Molecular Architecture of Salmonella Typhimurium Virus P22 Genome Ejection Machinery.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted ß-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.

Cryo-EM Structure of a Kinetically Trapped Dodecameric Portal Protein from the Pseudomonas-phage PaP3.

Portal proteins are dodecameric assemblies that occupy a unique 5-fold vertex of the icosahedral capsid of tailed bacteriophages and herpesviruses. The portal vertex interrupts the icosahedral symmetry, and in vivo, its assembly and incorporation in procapsid are controlled by the scaffolding protein. Ectopically expressed portal oligomers are polymorphic in solution, and portal rings built by a different number of subunits have been documented in the literature. In this paper, we describe the cryo-EM structure of the portal protein from the Pseudomonas-phage PaP3, which we determined at 3.4 Å resolution. Structural analysis revealed a dodecamer with helical rather than rotational symmetry, which we hypothesize is kinetically trapped. The helical assembly was stabilized by local mispairing of portal subunits caused by the slippage of crown and barrel helices that move like a lever with respect to the portal body. Removing the C-terminal barrel promoted assembly of undecameric and dodecameric rings with quasi-rotational symmetry, suggesting that the barrel contributes to subunits mispairing. However, ΔC-portal rings were intrinsically asymmetric, with most particles having one open portal subunit interface. Together, these data expand the structural repertoire of viral portal proteins to Pseudomonas-phages and shed light on the unexpected plasticity of the portal protein quaternary structure.