent experiments.
Table 4.
Mutagenesis and In-Fusion cloning primer sequences.