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Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcusxanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence. [ABSTRACT FROM AUTHOR]

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses.

ABSTRACT Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored. IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.

First discovered in bacteriophage HK97, biological chainmail is a highly stable system formed by concatenated protein rings. Each subunit of the ring contains the HK97-like fold, which is characterized by its submarine-like shape with a 5-stranded β sheet in the axial (A) domain, spine helix in the peripheral (P) domain, and an extended (E) loop. HK97 capsid consists of covalently-linked copies of just one HK97-like fold protein and represents the most effective strategy to form highly stable chainmail needed for dsDNA genome encapsidation. Recently, near-atomic resolution structures enabled by cryo electron microscopy (cryoEM) have revealed a range of other, more complex variants of this strategy for constructing dsDNA viruses. The first strategy, exemplified by P22-like phages, is the attachment of an insertional (I) domain to the core 5-stranded β sheet of the HK97-like fold. The atomic models of the Bordetella phage BPP-1 showcases an alternative topology of the classic HK97 topology of the HK97-like fold, as well as the second strategy for constructing stable capsids, where an auxiliary jellyroll protein dimer serves to cement the non-covalent chainmail formed by capsid protein subunits. The third strategy, found in lambda-like phages, uses auxiliary protein trimers to stabilize the underlying non-covalent chainmail near the 3-fold axis. Herpesviruses represent highly complex viruses that use a combination of these strategies, resulting in four-level hierarchical organization including a non-covalent chainmail formed by the HK97-like fold domain found in the floor region. A thorough understanding of these structures should help unlock the enigma of the emergence and evolution of dsDNA viruses and inform bioengineering efforts based on these viruses.

Abstract: One of the outstanding questions in biology today is the origin of viruses. We have discovered a protein in the hyperthermophile Sulfolobus solfataricus while following proteome regulation during viral infection that led to the discovery of a fossil provirus. Characterization of the wild type and recombinant protein revealed that it assembled into virus-like particles with a diameter of ~32nm. Sequence and structural analyses showed that the likely proviral capsid protein, Sso2749, is homologous to a protein from Pyrococcus furiosus that forms virus-like particles using the HK-97 major capsid protein fold. The SsP2-provirus appears mosaic and contains proteins with similarity to, among others, eukaryotic herpesviruses and tailed dsDNA bacteriophage families, reinforcing the hypothesis of a common ancestral gene pool across all three domains of life. This is the first description of the HK-97 fold in a crenarchaeal virus and the first direct genomic connection of linocin-like protein cages to a virus. [Copyright &y& Elsevier]

Abstract: Phage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a ∼200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold–capsid fusion) and λ (protease–scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12Å. The capsids have lambdoid T=7 geometry and are well modeled with the atomic structures of HK97 mcp and phage λ gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and λ. [Copyright &y& Elsevier]

Abstract: The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly. [Copyright &y& Elsevier]

The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into “Whiffleballs”, particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer. [Copyright &y& Elsevier]

We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change. We observe for all of the viral capsids that the nature of the modes is different. In particular for the T=7 virus, HK97, for which the shape of the capsid changes from spherical to faceted polyhedra, two modes are necessary to accomplish the conformational transition. In addition, we extend our study to viral capsids with other T numbers, and discuss the similarities and differences in the features of virus capsid conformational dynamics. We note that the pentamers generally have higher flexibility and propensity to move freely from the other capsomers, which facilitates the shape adaptation that may be important in the viral life cycle. [Copyright &y& Elsevier]

There is renewed interest in bacterial viruses (phages) as alternatives to antibiotics. All phage treatments to date have used virulent phages rather than temperate ones, as these can integrate into the genome of the bacterial host and lie dormant. However, temperate phages are abundant and easier to isolate. To make use of these entities, we leverage stressors known to awaken these dormant, integrated phages. Co-administration of the temperate phage HK97 with sub-inhibitory concentrations of the antibiotic ciprofloxacin results in bacterial eradication (≥8 log reduction) in vitro. This synergy is mechanistically distinct from phage-antibiotic-synergy described for virulent phages. Instead, the antibiotic specifically selects against bacteria in which the phage has integrated. As the interaction between temperate phages and stressors such as ciprofloxacin are known to be widespread, this approach may be broadly applicable and enable the use of temperate phages to combat bacterial infections. [Display omitted] • Temperate phage HK97 synergizes with ciprofloxacin to eradicate E. coli in vitro • The synergy is 4.5 × 105 greater than the multiplicative effects of its components • This is mechanistically distinct from traditional phage antibiotic synergy • Temperate phage-antibiotic synergy is driven by depletion of lysogens Most bacterial viruses (phages) cannot be used in therapy as they are temperate and go dormant in their hosts. Al-Anany et al. eradicate Escherichia coli through the synergistic effects of a temperate phage and signals that awaken it. This targets conserved pathways and may be broadly applicable for phage therapy. [ABSTRACT FROM AUTHOR]

Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored. IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization., (Copyright © 2017 Hua et al.)