DNA Heats Up: Energetics of Genome Ejection from Phage Revealed by Isothermal Titration Calorimetry

Most bacteriophages are known to inject their double-stranded DNA into bacteria upon receptor binding in an essentially spontaneous way. This downhill thermodynamic process from the intact virion to the empty viral capsid plus released DNA is made possible by the energy stored during active packaging of the genome into the capsid. Only indirect measurements of this energy have been available until now, using either single-molecule or osmotic suppression techniques. In this work, we describe for the first time the use of isothermal titration calorimetry to directly measure the heat released (or, equivalently, the enthalpy) during DNA ejection from phage λ, triggered in solution by a solubilized receptor. Quantitative analyses of the results lead to the identification of thermodynamic determinants associated with DNA ejection. The values obtained were found to be consistent with those previously predicted by analytical models and numerical simulations. Moreover, the results confirm the role of DNA hydration in the energetics of genome confinement in viral capsids.     

Osmotic pressure: resisting or promoting DNA ejection from phage?

Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.     

Bacteriophage T5 DNA ejection under pressure

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for λ and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and λ, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.     

Real-time imaging of DNA ejection from single phage particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.     

Is the In?Vitro Ejection of Bacteriophage DNA Quasistatic? A Bulk to Single Virus Study

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection—binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.     

Pressure built by DNA packing inside virions: enough to drive DNA ejection in vitro, largely insufficient for delivery into the bacterial cytoplasm

Tailed bacteriophage particles carry DNA highly pressurized inside the capsid. Challenge with their receptor promotes release of viral DNA. We show that addition of the osmolyte polyethylene glycol (PEG) has two distinct effects in bacteriophage SPP1 DNA ejection. One effect is to inhibit the trigger for DNA ejection. The other effect is to exert an osmotic pressure that controls the extent of DNA released in phages that initiate ejection. We carried out independent measurements of each effect, which is an essential requirement for their quantitative study. The fraction of phages that do not eject increased linearly with the external osmotic pressure. In the remaining phage particles ejection stopped after a defined amount of DNA was reached inside the capsid. Direct measurement of the size of non-ejected DNA by gel electrophoresis at different PEG concentrations in the latter sub-population allowed determination of the external osmotic pressure that balances the force powering DNA exit (47 atm for SPP1 wild-type). DNA exit stops when the ejection force mainly due to repulsion between DNA strands inside the SPP1 capsid equalizes the force resisting DNA insertion into the PEG solution. Considering the turgor pressure in the Bacillus subtilis cytoplasm the energy stored in the tight phage DNA packing is only sufficient to power entry of the first 17% of the SPP1 chromosome into the cell, the remaining 83% requiring application of additional force for internalization.     

Forces during bacteriophage DNA packaging and ejection

The conjunction of insights from structural biology, solution biochemistry, genetics, and single-molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes. One area that has been a beneficiary of these experimental techniques is the study of viruses. In this article we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle. We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome. Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection. The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure.     

Effects of salt concentrations and bending energy on the extent of ejection of phage genomes

Recent work has shown that pressures inside dsDNA phage capsids can be as high as many tens of atmospheres; it is this pressure that is responsible for initiation of the delivery of phage genomes to host cells. The forces driving ejection of the genome have been shown to decrease monotonically as ejection proceeds, and hence to be strongly dependent on the genome length. Here we investigate the effects of ambient salts on the pressures inside phage-λ, for the cases of mono-, di-, and tetravalent cations, and measure how the extent of ejection against a fixed osmotic pressure (mimicking the bacterial cytoplasm) varies with cation concentration. We find, for example, that the ejection fraction is halved in 30 mM Mg²⁺ and is decreased by a factor of 10 upon addition of 1 mM spermine. These effects are calculated from a simple model of genome packaging, using DNA-DNA repulsion energies as determined independently from x-ray diffraction measurements on bulk DNA solutions. By comparing the measured ejection fractions with values implied from the bulk DNA solution data, we predict that the bending energy makes the d-spacings inside the capsid larger than those for bulk DNA at the same osmotic pressure.     

Cryomicroscopy of human cytomegalovirus virions reveals more densely packed genomic DNA than in herpes simplex virus type 1

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic beta-herpesvirus) has a genome that is significantly larger (>50 %) than that of the alpha-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1.We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 A. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 A. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome.     

Dynamics of DNA ejection from bacteriophage

The ejection of DNA from a bacterial virus (i.e., phage) into its host cell is a biologically important example of the translocation of a macromolecular chain along its length through a membrane. The simplest mechanism for this motion is diffusion, but in the case of phage ejection a significant driving force derives from the high degree of stress to which the DNA is subjected in the viral capsid. The translocation is further sped up by the ratcheting and entropic forces associated with proteins that bind to the viral DNA in the host cell cytoplasm. We formulate a generalized diffusion equation that includes these various pushing and pulling effects and make estimates of the corresponding speedups in the overall translocation process. Stress in the capsid is the dominant factor throughout early ejection, with the pull due to binding particles taking over at later stages. Confinement effects are also investigated, in the case where the phage injects its DNA into a volume comparable to the capsid size. Our results suggest a series of in vitro experiments involving the ejection of DNA into vesicles filled with varying amounts of binding proteins from phage whose state of stress is controlled by ambient salt conditions or by tuning genome length.     

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. “Decoration” proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to ∼ 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage λ as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage λ capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to “idle” at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-γS only partially stabilizes the nucleocapsid, and a DNA is released in “quantized” steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.     

Uncoating the herpes simplex virus genome

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.     

Dynamics of bacteriophage genome ejection in vitro and in vivo

Bacteriophages, phages for short, are viruses of bacteria. The majority of phages contain a double-stranded DNA genome packaged in a capsid at a density of ～500 mg ml(-1). This high density requires substantial compression of the normal B-form helix, leading to the conjecture that DNA in mature phage virions is under significant pressure, and that pressure is used to eject the DNA during infection. A large number of theoretical, computer simulation and in vitro experimental studies surrounding this conjecture have revealed many--though often isolated and/or contradictory--aspects of packaged DNA. This prompts us to present a unified view of the statistical physics and thermodynamics of DNA packaged in phage capsids. We argue that the DNA in a mature phage is in a (meta)stable state, wherein electrostatic self-repulsion is balanced by curvature stress due to confinement in the capsid. We show that in addition to the osmotic pressure associated with the packaged DNA and its counterions, there are four different pressures within the capsid: pressure on the DNA, hydrostatic pressure, the pressure experienced by the capsid and the pressure associated with the chemical potential of DNA ejection. Significantly, we analyze the mechanism of force transmission in the packaged DNA and demonstrate that the pressure on DNA is not important for ejection. We derive equations showing a strong hydrostatic pressure difference across the capsid shell. We propose that when a phage is triggered to eject by interaction with its receptor in vitro, the (thermodynamic) incentive of water molecules to enter the phage capsid flushes the DNA out of the capsid. In vivo, the difference between the osmotic pressures in the bacterial cell cytoplasm and the culture medium similarly results in a water flow that drags the DNA out of the capsid and into the bacterial cell.     

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.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

Major capsid reinforcement by a minor protein in herpesviruses and phage

Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres. Our data reveal that the loosely formed capsid structure is reinforced post-assembly by the minor capsid protein UL25 in HSV-1 and gpD in bacteriophage λ. Using atomic force microscopy nano-indentation analysis, we show that the capsid becomes stiffer upon binding of UL25 and gpD due to increased structural stability. At the same time the force required to break the capsid increases by ∼70% for both herpes and phage. This demonstrates a universal and evolutionarily conserved function of the minor capsid protein: facilitating the retention of the pressurized viral genome in the capsid. Since all eight human herpesviruses have UL25 orthologs, this discovery offers new opportunities to interfere with herpes replication by disrupting the precise force balance between the encapsidated DNA and the capsid proteins crucial for viral replication.     

Ion-dependent DNA configuration in bacteriophage capsids

Bacteriophages densely pack their long double-stranded DNA genome inside a protein capsid. The conformation of the viral genome inside the capsid is consistent with a hexagonal liquid crystalline structure. Experiments have confirmed that the details of the hexagonal packing depend on the electrochemistry of the capsid and its environment. In this work, we propose a biophysical model that quantifies the relationship between DNA configurations inside bacteriophage capsids and the types and concentrations of ions present in a biological system. We introduce an expression for the free energy that combines the electrostatic energy with contributions from bending of individual segments of DNA and Lennard-Jones-type interactions between these segments. The equilibrium points of this energy solve a partial differential equation that defines the distributions of DNA and the ions inside the capsid. We develop a computational approach that allows us to simulate much larger systems than what is possible using the existing molecular-level methods. In particular, we are able to estimate bending and repulsion between the DNA segments as well as the full electrochemistry of the solution, both inside and outside of the capsid. The numerical results show good agreement with existing experiments and with molecular dynamics simulations for small capsids.     

DNA Ejection from an Archaeal Virus—A Single-Molecule Approach

The translocation of genetic material from the viral capsid to the cell is an essential part of the viral infection process. Whether the energetics of this process is driven by the energy stored within the confined nucleic acid or cellular processes pull the genome into the cell has been the subject of discussion. However, in vitro studies of genome ejection have been limited to a few head-tailed bacteriophages with a double-stranded DNA genome. Here we describe a DNA release system that operates in an archaeal virus. This virus infects an archaeon Haloarcula hispanica that was isolated from a hypersaline environment. The DNA-ejection velocity of His1, determined by single-molecule experiments, is comparable to that of bacterial viruses. We found that the ejection process is modulated by the external osmotic pressure (polyethylene glycol (PEG)) and by increased ion (Mg²⁺ and Na⁺) concentration. The observed ejection was unidirectional, randomly paused, and incomplete, which suggests that cellular processes are required to complete the DNA transfer.     

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.     

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.