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In the assembly pathway of tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses, a procapsid with a dodecameric portal for DNA delivery at a unique vertex is initially formed. Appropriate procapsid assembly requires the transient presence of multiple copies of a scaffolding protein (SP), which is absent in the mature virion. However, how the SP contributes to dodecameric portal formation, facilitates portal and coat protein incorporation, and is subsequently released remains unclear because of a lack of structural information. Here, we present the structure of the SP–portal complex within the procapsid of bacteriophage P22 at 3–9 Å resolutions. The AlphaFold2-predicted SP model fits well with the density map of the complex. The SP forms trimers and tetramers that interact to yield a dome-like complex on the portal. Two SP domains mediate multimerization. Each trimer interacts with two neighboring portal subunits. The SP has a loop-hook-like structure that aids in coat protein recruitment during viral assembly. The loops of those SP subunits on the portal are positioned in clefts between adjacent portal subunits. Conformational changes in the portal during phage maturation may trigger the disassembly and release of the SP complex. Our findings provide insights into SP-assisted procapsid assembly in bacteriophage P22 and suggest that this strategy is also implemented by other dsDNA viruses, including herpesviruses.

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

Molecular communication across physical barriers requires pores to connect the environments on either side and discriminate between the diffusants. Here we use porous virus-like particles (VLPs) derived from bacteriophage P22 to investigate the range of molecule sizes able to gain access to its interior. Although there are cryo-EM models of the VLP, they may not accurately depict the parameters of the molecules able to pass across the pores due to the dynamic nature of the P22 particles in the solution. After encapsulating the enzyme AdhD within the P22 VLPs, we use a redox reaction involving PAMAM dendrimer modified NADH/NAD+ to examine the size and charge limitations of molecules entering P22. Utilizing the three different accessible morphologies of the P22 particles, we determine the effective pore sizes of each and demonstrate that negatively charged substrates diffuse across more readily when compared to those that are neutral, despite the negatively charge exterior of the particles. Molecular transport across physical barriers requires pores that connect to the environment. Here, the authors report on a solution based dynamic study into the effects of size and charge on the transport through the pores of virus-like particles derived from bacteriophage P22 using an encapsulated redox system.

Bacteriophages deliver their genome into the host cell through large proteinaceous assemblies called portal proteins. The first high-resolution structure of a portal protein from the Podovirae family reveals a ~1.1-MDa assembly formed by 24 proteins and a second structure uncovers an unexpected barrel-shaped domain that is essential for infectivity. DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.

For successful infection, bacteriophages must overcome multiple barriers to transport their genome and proteins across the bacterial cell envelope. We use cryo-electron tomography to study the infection initiation of phage P22 in Salmonella enterica serovar Typhimurium, revealing how a channel forms to allow genome translocation into the cytoplasm. Our results show free phages that initially attach obliquely to the cell through interactions between the O antigen and two of the six tailspikes; the tail needle also abuts the cell surface. The virion then orients perpendicularly and the needle penetrates the outer membrane. The needle is released and the internal head protein gp7* is ejected and assembles into an extracellular channel that extends from the gp10 baseplate to the cell surface. A second protein, gp20, is ejected and assembles into a structure that extends the extracellular channel across the outer membrane into the periplasm. Insertion of the third ejected protein, gp16, into the cytoplasmic membrane probably completes the overall trans-envelope channel into the cytoplasm. Construction of a trans-envelope channel is an essential step during infection of Gram-negative bacteria by all short-tailed phages, because such virions cannot directly deliver their genome into the cell cytoplasm. Cryo-electron tomography was used to study the initial steps of infection of Salmonella enterica serovar Salmonella Typhimurium with phage P22 and reveals how the phage forms a channel through the host outer and inner membranes to translocate its genome into the bacterial cytoplasm.

Bacteriophages, herpesviruses, and other large double-stranded DNA (dsDNA) viruses contain molecular machines that pump DNA into preassembled procapsids, generating internal capsid pressures exceeding, by 10-fold, that of bottled champagne. A 17 angstrom resolution asymmetric reconstruction of the infectious P22 virion reveals that tightly spooled DNA about the portal dodecamer forces a conformation that is significantly different from that observed in isolated portals assembled from ectopically expressed protein. We propose that the tight dsDNA spooling activates the switch that signals the headful chromosome packing density to the particle exterior.

The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. IMPORTANCE While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ : the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes. While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ : the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.

Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 Å and 9.5 Å resolutions, respectively. These structures allowed visualization of α-helices and β-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.

Bacteriophage assembly frequently begins with the formation of a precursor capsid that serves as a DNA packaging machine. The DNA packaging is accompanied by a morphogenesis of the small round precursor capsid into a large polyhedral DNA-containing mature phage. In vitro, this transformation can be induced by heat or chemical treatment of P22 procapsids. In this work, we examine bacteriophage P22 morphogenesis by comparing three-dimensional structures of capsids expanded both in vitro by heat treatment and in vivo by DNA packaging. The heat-expanded capsid reveals a structure that is virtually the same as the in vivo expanded capsid except that the pentons, normally present at the icosahedral fivefold positions, have been released. The similarities of these two capsid structures suggest that the mechanism of heat expansion is similar to in vivo expansion. The loss of the pentons further suggests the necessity of specific penton-hexon interactions during expansion. We propose a model whereby the penton-hexon interactions are stabilized through interactions of DNA, coat protein, and other minor proteins. When considered in the context of other studies using chemical or heat treatment of capsids, our study indicates that penton release may be a common trend among double-stranded DNA containing viruses.

Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.