Maturation of phage T7 involves structural modification of both shell and inner core components

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.     

Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.     

DNA packaging of bacteriophage T4 proheads in vitro. Evidence that prohead expansion is not coupled to DNA packaging

We developed a system for DNA packaging of isolated bacteriophage T4 proheads in vitro and studied the role of prohead expansion in DNA packaging. Biologically active proheads have been purified from a number of packaging-deficient mutant extracts. The cleaved mature prohead is the active structural precursor for the DNA packaging reaction. Packaging of proheads requires ATP, Mg2+ and spermidine, and is stimulated by polyethylene glycol and dextran. Predominantly expanded proheads (ELPs) are produced at 37 degrees C and predominantly unexpanded proheads (ESPs) are produced at 20 degrees C. Both the expanded and unexpanded proheads are active in DNA packaging in vitro. This is based on the observations that (1) both ESPs and ELPs purified by chromatography on DEAE-Sephacel showed DNA packaging activity; (2) apparently homogeneous ELPs prepared by treatment with sodium dodecyl sulfate (which dissociates ESPs) retained significant biological activity; (3) specific precipitation of ELPs with anti-hoc immunoglobulin G resulted in loss of DNA packaging activity; and (4) ESPs upon expansion in vitro to ELPs retained packaging activity. Therefore, contrary to the models that couple DNA packaging to head expansion, in T4 the expansion and packaging appear to be independent, since the already expanded DNA-free proheads can be packaged in vitro. We therefore propose that the unexpanded to expanded prohead transition has evolved to stabilize the capsid and to reorganize the prohead shell functionally from a core-interacting to a DNA-interacting inner surface.     

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.     

Characterization of the bacteriophage T3 DNA packaging reaction in vitro in a defined system

The bacteriophage T3 DNA packaging system in vitro defined here is composed of purified proheads and two non-capsid proteins, the products of genes 18 and 19 (gp18 and gp19). In this system, a precursor complex (50 S complex) accumulates in the presence of adenosine 5'-O-(3'-thiotriphosphate) (ATP-gamma-S), a non-hydrolyzable analog of ATP. The 50 S complex is converted to a filled head in the presence of ATP. The conversion of the 50 S complex, formed by preincubation with ATP-gamma-S, to the mature head proceeds in a synchronous manner after the addition of ATP. The lag time for formation of mature heads from the 50 S complex is 1.8, 4.5 and 6.8 minutes at 30, 25 and 20 degrees C, respectively. DNA is translocated into the capsid at a constant rate of 5.7 x 10(3) base-pairs per minute at 20 degrees C. The conversion of the 50 S complex to the mature head exhibits a sigmoidal relationship with respect to the concentration of ATP, the concentration for half-maximal activity being about 20 microM. The transition of the prohead to the expanded capsid occurs at 20 degrees C at one minute 40 seconds after the initiation of DNA translocation, when one-fourth of the genome has been packaged into a prohead. At the same time, the capsid-DNA complex becomes stable to high concentrations of salt. When DNA translocation is interrupted by the addition of ATP-gamma-S, packaged DNA exists at 0 degrees C as well as at 20 degrees C but the exit of DNA stops after one-third of the genome is inside the capsid. After exit, DNA is retranslocated into the expanded capsid by the addition of ATP at a rate of about 5.7 x 10(3) base-pairs per minute at 20 degrees C. The decrease in concentration of ATP interrupts DNA translocation into the capsid but does not induce DNA exit. Interrupted DNA translocation may be reinitiated by the addition of ATP. DNA exit is not induced by the addition of ATP-gamma-S to mature heads or partially filled heads pretreated with DNase.     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.     

The role of capsid maturation on adenovirus priming for sequential uncoating

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.     

Maturation in Action: CryoEM Study of a Viral Capsid Caught during Expansion

Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate before any crosslink formation. This form displays 6-fold symmetric hexamers, but capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that coat subunit strain release acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions.     

Ion etching of bacteriophage lambda: evidence that the right end of the DNA is located at the outside of the phage DNA mass.

Bacteriophage lambda was etched in an Ar+ plasma under conditions in which the capsid and some of the DNA were eroded (by sputtering) from the particle surface. Analysis of the DNA remaining in etched phage demonstrated an enrichment in sequences derived from the left end and middle of the genome; sequences from the right end were selectively lost. The results suggest that the DNA in the mature phage is arranged with its left end toward the center and its right end toward the exterior of the overall DNA mass. Since the left end is the first to enter the phage prohead, the results are most compatible with the view that prohead filling also proceeds from the center to the exterior of the cavity. The suggested arrangement of lambda DNA is comparable to that observed in phage T4 and is consistent with the spiral-fold model of packaged DNA.     

Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein

The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions.     

An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.     

Double-stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease

Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus-encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.     

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.     

Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors

The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20, 40 and 20 + 40 were cloned into expression vectors under lambda pL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The data suggest that a specific gp20-gp40-membrane insertion structure constitutes the T4 prohead assembly initiation complex.     

Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.     

Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼ 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼ 10^− 4 of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T = 7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼ 15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (< 28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.