A pH-Responsive Virus-Like Particle as a Protein Cage for a Targeted Delivery.

A stimuli-responsive protein self-assembly offers promising utility as a protein nanocage for biotechnological and medical applications. Herein, the development of a virus-like particle (VLP) that undergoes a transition between assembly and disassembly under a neutral and acidic pH, respectively, for a targeted delivery is reported. The structure of the bacteriophage P22 coat protein is used for the computational design of coat subunits that self-assemble into a pH-responsive VLP. Subunit designs are generated through iterative computational cycles of histidine substitutions and evaluation of the interaction energies among the subunits under an acidic and neutral pH. The top subunit designs are tested and one that is assembled into a VLP showing the highest pH-dependent structural transition is selected. The cryo-EM structure of the VLP is determined, and the structural basis of a pH-triggered disassembly is delineated. The utility of the designed VLP is exemplified through the targeted delivery of a cytotoxic protein cargo into tumor cells in a pH-dependent manner. These results provide strategies for the development of self-assembling protein architectures with new functionality for diverse applications.

Molecular Architecture of Salmonella Typhimurium Virus P22 Genome Ejection Machinery.

Bacteriophage P22 is a prototypical member of the Podoviridae superfamily. Since its discovery in 1952, P22 has become a paradigm for phage transduction and a model for icosahedral viral capsid assembly. Here, we describe the complete architecture of the P22 tail apparatus (gp1, gp4, gp10, gp9, and gp26) and the potential location and organization of P22 ejection proteins (gp7, gp20, and gp16), determined using cryo-EM localized reconstruction, genetic knockouts, and biochemical analysis. We found that the tail apparatus exists in two equivalent conformations, rotated by ∼6° relative to the capsid. Portal protomers make unique contacts with coat subunits in both conformations, explaining the 12:5 symmetry mismatch. The tail assembles around the hexameric tail hub (gp10), which folds into an interrupted ß-propeller characterized by an apical insertion domain. The tail hub connects proximally to the dodecameric portal protein and head-to-tail adapter (gp4), distally to the trimeric tail needle (gp26), and laterally to six trimeric tailspikes (gp9) that attach asymmetrically to gp10 insertion domain. Cryo-EM analysis of P22 mutants lacking the ejection proteins gp7 or gp20 and biochemical analysis of purified recombinant proteins suggest that gp7 and gp20 form a molecular complex associated with the tail apparatus via the portal protein barrel. We identified a putative signal transduction pathway from the tailspike to the tail needle, mediated by three flexible loops in the tail hub, that explains how lipopolysaccharide (LPS) is sufficient to trigger the ejection of the P22 DNA in vitro.

Higher-Order VLP-Based Protein Macromolecular Framework Structures Assembled via Coiled-Coil Interactions.

Hierarchical organization is one of the fundamental features observed in biological systems that allows for efficient and effective functioning. Virus-like particles (VLPs) are elegant examples of a hierarchically organized supramolecular structure, where many subunits are self-assembled to generate the functional cage-like architecture. Utilizing VLPs as building blocks to construct two- and three-dimensional (3D) higher-order structures is an emerging research area in developing functional biomimetic materials. VLPs derived from P22 bacteriophages can be repurposed as nanoreactors by encapsulating enzymes and modular units to build higher-order catalytic materials via several techniques. In this study, we have used coiled-coil peptide interactions to mediate the P22 interparticle assembly into a highly stable, amorphous protein macromolecular framework (PMF) material, where the assembly does not depend on the VLP morphology, a limitation observed in previously reported P22 PMF assemblies. Many encapsulated enzymes lose their optimum functionalities under the harsh conditions that are required for the P22 VLP morphology transitions. Therefore, the coiled-coil-based PMF provides a fitting and versatile platform for constructing functional higher-order catalytic materials compatible with sensitive enzymes. We have characterized the material properties of the PMF and utilized the disordered PMF to construct a biocatalytic 3D material performing single- and multistep catalysis.

Encapsulation of Pseudomonas aeruginosa elastase inside the P22 virus-like particle for controlling enzyme-substrate interactions.

Controlling interactions between enzymes and interaction partners, such as substrates, is important for applications in cellular biology and molecular biochemistry. A strategy for controlling enzyme access with substrate interaction partners is to exploit encapsulation of enzymes inside nanoparticles to limit the accessibility of the enzymes to large macromolecules, but allow free exchange of small-molecule substrates. The research here evaluates the encapsulation of Pseudomonas aeruginosa elastase inside the bacteriophage P22 virus-like particle (VLP) to examine the ability to allow free soluble substrates access to the enzyme while blocking large macromolecular substrate interactions. The results show that the active elastase protease can be encapsulated inside the P22 VLP, which blocks its ability to disrupt cell monolayers, but allows soluble substrates to be catalytically cleaved, supporting the viability of this approach for future investigations.

Bioinspired Approaches to Self-Assembly of Virus-like Particles: From Molecules to Materials.

When viewed through the lens of materials science, nature provides a vast library of hierarchically organized structures that serve as inspiration and raw materials for new synthetic materials. The structural organization of complex bioarchitectures with advanced functions arises from the association of building blocks and is strongly supported by ubiquitous mechanisms of self-assembly, where interactions among components result in spontaneous assembly into defined structures. Viruses are exemplary, where a capsid structure, often formed from the self-assembly of many individual protein subunits, serves as a vehicle for the transport and protection of the viral genome. Higher-order assemblies of viral particles are also found in nature with unexpected collective behaviors. When the infectious aspect of viruses is removed, the self-assembly of viral particles and their potential for hierarchical assembly become an inspiration for the design and construction of a new class of functional materials at a range of different length scales.Salmonella typhimurium bacteriophage P22 is a well-studied model for understanding viral self-assembly and the construction of virus-like particle (VLP)-based materials. The formation of cage-like P22 VLP structures results from scaffold protein (SP)-directed self-assembly of coat protein (CP) subunits into icosahedral capsids with encapsulation of SP inside the capsid. Employing the CP-SP interaction during self-assembly, the encapsulation of guest protein cargos inside P22 VLPs can be achieved with control over the composition and the number of guest cargos. The morphology of cargo-loaded VLPs can be altered, along with changes in both the physical properties of the capsid and the cargo-capsid interactions, by mimicking aspects of the infectious P22 viral maturation. The structure of the capsid differentiates the inside cavity from the outside environment and serves as a protecting layer for the encapsulated cargos. Pores in the capsid shell regulate molecular exchange between inside and outside, where small molecules can traverse the capsid freely while the diffusion of larger molecules is limited by the pores. The interior cavity of the P22 capsid can be packed with hundreds of copies of cargo proteins (especially enzymes), enforcing intermolecular proximity, making this an ideal model system in which to study enzymatic catalysis in crowded and confined environments. These aspects highlight the development of functional nanomaterials from individual P22 VLPs, through biomimetic design and self-assembly, resulting in fabrication of nanoreactors with controlled catalytic behaviors.Individual P22 VLPs have been used as building blocks for the self-assembly of higher-order structures. This relies on a balance between the intrinsic interparticle repulsion and a tunable interparticle attraction. The ordering of VLPs within three-dimensional assemblies is dependent on the balance between repulsive and attractive interactions: too strong an attraction results in kinetically trapped disordered structures, while decreasing the attraction can lead to more ordered arrays. These higher-order assemblies display collective behavior of high charge density beyond those of the individual VLPs.The development of synthetic nanomaterials based on P22 VLPs demonstrates how the potential for hierarchical self-assembly can be applied to other self-assembling capsid structures across multiple length scales toward future bioinspired functional materials.

Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins.

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent.

Controlled Modular Multivalent Presentation of the CD40 Ligand on P22 Virus-like Particles Leads to Tunable Amplification of CD40 Signaling.

Ligands of the tumor necrosis factor superfamily (TNFSF) are appealing targets for immunotherapy research due to their integral involvement in stimulation or restriction of immune responses. TNFSF-targeted therapies are currently being developed to combat immunologically based diseases and cancer. A crucial determinant of effective TNFSF receptor binding and signaling is the trimeric quaternary structure of the ligand. Additionally, ligand multivalency is essential to propagate strong signaling in effector cells. Thus, designing a synthetic platform to display trimeric TNFSF ligands in a multivalent manner is necessary to further the understanding of ligand-receptor interactions. Viral nanocages have architectures that are amenable to genetic and chemical modifications of both their interior and exterior surfaces. Notably, the exterior surface of virus-like particles can be utilized as a platform for the modular multivalent presentation of target proteins. In this study, we build on previous efforts exploring the bacteriophage P22 virus-like particle for the exterior multivalent modular display of a potent immune-stimulating TNFSF protein, CD40 ligand (CD40L). Using a cell-based reporter system, we quantify the effects of tunable avidity on CD40 signaling by CD40L displayed on the surface of P22 nanocages. Multivalent presentation of CD40L resulted in a 53.6-fold decrease of the half maximal effective concentration (EC50) compared to free CD40L, indicating higher potency. Our results emphasize the power of using P22-based biomimetics to study ligand-receptor interactions within their proper structural context, which may contribute to the development of effective immune modulators.

NMR Mapping of Disordered Segments from a Viral Scaffolding Protein Enclosed in a 23 MDa Procapsid.

Scaffolding proteins (SPs) are required for the capsid shell assembly of many tailed double-stranded DNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, only one high-resolution structure is available for SPs within procapsids. Here, we use the inherent size limit of NMR to identify mobile segments of the 303-residue phage P22 SP free in solution and when incorporated into a ∼23 MDa procapsid complex. Free SP gives NMR signals from its acidic N-terminus (residues 1-40) and basic C-terminus (residues 264-303), whereas NMR signals from the middle segment (residues 41-263) are missing because of intermediate conformational exchange on the NMR chemical shift timescale. When SP is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix domain disappear because of binding to the procapsid interior. Signals from the N-terminal domain persist, indicating that this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus, coupled with its high content of negative charges, is likely important for dissociation and release of SP during the double-stranded DNA genome packaging step accompanying phage maturation.

Development of target-tunable P22 VLP-based delivery nanoplatforms using bacterial superglue.

Protein cage nanoparticles are widely used as targeted delivery nanoplatforms, because they have well-defined symmetric architectures, high biocompatibility, and enough plasticity to be modified to produce a range of different functionalities. Targeting peptides and ligands are often incorporated on the surface of protein cage nanoparticles. In this research, we adopted the SpyTag/SpyCatcher protein ligation system to covalently display target-specific affibody molecules on the exterior surface of bacteriophage P22 virus-like particles (VLP) and evaluated their modularity and efficacy of targeted delivery. We genetically introduced the 13 amino acid SpyTag peptide into the C-terminus of the P22 capsid protein to construct a target-tunable nanoplatform. We constructed two different SpyCatcher-fused affibody molecules as targeting ligands, SC-EGFRAfb and SC-HER2Afb, which selectively bind to EGFR and HER2 surface markers, respectively. We produced target-specific P22 VLP-based delivery nanoplatforms for the target cell lines by selectively combining SpyTagged P22 VLP and SC-fused affibody molecules. We confirmed its target-switchable modularity through cell imaging and verified the target-specific drug delivery efficacy of the affibody molecules displaying P22 VLP using cell viability assays. The P22 VLP-based delivery nanoplatforms can be used as multifunctional delivery vehicles by ligating other functional proteins, as well as affibody molecules. The interior cavity of P22 VLP can be also used to load cargoes like enzymes and therapeutic proteins. We anticipate that the nanoplatforms will provide new opportunities for developing target-specific functional protein delivery systems.

Of capsid structure and stability: The partnership between charged residues of E-loop and P-domain of the bacteriophage P22 coat protein.

Tailed dsDNA bacteriophages and herpesviruses form capsids using coat proteins that have the HK97 fold. In these viruses, the coat proteins first assemble into procapsids, which subsequently mature during DNA packaging. Generally interactions between the coat protein E-loop of one subunit and the P-domain of an adjacent subunit help stabilize both capsomers and capsids. Based on a recent 3.3 Šcryo-EM structure of the bacteriophage P22 virion, E-loop amino acids E52, E59 and E72 were suggested to stabilize the capsid through intra-capsomer salt bridges with the P-domain residues R102, R109 and K118. The glutamic acid residues were each mutated to alanine to test this hypothesis. The substitutions resulted in a WT phenotype and did not destabilize capsids; rather, the alanine substituted coat proteins increased the stability of procapsids and virions. These results indicate that different types of interactions must be used between the E-loop and P-domain to stabilize phage P22 procapsids and virions.

A Hydrophobic Network: Intersubunit and Intercapsomer Interactions Stabilizing the Bacteriophage P22 Capsid.

Double-stranded DNA (dsDNA) tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here, we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold.IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra- and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra- and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.

Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22.

Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

Conservation and Divergence of the I-Domain Inserted into the Ubiquitous HK97 Coat Protein Fold in P22-Like Bacteriophages.

Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel ß-barrels. Nevertheless, significant structural differences occur in loops connecting the ß-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions.IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded ß-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages.

Chemically Induced Morphogenesis of P22 Virus-like Particles by the Surfactant Sodium Dodecyl Sulfate.

In the infectious P22 bacteriophage, the packaging of DNA into the initially formed procapsid triggers a remarkable morphological transformation where the capsid expands from 58 to 62 nm. Along with the increase in size, this maturation also provides greater stability to the capsid and initiates the release of the scaffolding protein (SP). (2,4) In the P22 virus-like particle (VLP), this transformation can be mimicked in vitro by heating the procapsid particles to 65 °C or by treatment with sodium dodecyl sulfate (SDS). (5,6) Heating the P22 particles at 65 °C for 20 min is well established to trigger the transformation of P22 to the expanded (EX) P22 VLP but does not always result in a fully expanded population. Incubation with SDS resulted in a >80% expanded population for all P22 variants used in this work. This study elucidates the importance of the stoichiometric ratio between P22 subunits and SDS, the charge of the headgroup, and length of the carbon chain for the transformation. We propose a mechanism by which the expansion takes place, where both the negatively charged sulfate group and hydrophobic tail interact with the coat protein (CP) monomers within the capsid shell in a process that is facilitated by an internal osmotic pressure generated by an encapsulated macromolecular cargo.

Encapsulation of Active Enzymes within Bacteriophage P22 Virus-Like Particles.

Virus-like particles (VLPs) are nonpathogenic protein cage structures derived from viral coat proteins that have found utility in the area of biomaterials and nanotechnology. VLPs have been exploited as containers for the sequestration and encapsulation of a wide range of guest molecules in their hollow interiors. The robust nature of VLPs lend them as versatile scaffolds that can be exploited to provide protection to encapsulated guest molecules, such as enzymes which are often susceptible to inactivation and degradation, and for organization and construction of new nanomaterials incorporating the chemical properties of the guest molecules. In this chapter a background and methodology for the encapsulation of enzymes on the interior of the bacteriophage P22 derived VLP is described.

Synthesis of Cross-Linked 2-Aminoethyl Methacrylate in P22 Viral Capsid via Atom-Transfer Radical Polymerization.

Virus-like particles (VLPs) resemble viruses, but are devoid their genetic material, rendering them as noninfectious, hollow protein shells. VLPs are ideal templates to synthesize nanoparticles because they have homogeneous size and their empty cavity can provide a confined environment for selectively directed synthesis. Atom-transfer radical polymerization (ATRP) is well suited for directed synthesis of polymers inside VLPs. In addition to being rapid, monomer-promiscuous, and resulting in products with relatively low polydispersity, the simplicity of the ATRP initiator allows it to be readily modified for amending to biomolecules. This chapter describes the polymerization of 2-aminoethyl methacrylate (AEMA) via ATRP in a viral capsid derived from the bacteriophage P22.

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation.

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.

A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain.

P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.

Function of bacteriophage G7C esterase tailspike in host cell adsorption.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.