Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses

Abstract

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

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 Šradius 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 30ų 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.

Fig 4. Infection of CHO-K1 cells by the recombinant mutants rv^SAUVP3^158K, rv^SAUVP1^83K and rv^SAUVP1^158K 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.