Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive EV-A71 virulence in mice

Abstract

19Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological 20 complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly 21 understood. In mice, adaptation and virulence determinants have been mapped to mutations at 22 VP1-145, VP1-244 and VP2-149. We hypothesized that heparin-binding phenotype shapes 23 EV-A71 virulence in mice. We constructed six viruses with varying residues at VP1-98, VP1-24 145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 25 98E/145Q/149K, termed EQK) to generate KQK, KEK, EEK, EEI and EQI variants. We 26 demonstrated that the weak heparin-binder EEI was highly lethal in mice. The initially strong 27 heparin-binding EQI variant acquired an additional mutation VP1-K244E, which confers weak 28 heparin-binding phenotype resulting in elevated viremia and increased brain inflammation and 29 virus antigens in mice, with subsequent high virulence. EEI and EQI-K244E variants 30 inoculated into mice disseminated efficiently and displayed high viremia. Increasing 31 polymerase fidelity and impairing recombination of EQI attenuated virulence, suggesting the 32 importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico 33 docking and deep sequencing approaches, we inferred that virus population diversity is shaped 34 by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface 35 charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo 36 dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, 37 which ultimately results in increased neurovirulence. The dynamic switching from heparin-38 binding to weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71. 39 40 Author summary 41 Enterovirus A71 (EV-A71) is the primary cause of hand, foot and mouth disease, and it can 42 also infect the central nervous system and cause fatal outbreaks in young children. EV-A71 43 pathogenesis remains elusive. In this study, we demonstrated that EV-A71 variants with strong 44 affinity to heparan sulfate (heparin) have a growth advantage in tissue culture, but are 45 disadvantageous in vivo. When inoculated into mice, strong heparin-binding virus variants are 46 more likely to be adsorbed to peripheral tissues, resulting in impaired ability to disseminate 47 and being cleared from the bloodstream rapidly. The lower viremia level resulted in no 48 neuroinvasion. In contrast, weak heparin-binding variants show greater levels of viremia, 49 dissemination and subsequent neurovirulence in mice. We also provide evidence that the ability 50 of EV-A71 to bind heparin is mediated by electrostatic surface charges due to amino acids on 51 the virus capsid surface. In mice, EV-A71 undergoes adaptive mutation to acquire greater 52 negative surface charges, thus generating new virulent variants with weak heparin-binding 53 which allows greater viral spread. Our study underlines the...

Cellular receptors for enterovirus A71

“…Similar results were obtained by Fujii et al [85] using cynomolgus monkeys. More recently, Tee et al [86] generated a number of mutants that showed different degrees of heparin binding activity. They showed that weak heparin binders have a more virulent phenotype than strong heparin binders in a neonatal mouse model.…”