Role of DNA-DNA sliding friction and nonequilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers. We measure extremely low initial exit velocity, a regime of exponentially increasing velocity, stochastic pausing that dominates the kinetics and large dynamic heterogeneity. Measurements with variable applied force provide evidence that the initial velocity is controlled by DNA-DNA sliding friction, consistent with a Frenkel-Kontorova model for nanoscale friction. We confirm several aspects of the ejection dynamics predicted by theoretical models. Features of the pausing suggest that it is connected to the phenomenon of 'clogging' in soft matter systems. Our results provide evidence that DNA-DNA friction and clogging control the DNA exit dynamics, but that this friction does not significantly affect DNA packaging.

Role of DNA-DNA sliding friction and non-equilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers. We measure extremely low initial exit velocity, a regime of exponentially increasing velocity, stochastic pausing that dominates the kinetics, and large dynamic heterogeneity. Measurements with variable applied force provide evidence that the initial velocity is controlled by DNA-DNA sliding friction, consistent with a Frenkel-Kontorova model for nanoscale friction. We confirm several aspects of the ejection dynamics predicted by theoretical models. Features of the pausing suggest it is connected to the phenomenon of "clogging" in soft-matter systems. Our results provide evidence that DNA-DNA friction and clogging control the DNA exit dynamics, but that this friction does not significantly affect DNA packaging.

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

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.

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures.

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA.

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

Viral packaging ATPases utilize a glutamate switch to couple ATPase activity and DNA translocation.

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.

NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors.

Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

ATP/ADP modulates gp16-pRNA conformational change in the Phi29 DNA packaging motor.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA-gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a 'Z'-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

Slow and steady wins the race: physical limits on the rate of viral DNA packaging.

During the assembly of dsDNA viruses such as the tailed bacteriophages and herpesviruses, the viral chromosome is compacted to near crystalline density inside a preformed head shell. DNA translocation is driven by powerful ring ATPase motors that couple ATP binding, hydrolysis, and release to force generation and movement. Studies of the motor of the bacteriophage phi29 have revealed a complex mechanochemistry behind this process that slows as the head fills. Recent studies of the physical behavior of packaging DNA suggest that surprisingly long-time scales of relaxation of DNA inside the head and jamming phenomena during packaging create the physical need for regulation of the rate of packaging. Studies of DNA packaging in viral systems have, therefore, revealed fundamental insight into the complex behavior of DNA and the need for biological systems to accommodate these physical constraints.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

Experimental comparison of forces resisting viral DNA packaging and driving DNA ejection.

We compare forces resisting DNA packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from 0% to 80% filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation. Force resisting DNA packaging from ∼80% to 100% filling inferred from optical tweezers studies is also consistent with the predictions of this model. The striking agreement with these two different measurements suggests that the overall energetics of DNA packaging is well described by the model. However, since electron microscopy studies of phi29 do not reveal a spool conformation, our findings suggest that the spool model overestimates the role of bending rigidity and underestimates the role of intrastrand repulsion. Below ∼80% filling the inferred forces resisting packaging are unexpectedly lower than the inferred ejection forces, suggesting that in this filling range the forces are less accurately determined or strongly temperature dependent.

Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging.

In many viruses molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50-100 nm prohead shells1, 2. Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions3. Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems4-8. Experiments in which conditions are changed during packaging to switch DNA-DNA interactions between purely repulsive and net attractive reveal strongly history-dependent dynamics. An abrupt deceleration is usually observed before stalling, indicating that a transition in DNA conformation causes an abrupt increase in resistance. Our findings suggest that the concept of jamming can be extended to a single polymer molecule. However, compared with macroscopic samples of colloidal particles5 we find that single DNA molecules jam over a much larger range of densities. We attribute this difference to the nanoscale system size, consistent with theoretical predictions for jamming of attractive athermal particles.9, 10.

Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor.

Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

UNLABELLED: During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE: During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs.

Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging.

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.