Exploring picornavirus receptors and uncoating mechanisms

The Picornaviridae are a large family of non-enveloped RNA viruses that cause a wide range of human and veterinary diseases. The life cycle of a picornavirus begins with binding to its cognate receptor(s), which mediates attachment to a target cell, promotes endocytic uptake and in many cases destabilizes the capsid to stimulate uncoating; that is the delivery of the viral genome from the virus particle into the cytoplasm of the host cell. In this thesis, we aimed to identify novel picornavirus receptors and to investigate the impact of receptor binding on virion structure, focusing on the emerging human pathogens enterovirus D68 (EV-D68) and coxsackievirus A24 variant (CV-A24v), and the animal pathogen encephalomyocarditis virus (EMCV). EV-D68 is an emerging virus that causes outbreaks of respiratory disease and is associated with cases of acute flaccid paralysis in children. We explored EV-D68 receptor requirements via a genome-wide haploid genetic screen, which identified sialic acid (Sia) as an essential receptor and showed that EV-D68 can employ both α2,3- and α2,6-linked Sia as receptors. By determining a crystal structure of the EV-D68 prototype Fermon in complex with sialylated trisaccharides, we identified the Sia-binding site on the virion and revealed that Sia binding induces structural rearrangements in the viral capsid that result in ejection of the pocket factor, a fatty acid that regulates virus stability. These findings first showed that a glycan receptor can initiate enterovirus uncoating, via a similar mechanism as proteinaceous receptors. Although several EV-D68 strains strictly required Sia for infection, other strains could infect cells via a non-sialylated receptor. Using a haploid screen in Sia-deficient cells we showed that a Sia-independent strain could employ both Sia and sulfated glycosaminoglycans (sGAGs) as functional receptors. Remarkably, this screen did not identify the phospholipase PLA2G16, a recently discovered picornavirus host factor that promotes viral RNA translocation into the cytoplasm. We showed that binding of sGAGs, but not Sia, allowed the virus to circumvent PLA2G16. Cryo-EM analysis revealed that sGAG binding promotes virus uncoating, via extensive capsid rearrangements that enlarge the putative openings for genome release. These findings suggested that the need for PLA2G16 can be overcome by excessive receptor-mediated virion destabilization. We explored the receptor usage of EMCV, a commonly used model virus that causes fatal disease in domestic animals. Using a haploid genetic screen, we identified genes involved in FGF signaling as novel host factors and showed that the potential receptor ADAM9 plays a role in EMCV entry. We also investigated CV-A24v, the main cause of acute hemorrhagic conjunctivitis (AHC). Originally, CV-A24 was not associated with a human disease, but in 1970 a pathogenic “variant” emerged that caused AHC outbreaks and two pandemics. We showed that ICAM-1 is an essential CV-A24 receptor and, via a high-resolution cryo-EM structure, identified residues mediating contact between ICAM-1 and the virion. Moreover, we identified a capsid substitution that occurs in all pandemic CV-A24v strains and enhances the capacity of CV-A24v to bind Sia, revealing a possible link between Sia and viral adaptation to the eye