Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts

Abstract

Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses. Picornaviruses are important pathogens but their assembly is incompletely understood, preventing development of anti-viral drugs and vaccines. We report identification of multiple RNA stem–loops, with purine trinucleotide sequence motifs in each loop, distributed across the genomes of two enteroviruses, enterovirus-E and poliovirus. Each motif appears to have affinity for its cognate capsid protein. Cryo-EM reconstruction of enterovirus-E without imposed symmetry identifies many repeated CP-RNA contacts. These include stem-loops adjacent to the particle two-fold axes, which appear conserved between viruses, as are the amino acid side-chains that contact all the genomic RNA segments. Mutagenesis shows that these amino acids are essential for virion formation, consistent with an RNA-dependent assembly mechanism. These results suggest that all enteroviruses utilise a common assembly mechanism that requires defined CP-RNA contacts.