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Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

The tailed dsDNA bacteriophages or Caudovirales include three morphological families, Myoviridae, with contractile tails (example, phage T4), Siphoviridae with long, non-contractile tails (example, phage λ), and Podoviridae, with short, stumpy tails (examples, phages P22 and T7). Virions assemble via converging pathways. Capsids are built as empty procapsids and filled with genomic DNA; tails and tail fibers are made separately and tails are joined to capsids, followed by tail fibers. Tails and fibers identify hosts, then deliver the phage chromosome to the host where it is actively replicated, transcribed and translated to make progeny phage or enters a dormant, lysogenic state.

The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of capsid precursors called procapsids or proheads. Portals are essential and serve as the pore for DNA transit and the site of tail attachment; however, bacteriophage HK97 capsid proteins assemble efficiently without a portal when expressed from plasmids. Following portal co-expression, portals were incorporated into about half of the proheads that were made. In the absence of active capsid maturation protease, uncleaved proheads formed dimers, trimers, and tetramers of proheads during purification, but only if they had portals. These appeared bound to membrane-like fragments by their portals and could be disaggregated by detergents, supporting a role for membranes in their formation and in capsid assembly. The precursors to prohead oligomers were detected in cell extracts. These were able to bind to Octyl-Sepharose and could be released by detergent, while uncleaved proheads without portal or cleaved proheads with portal did not bind. Our results document a discrete change in the HK97 portal’s hydrophobicity induced by cleavage of the procapsid shell in which it is embedded. Additionally, we detected an increase in the rate of expansion induced by the presence of a portal complex in cleaved HK97 proheads. These results suggest that portals and capsids influence each other’s conformation during assembly. The formation of prohead oligomers also provides a rapid and sensitive assay for identification and analysis of portal incorporation mutants.

Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T = 7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the “E-loop,” that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153–R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153–R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153–R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly.

During maturation of the phage HK97 capsid, each of the 415 capsid subunits forms covalent bonds to neighboring subunits, stabilizing the capsid. Crosslinking is catalyzed not by a separate enzyme but by subunits of the assembled capsid in response to conformational rearrangements during maturation. This report investigates the catalytic mechanism. Earlier work established that the crosslinks are isopeptide (amide) bonds between side chains of a lysine on one subunit and an asparagine on another subunit, aided by a catalytic glutamate on a third subunit. The mature capsid structure suggests that the reaction may be facilitated by the arrival of a valine with the lysine to complete a hydrophobic pocket surrounding the glutamate, lysine and asparagine. We show that this valine has an essential role for efficient crosslinking, and that any of six other amino acids can successfully substitute for valine. Evidently none of the remaining 13 amino acids will work.

dsDNA Bacteriophages, some dsDNA archaeal viruses and the Herpesviruses share many features including a common capsid assembly pathway and coat protein fold. The coat proteins of these viruses, which have the HK97 fold, co-assemble with a free or attached scaffolding protein and other capsid proteins into a precursor capsid, known as a procapsid or prohead. The procapsid is a metastable state that increases in stability as a result of morphological changes that occur during the dsDNA packaging reaction. We review evidence from several systems indicating that proper contacts acquired in the assembly of the procapsid are critical to forming the correct morphology in the mature capsid.

The 102 residue N-terminal extension of the HK97 major capsid protein, the delta domain, is normally present during the assembly of immature HK97 procapsids, but it is removed during maturation like well-known internal scaffolding proteins of other tailed phages and herpesviruses. The delta domain also shares other unusual properties usually found in other viral and phage scaffolding proteins, including its location on the inside of the capsid, a high predicted and measured α-helical content, and an additional prediction for the ability to form parallel coiled-coils. Viral scaffolding proteins are essential for capsid assembly and phage viability, so we tested whether the HK97 delta domain was essential for capsid assembly. We studied the effects of deleting all or parts of the delta domain on capsid assembly and on complementation of capsid-protein-defective phage, and our results demonstrate that the delta domain is required for HK97 capsid assembly.

The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg(2+), DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 degrees C and 96 degrees C. In other conditions (absence of Mg(2+) and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit-probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.

We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.

Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.

As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local minima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross-linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in maturation. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable intermediates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conformations of its precursor and mature states., IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in capsid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of principle, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.

In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.

A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage λ, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous −1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous −2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.

Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli. These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. Analogous transformations also occur in unrelated viruses and protein complexes. We find that expansion and crosslinking happen concurrently during maturation at low pH. Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.

The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18 A and the maximum diameter is 550 A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow-growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~400 G-loops interacts with lysine 178 of the E-loop (extended loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly and, in some cases, induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral and hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles.

The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), double-stranded DNA bacteriophages of Escherichia coli and members of the lambdoid or lambda-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or, in some cases, at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome. The comparative analysis also allows some inferences about function of genes or other genetic elements. We give examples for the generalized recombination genes of HK97, HK022 and P22, and for a putative headtail adaptor protein of HK97 and HK022. We also use the comparative approach to identify a new class of genetic elements, the morons, which consist of a protein-coding region flanked by a putative delta 70 promoter and a putative factor-independent transcription terminator, all located between two genes that may be adjacent in a different phage. We argue that morons are autonomous genetic modules that are expressed from the repressed prophage. Sequence composition of the morons implies that they have entered the phages' genomes by horizontal transfer in relatively recent evolutionary time.

Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation. First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles. Maturation is initiated by proteolysis, producing a metastable particle primed for expansion—the major structural transition. We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo–electron microscopy. The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions. Three distinct intermediates were identified, which are comparable to transitional states in protein folding. The intermediates' structures reveal the molecular events occurring during expansion. Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process.

HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage λ, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 Å in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 Å resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been confirmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 Å. The cell dimensions after post-refinement were a = 580, b = 626, c = 788 Å, β = 90.0°. From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even reflections along the 0k0 lattice line indicate that the space group is P21. The rotation function was used to determine the orientation of the particles in the unit cell and to confirm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18° away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at (1\over4, 0, 1\over4) and the other particle at (3\over4, 1\over2, 3\over4). The particle position and orientation were confirmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 Å is under way.

The capsid shells of bacteriophage HK97 and several other phages contain polypeptides that are covalently linked into complexes so large that they do not enter polyacrylamide gels after denaturation. The enormous apparent size of these protein complexes in HK97 derives from a novel protein topology. HK97 subunits cross-link via isopeptide bonds into oligomers that are closed rings of five or six members. However, polypeptides from neighboring pentamer and hexamer rings intertwine before the covalent cross-links form. As a result, adjacent protein rings catenate into a network similar to chainmail armor. In vitro linking and unlinking experiments provide strong support for the chainmail model, which explains the unusual properties of these bacteriophages and may apply to other macromolecular structures.

Tailed double-stranded DNA bacteriophages and herpesviruses build capsids by co-assembling a major capsid protein with an internal scaffolding protein that then exits from the assembled structure either intact or after digestion in situ by a protease. In bacteriophage HK97, the 102-residue N-terminal delta domain of the major capsid protein is also removed by proteolysis after assembly and appears to perform the scaffolding function. We describe the HK97 protease that carries out these maturation cleavages. Insertion mutations at seven sites in the protease gene produced mutant proteins that assemble into proheads, and those in the N-terminal two-thirds were enzymatically inactive. Plasmid-expressed protease was rapidly cleaved in vivo but was stabilized by co-expression with the delta domain. Purified protease was found to be active during the assembly of proheads in vitro. Heterologous fusions to the intact protease or to C-terminal fragments targeted fusion proteins into proheads. We confirm that the catalytic activity resides in the N-terminal two-thirds of the protease polypeptide and suggest that the C-terminal one-fifth of the protein contains a capsid targeting signal. The implications of this arrangement are compared to capsid targeting systems in other phages, herpesviruses, and encapsulins.

Bacteriophage λ makes two proteins with overlapping amino acid sequences that are essential for tail assembly. These two proteins, gpG and gpGT, are related by a programmed translational frameshift that is conserved among diverse phages and functions in λ to ensure that gpG and the frameshift product gpGT are made in a molar ratio of approximately 30:1. Although both proteins are required and must be present in the correct ratio for assembly of functional tails, neither is present in mature tails. During λ tail assembly, major tail protein gpV polymerizes to form a long tube whose length is controlled by the tape measure protein gpH. We show that the “G” domains of gpG and gpGT bind to all or parts of tail length tape measure protein gpH and that the “T” domain of gpGT binds to major tail shaft subunit gpV, and present a model for how gpG and gpGT chaperone gpH and direct the polymerization of gpV to form a tail of the correct length.

The 42 kDa capsid protein of bacteriophage HK97 requires the GroEL and GroES chaperonin proteins of its Escherichia coli host to facilitate correct folding, both in vivo and in vitro. In the absence of GroES and ATP, denatured gp5 forms a stable complex with the 14 subunit GroEL molecule. We characterized the electrophoretic and biochemical properties of this complex. In electrophoresis on a native (nondenaturing) gel, the band of the gp5-GroEL complex shifts to a slower migrating position relative to uncomplexed GroEL. The results show that there is only one subunit of gp5 bound to each GroEL 14-mer and that the shift in band position is due primarily to a change in the overall charge of the complex relative to uncomplexed GroEL, and not to a change in size or shape. GroEL forms similar complexes with proteolytic fragments of gp5, with a series of sequence duplication derivatives of gp5, and with other proteins. Electrophoretic examination of these complexes shows that a band shift occurs with proteins larger than 31-33 kDa but not with smaller proteins. For those proteins that cause a band shift upon complex formation, the magnitude of the shift is correlated with the predicted if the charge of the complex were simply the sum of the charge of GroEL and the charge of the substrate protein. We suggest that binding of a substrate protein to GroEL is accompanied by a net binding of solution cations to the complex, but only in the case of proteins above a minimum size of 31-33 kDa. The gp5-GroEL complex is in an association/dissociation equilibrium, with a binding constant measured in the range of 11-17 microM-1.

Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

We report studies to determine which bacteriophage genes are required for assembly of phage HK97 proheads and what roles they play. We identify the gene encoding the major capsid protein of phage HK97 and report its DNA sequence, together with the DNA sequences of the two genes immediately upstream from it. When the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of the unprocessed 42 kDa capsid protein. No separately encoded scaffolding protein is required for this assembly. If the 25 kDa product of the next gene upstream is co-expressed with the capsid protein, the prohead structures that are produced undergo the normal morphogenetic cleavage, which removes 102 amino acids from the N terminus of each subunit, leaving 31 kDa subunits. The 25 kDa protein is therefore probably a phage-encoded protease. The third gene, upstream from the protease gene, encodes the portal protein. Presence of the portal protein is not required for assembly of the capsid protein. Analysis of the phenotypes of four single amino acid-substitution mutants in the capsid-protein gene leads to several insights into the functions of the capsid protein and its interactions with the putative protease.

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli , the delta domain guides 420 copies of the subunit into a procapsid with T = 7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I +P was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I +P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I +P was determined at 5.2 A resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I +P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.

The common laboratory strain of bacteriophage lambda--lambda wild type or lambda PaPa--carries a frameshift mutation relative to Ur-lambda, the original isolate. The Ur-lambda virions have thin, jointed tail fibers that are absent from lambda wild type. Two novel proteins of Ur-lambda constitute the fibers: the product of stf, the gene that is disrupted in lambda wild type by the frameshift mutation, and the product of gene tfa, a protein that is implicated in facilitating tail fiber assembly. Relative to lambda wild type, Ur-lambda has expanded receptor specificity and adsorbs to Escherichia coli cells more rapidly.

The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.

The lambda-like double-stranded (ds) DNA bacteriophage HK97 is a favourable system for studying viral capsid maturation since it can be assembled in Escherichia coli from the expression of just two viral gene products and maturation is easily triggered and analysed in vitro. Various mature viral capsid structures have been determined but until now no procapsids have been available for a dsDNA virus or bacteriophage. Gertsman et al. now report the high-resolution structure of the HK97 procapsid providing insight into the capsid assembly process leading to infectious virions. The knowledge gained from this structure is relevant for related viruses such as the human herpesvirus. This paper reports the high-resolution structure of the double-stranded DNA bacteriophage HK97 procapsid, providing insight into the capsid assembly process leading to infectious virions. The knowledge gained from this structure is relevant for related viruses such as human herpesviruses. Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging1. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 A resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 A (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol-1 of energy3. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold4.

The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching.

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.

Document S1. One figure and Supplemental Experimental ProceduresxDownload (.13 MB ) Document S1. One figure and Supplemental Experimental Procedures

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.

The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 A larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles "hop" over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.

Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle. Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the mature Head state. In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits. The final state, termed Head-II, is a 600 angstroms diameter icosahedral structure with catenated subunit rings. Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates. Here we extend the characterization of the acid-induced expansion of HK97 in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS. We find that the greatest changes in all observables occur at an early stage of maturation. Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity. These spectral changes reveal two kinetic phases of the expansion reaction. The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9. The second, slower phase reported by fluorescence is relatively insensitive to pH. Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase. Native agarose gel assays corroborated this finding. By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence. Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of beta-strand and beta-turn observed in the Head-II crystal structure. Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid.

HK97 Prohead II is an early intermediate in the maturation of HK97, a T = 7 dsDNA-tailed bacteriophage related to bacteriophage lambda. Previously, selected capsid-protein genes of HK97 were expressed in Escherichia coli and spontaneously assembled to form an icosahedral capsid that followed a maturation pathway closely similar to the authentic virion. The crystal structure of the mature HK97 capsid (Head II) made in this way was reported at 3.5 A resolution. Additional high-resolution structures of intermediates are needed to understand the maturation mechanism. The crystal structure of expressed Prohead II will elucidate the early steps of HK97 assembly. Crystals of the Prohead II mutant W336F were grown in 0.1 M HEPES pH 7.5, 0.2 M CaCl(2) and 2-3% PEG 4000 at a Prohead II concentration of 16.5 mg ml(-1). It was not possible to grow high-quality crystals of wild-type Prohead II. Diffraction was observed to 5 A resolution from these crystals on beamline 14BM-C at the Advanced Photon Source and data were collected to 5.5 A with a completeness of 77%. The space group was P2(1)3, with unit-cell parameter a = 707.0 A and four particles in the unit cell. The particles are on the body diagonals of the cubic cell, with icosahedral threefold axes coincident with crystallographic threefold axes. Self-rotation function and locked-rotation function analysis determined the particle orientation and a one-dimensional R-factor search along the body diagonal indicated that the particle centers were close to (1/4, 1/4, 1/4) and symmetry-related positions. Molecular-replacement averaging and phase extension are under way.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.