Flexible structural arrangement and DNA-binding properties of protein p6 from Bacillus subtillis phage φ29.

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.

Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment.

Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

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.

Retained Stability of the RNA Structure in DNA Packaging Motor with a Single Mg2+ Ion Bound at the Double Mg-Clamp Structure.

It has been known that the binding of two Mg2+ ions to the phosphate groups of RNA is essential for enhancing the stability of a three-way junction oligomeric prohead RNA (3WJ-pRNA). Here, we investigate the coupling between binding of two Mg2+ ions and the stability of the complex structure by calculating the rupture force of 3WJ-pRNA using steered molecular dynamics simulations. Our simulations showed that the mechanical stability of the pRNA structure is largely retained with a single Mg2+ ion bound. Because of a strong electrostatic repulsion between the adjacent Mg2+ ions, the rupture force with two Mg2+ ions bound is smaller than the sum of the rupture forces with either one of Mg2+ ions bound. Our results suggest that binding of the second Mg2+ ion could be redundant for maintaining the rigidity of 3WJ-pRNA, which allows 3WJ-pRNA to have a flexible working range of Mg2+ concentration in a cell.

Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein.

Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module, here we identified eight gene products that attenuated B. subtilis growth. Of the eight phage gene products that attenuated bacterial growth, a 25-kDa protein called Gp53 was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis Our results further reveal that Gp53 is a phage-encoded adaptor-like protein that modulates the activity of the ClpCP protease to enable efficient SPO1 phage progeny development. In summary, our findings indicate that the bacterial ClpCP protease is the target of xenogeneic (dys)regulation by a SPO1 phage-derived factor and add Gp53 to the list of antibacterial products that target bacterial protein degradation and therefore may have utility for the development of novel antibacterial agents.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Permeability modes in fluctuating lipid membranes with DNA-translocating pores.

Membrane pores can significantly alter not only the permeation dynamics of biological membranes but also their elasticity. Large membrane pores able to transport macromolecular contents represent an interesting model to test theoretical predictions that assign active-like (non-equilibrium) behavior to the permeability contributions to the enhanced membrane fluctuations existing in permeable membranes [Maneville et al. Phys. Rev. Lett. 82, 4356 (1999)]. Such high-amplitude active contributions arise from the forced transport of solvent and solutes through the open pores, which becomes even dominant at large permeability. In this paper, we present a detailed experimental analysis of the active shape fluctuations that appear in highly permeable lipid vesicles with large macromolecular pores inserted in the lipid membrane, which are a consequence of transport permeability events occurred in an osmotic gradient. The experimental results are found in quantitative agreement with theory, showing a remarkable dependence with the density of membrane pores and giving account of mechanical compliances and permeability rates that are compatible with the large size of the membrane pore considered. The presence of individual permeation events has been detected in the fluctuation time-series, from which a stochastic distribution of the permeation events compatible with a shot-noise has been deduced. The non-equilibrium character of the membrane fluctuations in a permeation field, even if the membrane pores are mere passive transporters, is clearly demonstrated. Finally, a bio-nano-technology outlook of the proposed synthetic concept is given on the context of prospective uses as active membrane DNA-pores exploitable in gen-delivery applications based on lipid vesicles.

Directional mechanical stability of Bacteriophage φ29 motor's 3WJ-pRNA: Extraordinary robustness along portal axis.

The molecular motor exploited by bacteriophage φ29 to pack DNA into its capsid is regarded as one of the most powerful mechanical devices present in viral, bacterial, and eukaryotic systems alike. Acting as a linker element, a prohead RNA (pRNA) effectively joins the connector and ATPase (adenosine triphosphatase) components of the φ29 motor. During DNA packing, this pRNA needs to withstand enormous strain along the capsid's portal axis-how this remarkable stability is achieved remains to be elucidated. We investigate the mechanical properties of the φ29 motor's three-way junction (3WJ)-pRNA using a combined steered molecular dynamics and atomic force spectroscopy approach. The 3WJ exhibits strong resistance to stretching along its coaxial helices, demonstrating its super structural robustness. This resistance disappears, however, when external forces are applied to the transverse directions. From a molecular standpoint, we demonstrate that this direction-dependent stability can be attributed to two Mg clamps that cooperate and generate mechanical resistance in the pRNA's coaxial direction. Our results suggest that the asymmetric nature of the 3WJ's mechanical stability is entwined with its biological function: Enhanced rigidity along the portal axis is likely essential to withstand the strain caused by DNA condensation, and flexibility in other directions should aid in the assembly of the pRNA and its association with other motor components.

The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region.

Bacteriophage endolysin is one of the most promising antibiotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidoglycans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in catalytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall binding domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to obtain an engineered lysin that can lyse bacteria from the outside. Our results showed that lysep3-D8 could lyse both Gramnegative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 µg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bactericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 µg/ml. The cell wall binding domain of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic domain to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

Thermodynamic stabilities of three-way junction nanomotifs in prohead RNA.

The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure-function hypotheses and guide the rational design of functional RNA supramolecular assemblies.

Three-step channel conformational changes common to DNA packaging motors of bacterial viruses T3, T4, SPP1, and Phi29.

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for the genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the "One-way revolving" mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. The data suggest that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation for DNA movement in the reverse direction during ejection.

The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration.

Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Šcrystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction.

Complete genome sequence of the cold-active bacteriophage VMY22 from Bacillus cereus.

The cold-active bacteriophage VMY22, belonging to the Podoviridae family, was isolated from Mingyong Glacier in China. Sequence analysis revealed that the genome is 18,609 bp long, with an overall G + C content of 36.4 mol%, and 25 open reading frames (ORFs). The sequence contains 46 potential promoters, 6 transcription terminators, and no tRNAs. Most of the ORFs show a high degree of similarity to B103 (NC_004165). Two noteworthy findings were made. First, one of the predicted proteins, ORF 19, shows high sequence similarity to the bacteriocin biosynthesis protein from Bacillus cereus. From this information, we propose that the VMY22 phage is at an intermediate phase in its coevolution with its bacterial host. Second, seven of the hypothetical proteins appear to be unique to this cold-active B. cereus phage (i.e., not found in temperate-active B. cereus phages). These observations add to our current knowledge about the coevolution of bacteriophages and their hosts. The identification of a novel group of gene and protein structures and functions will lead to a better understanding of cold-adaptation mechanisms in bacteria and their bacteriophages.

Quantitative nanoscale electrostatics of viruses.

Electrostatics is one of the fundamental driving forces of the interaction between biomolecules in solution. In particular, the recognition events between viruses and host cells are dominated by both specific and non-specific interactions and the electric charge of viral particles determines the electrostatic force component of the latter. Here we probe the charge of individual viruses in liquid milieu by measuring the electrostatic force between a viral particle and the Atomic Force Microscope tip. The force spectroscopy data of co-adsorbed ϕ29 bacteriophage proheads and mature virions, adenovirus and minute virus of mice capsids is utilized for obtaining the corresponding density of charge for each virus. The systematic differences of the density of charge between the viral particles are consistent with the theoretical predictions obtained from X-ray structural data. Our results show that the density of charge is a distinguishing characteristic of each virus, depending crucially on the nature of the viral capsid and the presence/absence of the genetic material.

RNA Nanoparticles Derived from Three-Way Junction of Phi29 Motor pRNA Are Resistant to I-125 and Cs-131 Radiation.

Radiation reagents that specifically target tumors are in high demand for the treatment of cancer. The emerging field of RNA nanotechnology might provide new opportunities for targeted radiation therapy. This study investigates whether chemically modified RNA nanoparticles derived from the packaging RNA (pRNA) three-way junction (3WJ) of phi29 DNA-packaging motor are resistant to potent I-125 and Cs-131 radiation, which is a prerequisite for utilizing these RNA nanoparticles as carriers for targeted radiation therapy. pRNA 3WJ nanoparticles were constructed and characterized, and the stability of these nanoparticles under I-125 and Cs-131 irradiation with clinically relevant doses was examined. RNA nanoparticles derived from the pRNA 3WJ targeted tumors specifically and they were stable under irradiation of I-125 and Cs-131 with clinically relevant doses ranging from 1 to 90 Gy over a significantly long time up to 20 days, while control plasmid DNA was damaged at 20 Gy or higher.

Continuous allosteric regulation of a viral packaging motor by a sensor that detects the density and conformation of packaged DNA.

We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼ 70% and then rises sharply to ∼ 23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.

The collagen-like protein gp12 is a temperature-dependent reversible binder of SPP1 viral capsids.

Icosahedral capsids of viruses are lattices of defined geometry and homogeneous size. The (quasi-)equivalent organization of their protein building blocks provides, in numerous systems, the binding sites to assemble arrays of viral polypeptides organized with nanometer precision that protrude from the capsid surface. The capsid of bacterial virus (bacteriophage) SPP1 exposes, at its surface, the 6.6-kDa viral polypeptide gp12 that binds to the center of hexamers of the major capsid protein. Gp12 forms an elongated trimer with collagen-like properties. This is consistent with the fold of eight internal GXY repeats of gp12 to build a stable intersubunit triple helix in a prokaryotic setting. The trimer dissociates and unfolds at near physiological temperatures, as reported for eukaryotic collagen. Its structural organization is reacquired within seconds upon cooling. Interaction with the SPP1 capsid hexamers strongly stabilizes gp12, increasing its Tm to 54 °C. Above this temperature, gp12 dissociates from its binding sites and unfolds reversibly. Multivalent binding of gp12 trimers to the capsid is highly cooperative. The capsid lattice also provides a platform to assist folding and association of unfolded gp12 polypeptides. The original physicochemical properties of gp12 offer a thermoswitchable system for multivalent binding of the polypeptide to the SPP1 capsid surface.

Kinetic mechanism at the branchpoint between the DNA synthesis and editing pathways in individual DNA polymerase complexes.

Exonucleolytic editing of incorrectly incorporated nucleotides by replicative DNA polymerases (DNAPs) plays an essential role in the fidelity of DNA replication. Editing requires that the primer strand of the DNA substrate be transferred between the DNAP polymerase and exonuclease sites, separated by a distance that is typically on the order of ~30 Å. Dynamic transitions between functional states can be quantified with single-nucleotide spatial precision and submillisecond temporal resolution from ionic current time traces recorded when individual DNAP complexes are held atop a nanoscale pore in an electric field. In this study, we have exploited this capability to determine the kinetic relationship between the translocation step and primer strand transfer between the polymerase and exonuclease sites in complexes formed between the replicative DNAP from bacteriophage Φ29 and DNA. We demonstrate that the pathway for primer strand transfer from the polymerase to exonuclease site initiates prior to the translocation step, while complexes are in the pre-translocation state. We developed a mathematical method to determine simultaneously the forward and reverse translocation rates and the rates of primer strand transfer in both directions between the polymerase and the exonuclease sites, and we have applied it to determine these rates for Φ29 DNAP complexes formed with a DNA substrate bearing a fully complementary primer-template duplex. This work provides a framework that will be extended to determine the kinetic mechanisms by which incorporation of noncomplementary nucleotides promotes primer strand transfer from the polymerase site to the exonuclease site.

Insights into the structure and assembly of the bacteriophage 29 double-stranded DNA packaging motor.

UNLABELLED: The tailed double-stranded DNA (dsDNA) bacteriophage 29 packages its 19.3-kbp genome into a preassembled procapsid structure by using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near-crystalline densities within the confined space of the capsid requires that the packaging motor work against significant entropic, enthalpic, and DNA-bending energies. The motor consists of three phage-encoded components: the dodecameric connector protein gp10, an oligomeric RNA molecule known as the prohead RNA (pRNA), and the homomeric ring ATPase gp16. Although atomic resolution structures of the connector and different pRNA subdomains have been determined, the mechanism of self-assembly and the resulting stoichiometry of the various motor components on the phage capsid have been the subject of considerable controversy. Here a subnanometer asymmetric cryoelectron microscopy (cryo-EM) reconstruction of a connector-pRNA complex at a unique vertex of the procapsid conclusively demonstrates the pentameric symmetry of the pRNA and illuminates the relative arrangement of the connector and the pRNA. Additionally, a combination of biochemical and cryo-EM analyses of motor assembly intermediates suggests a sequence of molecular events that constitute the pathway by which the motor assembles on the head, thereby reconciling conflicting data regarding pRNA assembly and stoichiometry. Taken together, these data provide new insight into the assembly, structure, and mechanism of a complex molecular machine. IMPORTANCE: Viruses consist of a protein shell, or capsid, that protects and surrounds their genetic material. Thus, genome encapsidation is a fundamental and essential step in the life cycle of any virus. In dsDNA viruses, powerful molecular motors essentially pump the viral DNA into a preformed protein shell. This article describes how a viral dsDNA packaging motor self-assembles on the viral capsid and provides insight into its mechanism of action.