Alice-Roza Eruera

Picornavirus cell entry requires a series of capsid protein conformational changes leading to genome uncoating. For enteroviruses, receptor binding triggers the transition from a full (F) capsid to an altered (A) particle before releasing its genome and finally converting it into an empty (E) particle. In contrast, non-enteroviruses, such as Aphthovirus, Cardiovirus, or Seneca Valley virus, release their genomes by dissociating the capsid into pentamers. While the existence of a transient A-particle for non-enteroviruses was previously speculated, it has never been directly observed using structural methods. Seneca Valley virus (SVV) is an oncolytic picornavirus that selectively targets cancer cells by recognizing Tumor endothelial marker 8 (TEM8) as the host receptor. SVV disassembles into pentamers at acidic pH, suggesting that the acidic environment of the endosome could cause capsid disassembly. We used cryo-electron microscopy to investigate SVV under acidic conditions and in complex with TEM8 at physiological pH, identifying multiple uncoating intermediates. These include an altered-particle, an empty-rotated particle (E ^R ), and a series of open particles expelling the coiled genome. The A-particle is expanded, displays reduced interactions between capsid proteins, a reorganized genome, and has a poorly resolved VP1 N-terminus, VP2 N-terminus, and VP4. The E ^R particle has rotated pentamers, reduced contacts within the particle, lacks the genome, VP1 and VP2 N-termini, and VP4. Our work provides an understanding of transient SVV structural states and supports the existence of an intermediate SVV A-particle. These findings could help optimize SVV for oncolytic therapy. IMPORTANCE: Seneca Valley virus (SVV) is a non-enterovirus picornavirus with specific tumor tropism mediated by the receptor Tumor endothelial marker 8, also known as Anthrax toxin receptor 1. Using cryo-electron microscopy, it was possible to identify multiple structural states of SVV. We demonstrate that SVV capsids transition from full particles to altered (A) particles and then to empty-rotated (E ^R ) particles, with receptor binding and acidic pH driving these conformational changes, respectively. This study also identifies open particles with expelled genomes. Comparisons between A- and E ^R -particles reveal that peptide segments of VP1, VP2, and VP4 could potentially play a role in genome delivery. Future work can explore the formation of these structural states in vivo .

Picornavirus cell entry involves a series of capsid conformational changes that ultimately result in genome uncoating. For enteroviruses, receptor binding triggers a transition from full capsids into altered particles before releasing their genome and forming empty particles. In contrast, non-enteroviruses like Cardioviruses and Senecaviruses release their genome through dissociation into pentamers. Seneca Valley Virus (SVV), a clade I single-stranded RNA oncolytic virus that specifically targets and kills cancer cells by binding to Anthrax Toxin Receptor 1 (ANTXR1). Currently, SVV is being developed as an oncolytic agent against various cancers. Previous research from our lab demonstrated that SVV disassembles into pentamers at acidic pH, indicating that the acidic environment of the late endosome is essential for capsid disassembly and genome release. In this study, we utilized cryogenic-electron microscopy (cryo-EM) and single-particle analysis to investigate SVV structure under acidic conditions and in complex with ANTXR1 at physiological conditions. This enabled us to identify several uncoating intermediates and propose a mechanism for genome release, contributing to a deeper understanding of the infection mechanisms employed by non-enterovirus picornaviruses.

Coliphage N4 is a representative species of the Schitoviridae family of bacteriophages. Originally structurally studied in 2008, the capsid structure was solved to 14 Å to reveal an interesting arrangement of Ig-like decoration proteins across the surface of the capsid. Herein, we present a high-resolution N4 structure, reporting a 2.45 Å map of the capsid obtained via single particle cryogenic-electron microscopy. Structural analysis of the major capsid proteins (MCPs) and decoration proteins (gp56 and gp17) of phage N4 reveals a pattern of interactions across the capsid that are mediated by structurally homologous domains of gp17. In this study, an analysis of the complex interface contacts allows us to confirm that the gp17 Ig-like decoration proteins of N4 are likely employed by the virus to increase the capsid’s structural integrity.

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single particle cryo-EM. The core consists of tetrameric and octameric ejection proteins which form a ∼1.5 MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 Å and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single particle cryo-EM. The core consists of tetrameric and octameric ejection proteins which form a ∼1.5 MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 Å and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages. Significance Statement This study resolves the cryo-EM structure of bacteriophage ΦM1, which possesses several unique and interesting structural elements, including a pair of distinct decoration proteins that are underreported in tailed DNA phages. Significantly, we also report the internal ‘ejectosome’ proteins of ΦM1, which are highly non-conserved with previously solved proteins to date and demonstrate the structural diversity of ejection proteins. The ejectosome reveals a DNA spooling phenomenon whereby the viral genome wraps around the ejectosome within the capsid, which has never been reported before. We provide a clear, step-by-step method for the technically challenging reconstruction of ejectosomes using standard, open-source software.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC₅₀ = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.