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.

Melanie Chitray,Francois F Maree,Guntram Paul,Sonja Maree,Abhay Kotecha,Elizabeth E Fry,Peninah Nsamba,David I Stuart,Jingshan Ren,Jacques Theron,Tovhowani Ramulongo,Richard J Kuhn

doi:10.1371/journal.ppat.1008828

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.

Highlights

Foot-and-mouth disease virus (FMDV) is a small, non-enveloped, icosahedral virus with a polyadenylated, single-stranded, positive-sense RNA genome belonging to the Aphthovirus genus in the Picornaviridae family [1]
Current vaccines are produced by chemical inactivation of virus grown in large-scale production cultures of Baby hamster kidney (BHK)-21 cells
The significance of our research is in identifying amino acid residues responsible for enhancing cell culture adaptation of FMD field viruses

Summary

Introduction

Foot-and-mouth disease virus (FMDV) is a small, non-enveloped, icosahedral virus with a polyadenylated, single-stranded, positive-sense RNA genome belonging to the Aphthovirus genus in the Picornaviridae family [1]. FMDV naturally infects epithelial cells and to date studies have shown that FMDV can adhere to any of four members of the αV subgroup of the integrin family of cellular receptors, i.e. αVβ1, αVβ3, αVβ6 and αVβ8 [4,5,6,7,8,9,10,11] Attachment to these receptors is mediated via a highly conserved Arg-Gly-Asp (RGD) motif [12,13,14,15], located within the structurally disordered βGβH loop of VP1 [2,16,17]. Following FMDV-receptor interactions, the virus is internalized and the viral genome is released into the cytosol via acid-induced capsid dissociation [18,19]

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Conclusion