Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

ATP serves as a nucleotide switch coupling the genome maturation and packaging motor complexes of a virus assembly machine.

The assembly of double-stranded DNA viruses, from phages to herpesviruses, is strongly conserved. Terminase enzymes processively excise and package monomeric genomes from a concatemeric DNA substrate. The enzymes cycle between a stable maturation complex that introduces site-specific nicks into the duplex and a dynamic motor complex that rapidly translocates DNA into a procapsid shell, fueled by ATP hydrolysis. These tightly coupled reactions are catalyzed by terminase assembled into two functionally distinct nucleoprotein complexes; the maturation complex and the packaging motor complex, respectively. We describe the effects of nucleotides on the assembly of a catalytically competent maturation complex on viral DNA, their effect on maturation complex stability and their requirement for the transition to active packaging motor complex. ATP plays a major role in regulating all of these activities and may serve as a 'nucleotide switch' that mediates transitions between the two complexes during processive genome packaging. These biological processes are recapitulated in all of the dsDNA viruses that package monomeric genomes from concatemeric DNA substrates and the nucleotide switch mechanism may have broad biological implications with respect to virus assembly mechanisms.

Haemolysin coregulated protein is an exported receptor and chaperone of type VI secretion substrates.

Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

Evidence that a catalytic glutamate and an 'Arginine Toggle' act in concert to mediate ATP hydrolysis and mechanochemical coupling in a viral DNA packaging motor.

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine 'toggles' between interacting with a glutamate residue in the 'lid' subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an 'open' state to a 'closed' state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.

Physical and Functional Characterization of a Viral Genome Maturation Complex.

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

Phage lambda capsids as tunable display nanoparticles.

Nanoparticle technologies provide a powerful tool for the development of reagents for use in both therapeutic and diagnostic, or "theragnostic" biomedical applications. Two broad classes of particles are under development, viral and synthetic systems, each with their respective strengths and limitations. Here we adapt the phage lambda system to construct modular "designer" nanoparticles that blend these two approaches. We have constructed a variety of modified "decoration" proteins that allow site-specific modification of the shell with both protein and nonproteinaceous ligands including small molecules, carbohydrates, and synthetic display ligands. We show that the chimeric proteins can be used to simultaneously decorate the shell in a tunable surface density to afford particles that are physically homogeneous and that can be manufactured to display a variety of ligands in a defined composition. These designer nanoparticles set the stage for development of lambda as a theragnostic nanoparticle system.

Lysine addressability and mammalian cell interactions of bacteriophage λ procapsids.

Chemically or genetically modified virus particles, termed viral nanoparticles (VNPs), are being explored in applications such as drug delivery, vaccine development, and materials science. Each virus platform has inherent properties and advantages based on its structure, molecular composition, and biomolecular interactions. Bacteriophage λ was studied for its lysine addressability, stability, cellular uptake, and the ability to modify its cellular uptake. λ procapsids could be labeled primarily at a single residue on the gpE capsid protein as determined by tandem mass spectrometry, providing a unique attachment site for further capsid modification. Bioconjugation of transferrin to the procapsids mediated specific interaction with transferrin receptor-expressing cells. These studies demonstrate the utility of bacteriophage λ procapsids and their potential use as targeted drug delivery vehicles.

Energy-independent helicase activity of a viral genome packaging motor.

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.

Characterization of a Primordial Major Capsid-Scaffolding Protein Complex in Icosahedral Virus Shell Assembly.

Capsid assembly pathways are strongly conserved in the complex dsDNA viruses, where major capsid proteins (MCP) self-assemble into icosahedral procapsid shells, chaperoned by a scaffolding protein. Without a scaffold, the capsid proteins aggregate and form aberrant structures. This, coupled with the rapid co-polymerization of MCP and scaffolding proteins, has thwarted characterization of the earliest steps in shell assembly. Here we interrogate the structure and biophysical properties of a soluble, assembly-deficient phage lambda major capsid protein, MCP(W308A). The mutant protein is folded, soluble to high concentrations and binds to the scaffolding protein in an apparent SP2:MCP(W308A)1 stoichiometry but does not assemble beyond this initiating complex. The MCP(W308A) crystal structure was solved to 2.7 Å revealing the canonical HK97 fold in a "pre-assembly" conformation featuring the conserved N-arm and E-loops folded into the body of the protein. Structural, biophysical and computational analyses suggest that MCP(W308A) is thermodynamically trapped in this pre-assembly conformation precluding self-association interactions required for shell assembly. A model is described wherein dynamic interactions between MCP proteins play an essential role in high fidelity viral shell assembly. Scaffold-chaperoned MCP polymerization is a strongly conserved process in all the large dsDNA viruses and our results provide insight into this primordial complex in solution and have broad biological significance in our understanding of virus assembly mechanisms.

A Designer Nanoparticle Platform for Controlled Intracellular Delivery of Bioactive Macromolecules: Inhibition of Ubiquitin-Specific Protease 7 in Breast Cancer Cells.

Biological therapeutics represent an increasing and critical component of newly approved drugs; however, the inability to deliver biologics intracellularly in a controlled manner remains a major limitation. We have developed a semi-synthetic, tunable phage-like particle (PLP) platform derived from bacteriophage λ. The shell surface can be decorated with small-molecule, biological and synthetic moieties, alone or in combination and in defined ratios. Here, we demonstrate that the platform can be used to deliver biological macromolecules intracellularly and in a controlled manner. Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that has been widely recognized as an ideal target for the treatment of a variety of cancers. Recently, UbV.7.2, a novel biologic derived from the ubiquitin scaffold, was developed for inhibition of USP7, but issues remain in achieving efficient and controlled intracellular delivery of the biologic. We have shown that decoration of PLPs with trastuzumab (Trz), a HER2-targeted therapeutic used in the treatment of various cancers, results in specific targeting and uptake of Trz-PLPs into HER2-overexpressing breast cancer cells. By simultaneously decorating PLPs with Trz and UbV.7.2, we now show that these particles are also internalized by HER2-positive cells, thus providing a means for intracellular delivery of the biologic in a controlled fashion. Internalized particles retain USP7 inhibition activity of UbV.7.2 and alter the metabolic and proteomic landscapes of these cells. This study demonstrates that the λ "designer nanoparticles" represent a powerful system for the intracellular delivery of biologics in a defined dose.

Atomic-Layer Deposition Processes Applied to Phage λ and a Phage-like Particle Platform Yield Thermostable, Single-Shot Vaccines.

Especially in developing countries, the impact of vaccines can be limited by logistical obstacles associated with multiple dose regimens, pathogen variants, and challenges imposed by requirements for maintaining vaccines at low temperatures during shipping and storage. Thus, there is a need for vaccines that can be flexibly modified to address evolving pathogen landscapes, are stable outside of narrow "cold-chain" temperatures and require administration of only single doses. Here we demonstrate in proof-of-concept studies a vaccine platform that addresses these impediments to more widespread use of vaccines. The platform relies on bacteriophage-derived phage-like-particles (PLPs) that utilize a "plug-and-play" antigen delivery system that allows for fast, easy alteration of antigens on the surface of the PLPs. Thermostability of PLP-based vaccines can be achieved by embedding the PLPs within glassy particles produced by spray drying, and nanoscopic aluminum oxide layers applied using atomic layer deposition (ALD) can serve to control release of antigen in vivo, yielding vaccine formulations that elicit strong immune responses after administration of single doses. Bacteriophage λ was stabilized by spray drying to form powders that were incubated at 37 °C for up to a year without loss of infectious activity. PLPs derived from bacteriophage λ were expressed and purified from E. coli cultures, and an in vitro conjugation strategy was used to decorate specific PLP surface sites with T4-lysozyme, a model vaccine antigen. The resulting T4-lysozyme:PLP complexes (Lys-PLPs) were embedded in glassy dry powders formed by spray drying and coated with nanometer-thick layers of alumina deposited by ALD in a fluidized bed reactor. Alumina-coated Lys-PLP vaccines were stable for a least a month at 50 °C, and single doses of the alumina-coated vaccines elicited immune responses that were indistinguishable from responses generated by conventional two-dose, prime-and-boost dosing regimens of alum-adjuvanted Lys-PLP vaccines.

Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease.

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the United States within 1 year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a "designer nanoparticle" platform using phage-like particles (PLPs) derived from bacteriophage lambda for a multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor-binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs and RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose-response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.

Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity.

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage lambda gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.

Viral genome packaging machines: Structure and enzymology.

Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging pathways that these viruses use to catalyze ATP-driven translocation of the viral genome into a preassembled procapsid shell. One pathway is used by ϕ29-like phages and adenoviruses, which replicate and subsequently package a monomeric, unit-length genome covalently attached to a virus/phage-encoded protein at each 5'-end of the dsDNA genome. In a second, more ubiquitous packaging pathway characterized by phage lambda and the herpesviruses, the viral DNA is replicated as multigenome concatemers linked in a head-to-tail fashion. Genome packaging in these viruses thus requires excision of individual genomes from the concatemer that are then translocated into a preassembled procapsid. Hence, the ATPases that power packaging in these viruses also possess nuclease activities that cut the genome from the concatemer at the beginning and end of packaging. This review focuses on proposed mechanisms of genome packaging in the dsDNA viruses using unit-length ϕ29 and concatemeric λ genome packaging motors as representative model systems.

Targeted Intracellular Delivery of Trastuzumab Using Designer Phage Lambda Nanoparticles Alters Cellular Programs in Human Breast Cancer Cells.

| Several diseases exhibit a high degree of heterogeneity and diverse reprogramming of cellular pathways. To address this complexity, additional strategies and technologies must be developed to define their scope and variability with the goal of improving current treatments. Nanomedicines derived from viruses are modular systems that can be easily adapted for combinatorial approaches, including imaging, biomarker targeting, and intracellular delivery of therapeutics. Here, we describe a "designer nanoparticle" system that can be rapidly engineered in a tunable and defined manner. Phage-like particles (PLPs) derived from bacteriophage lambda possess physiochemical properties compatible with pharmaceutical standards, and in vitro particle tracking and cell targeting are accomplished by simultaneous display of fluorescein-5-maleimide (F5M) and trastuzumab (Trz), respectively (Trz-PLPs). Trz-PLPs bind to the oncogenically active human epidermal growth factor receptor 2 (HER2) and are internalized by breast cancer cells of the HER2 overexpression subtype, but not by those lacking the HER2 amplification. Compared to treatment with Trz, robust internalization of Trz-PLPs results in higher intracellular concentrations of Trz, prolonged inhibition of cell growth, and modulated regulation of cellular programs associated with HER2 signaling, proliferation, metabolism, and protein synthesis. Given the implications to cancer pathogenesis and that dysregulated signaling and metabolism can lead to drug resistance and cancer cell survival, the present study identifies metabolic and proteomic liabilities that could be exploited by the PLP platform to enhance therapeutic efficacy. The lambda PLP system is robust and rapidly modifiable, which offers a platform that can be easily "tuned" for broad utility and tailored functionality.

Kinetic analysis of the genome packaging reaction in bacteriophage lambda.

Bacteriophage lambda is a double-stranded DNA virus that infects the Escherichia coli bacterium. lambda genomic DNA is replicated via rolling circle replication, resulting in multiple genomes linked head to tail at the cos site. To insert a single lambda genome into the viral capsid, the lambda terminase enzyme introduces symmetric nicks, 12 bp apart, at the cos site, and then promotes a strand separation reaction, releasing the tail end of the previous genome and leaving a binary complex consisting of lambda terminase bound to the head end of the adjacent genome. Next, the genome is translocated into the interior of the capsid particle, in a process that requires ATP hydrolysis by lambda terminase. Even though DNA packaging has been studied extensively, currently no bulk assays are available that have been optimized to report directly on DNA translocation. Rather, these assays are sensitive to assembly steps reflecting formation of the active, DNA packaging machine. In this work, we have modified the DNase protection assay commonly used to study DNA packaging in several bacteriophage systems, such that it reports directly on the kinetics of the DNA packaging reaction. We have analyzed our DNA packaging data according to an N-step sequential minimal kinetic model and have estimated an overall packaging rate of 119 +/- 8 bp/s, at 4 degrees C and 1 mM ATP. Furthermore, we have measured an apparent step size for the this reaction (m(obs)) of 410 +/- 150 bp. The magnitude of this value indicates that our assay is most likely sensitive to both mechanical steps associated with DNA insertion as well as occasional slow steps that are repeated every >410 bp. These slow steps may be reflective of the pausing events observed in recent single-molecule studies of DNA packaging in bacteriophage lambda [Fuller, D. N., et al. (2007) J. Mol. Biol. 373, 1113-1122]. Finally, we show that either ATP or ADP is required for terminase cutting at cos, to generate the active, DNA packaging complex.

Functional Dissection of a Viral DNA Packaging Machine's Walker B Motif.

Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.

The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity.

Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.

Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations.

Molecular motors drive genome packaging into preformed procapsids in many double-stranded (ds)DNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage lambda. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage phi29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged approximately 600 bp/s, which is 3.5-fold higher than phi29, and the motor processivity was also threefold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging, and much greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid, which lacks an accessory protein (gpD).

Bacteriophage lambda: The path from biology to theranostic agent.

Viral particles provide an attractive platform for the engineering of semisynthetic therapeutic nanoparticles. They can be modified both genetically and chemically in a defined manner to alter their surface characteristics, for targeting specific cell types, to improve their pharmacokinetic features and to attenuate (or enhance) their antigenicity. These advantages derive from a detailed understanding of virus biology, gleaned from decades of fundamental genetic, biochemical, and structural studies that have provided mechanistic insight into virus assembly pathways. In particular, bacteriophages offer significant advantages as nanoparticle platforms and several have been adapted toward the design and engineering of "designer" nanoparticles for therapeutic and diagnostic (theranostic) applications. The present review focuses on one such virus, bacteriophage lambda; I discuss the biology of lambda, the tools developed to faithfully recapitulate the lambda assembly reactions in vitro and the observations that have led to cooptation of the lambda system for nanoparticle design. This discussion illustrates how a fundamental understanding of virus assembly has allowed the rational design and construction of semisynthetic nanoparticles as potential theranostic agents and illustrates the concept of benchtop to bedside translational research. This article is categorized under: Biology-Inspired Nanomaterials> Protein and Virus-Based Structures Biology-Inspired Nanomaterials> Nucleic Acid-Based Structures.