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.

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors.

Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability, and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside VLPs can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here, we present a set of acceptor vectors that provide a quick and efficient way to build, test, and optimize cargo loading inside P22 VLPs. We prototyped the system using a yellow fluorescent protein and then applied it to mevalonate kinases (MKs), a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different MKs required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimize cargo enzyme behavior in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behavior.

Molecular exclusion limits for diffusion across a porous capsid.

Molecular communication across physical barriers requires pores to connect the environments on either side and discriminate between the diffusants. Here we use porous virus-like particles (VLPs) derived from bacteriophage P22 to investigate the range of molecule sizes able to gain access to its interior. Although there are cryo-EM models of the VLP, they may not accurately depict the parameters of the molecules able to pass across the pores due to the dynamic nature of the P22 particles in the solution. After encapsulating the enzyme AdhD within the P22 VLPs, we use a redox reaction involving PAMAM dendrimer modified NADH/NAD+ to examine the size and charge limitations of molecules entering P22. Utilizing the three different accessible morphologies of the P22 particles, we determine the effective pore sizes of each and demonstrate that negatively charged substrates diffuse across more readily when compared to those that are neutral, despite the negatively charge exterior of the particles.

Synthetic Virus-like Particles for Glutathione Biosynthesis.

Protein-based nanocompartments found in nature have inspired the development of functional nanomaterials for a range of applications including delivery of catalytic activities with therapeutic effects. As glutathione (GSH) plays a vital role in metabolic adaptation and many diseases are associated with its deficiency, supplementation of GSH biosynthetic activity might be a potential therapeutic when delivered directly to the disease site. Here, we report the successful design and production of active nanoreactors capable of catalyzing the partial or complete pathway for GSH biosynthesis, which was realized by encapsulating essential enzymes of the pathway inside the virus-like particle (VLP) derived from the bacteriophage P22. These nanoreactors are the first examples of nanocages specifically designed for the biosynthesis of oligomeric biomolecules. A dense packing of enzymes is achieved within the cavities of the nanoreactors, which allows us to study enzyme behavior, in a crowded and confined environment, including enzymatic kinetics and protein stability. In addition, the biomedical utility of the nanoreactors in protection against oxidative stress was confirmed using an in vitro cell culture model. Given that P22 VLP capsid was suggested as a potential liver-tropic nanocarrier in vivo, it will be promising to test the efficacy of these GSH nanoreactors as a novel treatment for GSH-deficient hepatic diseases.

Associations between antibiotic resistance and bacteriophage resistance phenotypes in laboratory and clinical strains of Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages have received great attention as an alternative over antibiotics due to the host specificity. Therefore, this study was designed to evaluate the associations between bacteriophage-insensitive (BI) and antibiotic-resistant mutants of Salmonella Typhimurium strains. Bacteriophage-sensitive (BS) Salmonella enterica serovar Typhimurium ATCC 19585 (BSSTWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (BSSTCIP), S. Typhimurium KCCM 40253 (BSSTLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (BSSTMDR) were used to induce the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR), which were characterized by measuring mutant frequency lysogenic induction, phage adsorption, antibiotic susceptibility, and differential gene expression. The numbers of BSSTWT, BSSTCIP, and BSSTLAB were reduced by P22 (>3 log), while the least lytic activity was observed for BSSTMDR, suggesting alteration in bacteriophage-binding receptors on the surface of multidrug-resistant strain. BSSTWT treated with P22 showed the large variation in the cell state (CV>40%) and highest mutant frequency (62%), followed by 25% for BSSTCIP. The least similarities between BSSTWT and BISTWT were observed for P22 and PBST-13 (<12%). The relative expression levels of bacteriophage-binding receptor-related genes (btuB, fhuA, fliK, fljB, ompC, ompF, rfaL, and tolC) were decreased in BISTCIP and BISTMDR. These results indicate that the bacteriophage resistance is highly associated with the antibiotic resistance. The findings in this study could pave the way for the application of bacteriophages as an alternative to control antibiotic-resistant bacteria.

Bi-enzymatic virus-like bionanoreactors for the transformation of endocrine disruptor compounds.

Endocrine disruptor compounds (EDCs) are pollutants able to alter both hormone synthesis and their regulation in animals and humans, thus, EDCs represent a risk for public health and for the environment. Cytochrome P450 enzymes (CYPs) are involved in the detoxification of a wide range of compounds, and it has been established that these enzymes produce the initial biotransformation of many EDCs. In this work, a bionanoreactor based on the encapsulation of an enhanced peroxygenase CYPBM321B3 inside the capsid of bacteriophage P22 virus-like particles (VLPs) was designed and characterized. VLPs were functionalized with glucose oxidase to generate in situ hydrogen peroxide necessary to activate the transformation of bisphenol A, nonylphenol, 17ß-estradiol, triclosan, and resorcinol. Catalytic parameters, as well as the chemical nature of reaction products are presented. The enzymatic nanoreactors showed specific activities varying from 0.175 to 0.456 min-1 in the transformation of these EDCs, which are equivalent to 22-77% of the activity obtained with free CYP. The capacity to transform structurally diverse compounds, easy production and glucose fueled catalytic activity make these enzymatic nanoreactors an interesting platform for enzyme delivery in the biomedical field.

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.

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex.

The gene cro promotes lytic growth of phages through binding of Cro protein dimers to regulatory DNA sites. Most Cro proteins are one-to-one orthologs, yet their sequence, structure and binding site sequences are quite divergent across lambdoid phages. We report the cocrystal structure of bacteriophage N15 Cro with a symmetric consensus site. We contrast this complex with an orthologous structure from phage λ, which has a dissimilar binding site sequence and a Cro protein that is highly divergent in sequence, dimerization interface and protein fold. The N15 Cro complex has less DNA bending and smaller DNA-induced changes in protein structure. N15 Cro makes fewer direct contacts and hydrogen bonds to bases, relying mostly on water-mediated and Van der Waals contacts to recognize the sequence. The recognition helices of N15 Cro and λ Cro make mostly nonhomologous and nonanalogous contacts. Interface alignment scores show that half-site binding geometries of N15 Cro and λ Cro are less similar to each other than to distantly related CI repressors. Despite this divergence, the Cro family shows several code-like protein-DNA sequence covariations. In some cases, orthologous genes can achieve a similar biological function using very different specific molecular interactions.

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.

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).

Loading the dice: The orientation of virus-like particles adsorbed on titanate assisted organosilanized surfaces.

The organization of virus-like particles (VLPs) on surfaces is a relevant matter for both fundamental and biomedical sciences. In this work, the authors have tailored surfaces with different surface tension components aiming at finding a relationship with the affinity of the different geometric/surface features of icosahedral P22 VLPs. The surfaces have been prepared by titanate assisted organosilanization with glycidyloxy, amino, and perfluoro silanes. Vibrational and photoelectron spectroscopies have allowed identifying the different functional groups of the organosilanes on the surfaces. Atomic force microscopy (AFM) showed that, irrespective of the organosilane used, the final root mean square roughness remains below 1 nm. Contact angle analyses confirm the effective formation of a set of surface chemistries exhibiting different balance among surface tension components. The study of the adsorption of P22 VLPs has involved the analysis of the dynamics of virus immobilization by fluorescence microscopy and the interpretation of the final VLP orientation by AFM. These analyses give rise to statistical distributions pointing to a higher affinity of VLPs toward perfluorinated surfaces, with a dominant fivefold conformation on this hydrophobic surface, but threefold and twofold symmetries dominating on hydrophilic surfaces. These results can be explained in terms of a reinforced hydrophobic interaction between the perfluorinated surface and the dominating hydrophobic residues present at the P22 pentons.

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.

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.

In Vivo Packaging of Protein Cargo Inside of Virus-Like Particle P22.

Protein cages are ubiquitous in nature and have been manipulated to encapsulate a range of nonnative cargos including organic, inorganic, and small molecules. Many protein cages are derived from virus capsids that have been rendered noninfectious through the preferential production and use of proteins that are solely involved in capsid assembly, but which do not encapsulate genetic material and therefore do not contribute to infectivity. Here, we describe the production of protein cargo(s) encapsulated inside of P22 virus-like particles (VLPs), derived from bacteriophage P22. This is achieved via genetic fusion of the cargo to a scaffolding protein, which becomes encapsulated in the P22 VLP during templated assembly of the protein cage.

High-resolution structure of podovirus tail adaptor suggests repositioning of an octad motif that mediates the sequential tail assembly.

The sophisticated tail structures of DNA bacteriophages play essential roles in life cycles. Podoviruses P22 and Sf6 have short tails consisting of multiple proteins, among which is a tail adaptor protein that connects the portal protein to the other tail proteins. Assembly of the tail has been shown to occur in a sequential manner to ensure proper molecular interactions, but the underlying mechanism remains to be understood. Here, we report the high-resolution structure of the tail adaptor protein gp7 from phage Sf6. The structure exhibits distinct distribution of opposite charges on two sides of the molecule. A gp7 dodecameric ring model shows an entirely negatively charged surface, suggesting that the assembly of the dodecamer occurs through head-to-tail interactions of the bipolar monomers. The N-terminal helix-loop structure undergoes rearrangement compared with that of the P22 homolog complexed with the portal, which is achieved by repositioning of two consecutive repeats of a conserved octad sequence motif. We propose that the conformation of the N-terminal helix-loop observed in the Sf6-gp7 and P22 portal:gp4 complex represents the pre- and postassembly state, respectively. Such motif repositioning may serve as a conformational switch that creates the docking site for the tail nozzle only after the assembly of adaptor protein to the portal. In addition, the C-terminal portion of gp7 shows conformational flexibility, indicating an induced fit on binding to the portal. These results provide insight into the mechanistic role of the adaptor protein in mediating the sequential assembly of the phage tail.

Not a barrier but a key: How bacteriophages exploit host's O-antigen as an essential receptor to initiate infection.

Tailed bacteriophages specific for Gram-negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events that orchestrate phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O-antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O-antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O-antigen specific host attachment must not always come along with O-antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O-antigen but does not degrade it, whereas rough strains or strains lacking O-antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host's O-antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O-antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps.

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain.

The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the ß-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.

Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.

Bacteriophage P22 ejects all of its internal proteins before its genome.

Double-stranded DNA bacteriophages are highly pressurized, providing a force driving ejection of a significant fraction of the genome from its capsid. In P22-like Podoviridae, internal proteins ("E proteins") are packaged into the capsid along with the genome, and without them the virus is not infectious. However, little is known about how and when these proteins come out of the virus. We employed an in vitro osmotic suppression system with high-molecular-weight polyethylene glycol to study P22 E protein release. While slow ejection of the DNA can be triggered by lipopolysaccharide (LPS), the rate is significantly enhanced by the membrane protein OmpA from Salmonella. In contrast, E proteins are not ejected unless both OmpA and LPS are present and their ejection when OmpA is present is largely complete before any genome is ejected, suggesting that E proteins play a key role in the early stage of transferring P22 DNA into the host.