E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole E217 virion before and after DNA ejection at 3.1 Å and 4.5 Å resolution, respectively, determined using cryogenic electron microscopy (cryo-EM). We identify and build de novo structures for 19 unique E217 gene products, resolve the tail genome-ejection machine in both extended and contracted states, and decipher the complete architecture of the baseplate formed by 66 polypeptide chains. We also determine that E217 recognizes the host O-antigen as a receptor, and we resolve the N-terminal portion of the O-antigen-binding tail fiber. We propose that E217 design principles presented in this paper are conserved across PB1-like Myoviridae phages of the Pbunavirus genus that encode a ~1.4 MDa baseplate, dramatically smaller than the coliphage T4.

The genome packaging motor of bacteriophages and herpesviruses is built by two terminase subunits, known as large (TerL) and small (TerS), both essential for viral genome packaging. TerL structure, composition, and assembly to an empty capsid, as well as the mechanisms of ATP-dependent DNA packaging, have been studied in depth, shedding light on the chemo-mechanical coupling between ATP hydrolysis and DNA translocation. Instead, significantly less is known about the small terminase subunit, TerS, which is dispensable or even inhibitory in vitro, but essential in vivo. By taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of phage TerSs, in this review, we take an inventory of known TerSs studied to date. Our analysis suggests that TerS evolved and diversified into a flexible molecular framework that can conserve biological function with minimal sequence and quaternary structure conservation to fit different packaging strategies and environmental conditions.

Bacterial viruses (or bacteriophages) have developed formidable ways to deliver their genetic information inside bacteria, overcoming the complexity of the bacterial-cell envelope. In short-tailed phages of the Podoviridae superfamily, genome ejection is mediated by a set of mysterious internal virion proteins, also called ejection or pilot proteins, which are required for infectivity. The ejection proteins are challenging to study due to their plastic structures and transient assembly and have remained less characterized than classical components such as the phage coat protein or terminase subunit. However, a spate of recent cryo-EM structures has elucidated key features underscoring these proteins’ assembly and conformational gymnastics that accompany their expulsion from the virion head through the portal protein channel into the host. In this review, we will use a phage-T7-centric approach to critically review the status of the literature on ejection proteins, decipher the conformational changes of T7 ejection proteins in the pre- and post-ejection conformation, and predict the conservation of these proteins in other Podoviridae. The challenge is to relate the structure of the ejection proteins to the mechanisms of genome ejection, which are exceedingly complex and use the host’s machinery.

Sf6 is a bacterial virus that infects the human pathogen Shigella flexneri. Here, we describe the cryo–electron microscopy structure of the Sf6 tail machine before DNA ejection, which we determined at a 2.7-angstrom resolution. We built de novo structures of all tail components and resolved four symmetry-mismatched interfaces. Unexpectedly, we found that the tail exists in two conformations, rotated by ~6° with respect to the capsid. The two tail conformers are identical in structure but differ solely in how the portal and head-to-tail adaptor carboxyl termini bond with the capsid at the fivefold vertex, similar to a diamond held over a five-pronged ring in two nonidentical states. Thus, in the mature Sf6 tail, the portal structure does not morph locally to accommodate the symmetry mismatch but exists in two energetic minima rotated by a discrete angle. We propose that the design principles of the Sf6 tail are conserved across P22-like Podoviridae.

Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/β1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms exhibit significant variation in stability from hours to days at room temperature. In particular, the refractory allele TEP1*R1 is significantly more stable than the susceptible allele TEP1*S1. This raises the question of whether the stability of cleaved TEP1 is linked to allelic variation and varying immunophenotypes. We have analyzed the stability of the cleaved form of additional TEP1 alleles and constructs. We show that stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains of TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1_cut/LRIM1/APL1C complex is related to the conformational change that induces the precipitation of cleaved TEP1.