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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.

The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid (or procapsid) to power genome encapsidation. Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonella, yet, less is known about the packaging motor of Pseudomonas-phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but assembles into a nonamer in its high-affinity heparin-binding conformation. The atomic structure of Pseudomonas phage PaP3 TerS, the first complete structure for a TerS from a cos phage, reveals nine helix-turn-helix (HTH) motifs asymmetrically arranged around a β-stranded channel, too narrow to accommodate DNA. PaP3 TerS binds DNA in a sequence-specific manner in vitro. X-ray scattering and molecular modeling suggest TerS adopts an open conformation in solution, characterized by dynamic HTHs that move around an oligomerization core, generating discrete binding crevices for DNA. We propose a model for sequence-specific recognition of packaging initiation sites by lateral interdigitation of DNA.

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