Structural rearrangements between portal protein subunits are essential for viral DNA translocation

Transport of DNA into preformed procapsids is a general strategy for genome packing inside virus particles. In most viruses, this task is accomplished by a complex of the viral packaging ATPase with the portal protein assembled at a specialized vertex of the procapsid. Such molecular motor translocates DNA through the central tunnel of the portal protein. A central question to understand this mechanism is whether the portal is a mere conduit for DNA or whether it participates actively on DNA translocation. The most constricted part of the bacteriophage SPP1 portal tunnel is formed by twelve loops, each contributed from one individual subunit. The position of each loop is stabilized by interactions with helix alpha-5, which extends into the portal putative ATPase docking interface. Here, we have engineered intersubunit disulfide bridges between alpha-5s of adjacent portal ring subunits. Such covalent constraint blocked DNA packaging, whereas reduction of the disulfide bridges restored normal packaging activity. DNA exit through the portal in SPP1 virions was unaffected. The data demonstrate that mobility between alpha-5 helices is essential for the mechanism of viral DNA translocation. We propose that the alpha-5 structural rearrangements serve to coordinate ATPase activity with the positions of portal tunnel loops relative to the DNA double helix.     

Direct Interaction of the Bacteriophage SPP1 Packaging ATPase with the Portal Protein

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.     

Role of gene 10 protein in the hierarchical assembly of the bacteriophage P22 portal vertex structure

The portal vertex structure of the phage P22 is a 2.8 MDa molecular machine that mediates attachment and injection of the viral genome into the host Salmonella enterica serovar Typhimurium. Five proteins form this molecular machine: the portal protein, gp1; the tail-spike, gp9; the tail-needle, gp26, and the tail accessory factors, gp4 and gp10. In order to understand the assembly of the portal vertex structure, we have isolated the gene encoding tail accessory factor gp10 and defined its structural composition and assembly within the portal vertex structure. In solution, monomeric gp10 is a beta-sheet-rich protein with a stable conformational structure, which spontaneously assembles into hexamers, likely via a dimeric intermediate. This oligomerization enhances the structural stability of the protein, which then becomes competent for assembly to a preformed portal protein:gp4 complex, and acts as a structural adaptor bridging the nascent phage tail to gp26 and gp9. Notably, in vitro purified tail accessory factors gp4, gp10, and gp26 do not significantly interact with each other in solution, but their assembly takes place efficiently when these factors are added sequentially onto an immobilized portal protein. This suggests that the assembly of the P22 tail is a highly sequential and cooperative process, likely mediated by structural rearrangements in the assembly components. The assembled portal vertex structure represents both a membrane-binding and penetrating device as well as a plug that retains the pressurized phage DNA inside the capsid.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.     

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.     

Modulation of the viral ATPase activity by the portal protein correlates with DNA packaging efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.     

The high-resolution functional map of bacteriophage SPP1 portal protein

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.     

Gal repressosome contains an antiparallel DNA loop

Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA.     

The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing to Mg(II)-mediated oligomerization and biological function

During the late stages of lambda bacteriophage assembly, the protein gpU terminates tail polymerization and participates at the interface between the mature capsid and tail components. When it engages the lambda tail, gpU undergoes a monomer-hexamer transition to achieve its biologically active form. Towards understanding how gpU participates in multiple protein-protein interactions, we have solved the structure of gpU in its monomeric state using NMR methods. The structure reveals a mixed alpha/beta motif with several dynamic loops at the periphery. Addition of 20 mM MgCl(2) is known to oligomerize gpU in the absence of its protein partners. Multiple image analysis of electron micrographs revealed ring-like structures of magnesium ion saturated gpU with a 30 A pore, consistent with its function as a portal for the passage of viral DNA into the host bacterium. The ability of magnesium ions to promote oligomerization was lost when substitutions were made at a cluster of acidic amino acids in the vicinity of helix alpha2 and the beta1-beta2 loop. Furthermore, substitutions at these sites abolished the biological activity of gpU.     

Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.     

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.     

Bacteriophage p22 portal vertex formation in vivo

Bacteriophage with double-stranded, linear DNA genomes package DNA into pre-assembled icosahedral procapsids through a unique vertex. The packaging vertex contains an oligomeric ring of a portal protein that serves as a recognition site for the packaging enzymes, a conduit for DNA translocation, and the site of tail attachment. Previous studies have suggested that the portal protein of bacteriophage P22 is not essential for shell assembly; however, when assembled in the absence of functional portal protein, the assembled heads are not active in vitro packaging assays. In terms of head assembly, this raises an interesting question: how are portal vertices defined during morphogenesis if their incorporation is not a requirement for head assembly? To address this, the P22 portal gene was cloned into an inducible expression vector and transformed into the P22 host Salmonella typhimurium to allow control of the dosage of portal protein during infections. Using pulse-chase radiolabeling, it was determined that the portal protein is recruited into virion during head assembly. Surprisingly, over-expression of the portal protein during wild-type P22 infection caused a dramatic reduction in the yield of infectious virus. The cause of this reduction was traced to two potentially related phenomena. First, excess portal protein caused aberrant head assembly resulting in the formation of T=7 procapsid-like particles (PLPs) with twice the normal amount of portal protein. Second, maturation of the PLPs was blocked during DNA packaging resulting in the accumulation of empty PLPs within the host. In addition to PLPs with normal morphology, smaller heads (apparently T=4) and aberrant spirals were also produced. Interestingly, maturation of the small heads was relatively efficient resulting in the formation of small mature particles that were tailed and contained a head full of DNA. These data suggest that incorporation of portal vertices into heads occurs during growth of the coat lattice at decision points that dictate head assembly fidelity.     

Initiation of P22 procapsid assembly in vivo

The procapsids of all double-stranded DNA phages have a unique portal vertex, which is the locus of DNA packaging and DNA injection. Procapsid assembly is also initiated at this vertex, which is defined by the presence of a cyclic dodecamer of the portal protein. Assembly of the procapsid shell of phage P22 requires the gene 5 coat protein and the gene 8 scaffolding protein. We report here that removal of gene product (gp) 1 portal protein of P22 by mutation does not slow the rate of polymerization of coat and scaffolding subunits into shells, indicating that the portal ring is dispensable for shell initiation. Mutant scaffolding subunits specified by tsU172 copolymerize with coat subunits into procapsids at restrictive temperature, and also correctly autoregulate their synthesis. However, the shell structures formed from the temperature-sensitive scaffolding subunits fail to incorporate the portal ring and the three minor DNA injection proteins. This mutation identifies a domain of the scaffolding protein specifically involved in organization of the portal vertex. The results suggest that it is a complex of the scaffolding protein that initiates procapsid assembly and organizes the portal ring.     

Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro

The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid. The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit. In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head. Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown. The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein. Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h. Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization. Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer. A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS. In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.     

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.     

Overexpression of miR-128 specifically inhibits the truncated isoform of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells

Background

The CTCF insulator protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. The protein has been hypothesized to organize the human genome by forming DNA loops.

Results

In this paper, we report biochemical evidence to support the role for CTCF in forming DNA loops. We have measured DNA bending by CTCF at the chicken HS4 β-globin FII insulator element in vitro and have observed a unique DNA structure with aberrant electrophoretic mobility which we believe to be a DNA loop. CTCF is able to form this unusual DNA structure at two other binding sites: the c-myc P2 promoter and the chicken F1 lysozyme gene silencer. We also demonstrate that the length though not the sequence of the DNA downstream of the binding site is important for the ability of CTCF to form this unusual DNA structure. We hypothesize that a single CTCF protein molecule is able to act as a "looper" possibly through the use of several of its zinc fingers.

Conclusions

CTCF is able to form an unusual DNA structure through the zinc finger domain of the protein. This unusual DNA structure is formed in a directional manner by the CTCF protein. The findings described in this paper suggest mechanisms by which CTCF is able to form DNA loops, organize the mammalian genome and function as an insulator protein.     

Structural roles of subunit cysteines in the folding and assembly of the DNA packaging machine (portal) of bacteriophage P22

The DNA packaging machine (portal assembly) of bacteriophage P22 is constructed from 12 copies of a multidomain 725-residue subunit comprising a complex alpha/beta fold. The portal subunit contains four cysteines (Cys 153, Cys 173, Cys 283, and Cys 516), which produce distinctive Raman markers in the spectral interval 2500-2600 cm(-1) originating from S-H bond-stretching vibrations diagnostic of S-H...X hydrogen-bonding interactions. The Raman spectrum is unique in the capability to characterize cysteine sulfhydryl interactions in proteins and shows that portal cysteine environments are significantly altered by assembly (Rodriguez-Casado et al. (2001) Biochemistry 40, 13583-13591). We have employed site-directed mutagenesis, size-exclusion chromatography, and Raman difference spectroscopy to characterize the roles of portal cysteines in subunit folding and dodecamer assembly. The stability of the portal monomer is severely reduced by a Cys --> Ser point mutation introduced at either residue 173 or 516. In the case of C516S, the destabilized monomer still forms portal rings, as visualized by negative-stain electron microscopy, whereas portal ring formation cannot be detected for C173S, which forms aberrant aggregates. The C283S mutant is a hyperstable monomer that is defective in portal ring formation. Interestingly, Cys 283 is suggested by secondary structure homology with the phi29 portal to be within a domain involved in DNA translocation. Conversely, the phenotype of the C153S mutant is close to that of the wild-type protein, implying that the sulfhydryl moiety of Cys 153 is not essential to formation of the native subunit fold and productive assembly dynamics. The present results demonstrate that cysteines of the P22 portal protein span a wide range of sulfhydryl hydrogen-bonding strengths in the wild-type assembly, that three of the four sulfhydryls play key roles in portal protein stability and assembly kinetics, and that substitution of a mutant seryl interaction (O-H...X) for a wild-type cysteinyl interaction (S-H...X) can either stabilize or destabilize the native fold depending upon sequence context.     

A P22 scaffold protein mutation increases the robustness of head assembly in the presence of excess portal protein

Bacteriophage with linear, double-stranded DNA genomes package DNA into preassembled protein shells called procapsids. Located at one vertex in the procapsid is a portal complex composed of a ring of 12 subunits of portal protein. The portal complex serves as a docking site for the DNA packaging enzymes, a conduit for the passage of DNA, and a binding site for the phage tail. An excess of the P22 portal protein alters the assembly pathway of the procapsid, giving rise to defective procapsid-like particles and aberrant heads. In the present study, we report the isolation of escape mutant phage that are able to replicate more efficiently than wild-type phage in the presence of excess portal protein. The escape mutations all mapped to the same phage genome segment spanning the portal, scaffold, coat, and open reading frame 69 genes. The mutations present in five of the escape mutants were determined by DNA sequencing. Interestingly, each mutant contained the same mutation in the scaffold gene, which changes the glycine at position 287 to glutamate. This mutation alone conferred an escape phenotype, and the heads assembled by phage harboring only this mutation had reduced levels of portal protein and exhibited increased head assembly fidelity in the presence of excess portal protein. Because this mutation resides in a region of scaffold protein necessary for coat protein binding, these findings suggest that the P22 scaffold protein may define the portal vertices in an indirect manner, possibly by regulating the fidelity of coat protein polymerization.     

Peering down the barrel of a bacteriophage portal: the genome packaging and release valve in p22

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 ? resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et?al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 ?, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins.     

A conformational switch in bacteriophage p22 portal protein primes genome injection

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.