Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Complete Virion Simulated: All-Atom Model of an MS2 Bacteriophage with Native Genome.

For the first time, a complete all-atom molecular dynamics (MD) model of a virus, bacteriophage MS2, in its entirety, including a protein outer shell, native genomic RNA with necessary divalent ions, and surrounding explicit aqueous solution with ions at physiological concentration, was built. The model is based on an experimentally measured cryo-EM structure, which was substantially augmented by reconstructing missing or low-resolution parts of the measured density (where the atomistic structure cannot be fit unambiguously). The model was tested by a quarter of a microsecond MD run, and various biophysical characteristics are obtained and analyzed. The developed methodology of building the model can be used for reconstructing other large biomolecular structures when experimental data are fragmented and/or of varying resolution, while the model itself can be used for studying the biology of MS2, including the dynamics of its interaction with the host bacteria.

The presence of RNA cargo is suspected to modify the surface hydrophobicity of the MS2 phage.

The surface hydrophobicity of native or engineered non-enveloped viruses and virus-like particles (VLPs) is a key parameter regulating their fate in living and artificial aqueous systems. Its modulation is mainly depending on the structure and environment of particles. Nevertheless, unexplained variations have been reported between structurally similar viruses and with pH. This indicates that some modulating factors of their hydrophobicity remain to be identified. Herein we investigate the potential involvement of RNA cargo in the MS2 phage used as non-enveloped RNA virus model, by examining the SDS-induced electrophoretic mobility shift (SEMS) determined for native MS2 virions and corresponding RNA-free VLPs at various pH. Interestingly, the SEMS of VLPs was larger and more variable from pH 5 to 9 compared to native virions. These observations are discussed in term of RNA-dependent changes in surface hydrophobicity, suggesting that RNA cargo may be a major modulator/regulator of this viral parameter.

Estimation of Nanoparticle's Surface Electrostatic Potential in Solution Using Acid-Base Molecular Probes. III. Experimental Hydrophobicity/Hydrophilicity and Charge Distribution of MS2 Virus Surface.

MS2 bacteriophage is often used as a model for evaluating pathogenic viruses' behavior in aqueous solution. However, the questions of the virus surface's hydrophilic/hydrophobic balance, the charge distribution, and the binding mechanism are open. Using the dynamic light scattering method and laser Doppler electrophoresis, the hydrodynamic diameter and the ζ-potential of the virus particles were measured at their concentration of 5 × 1011 particles per mL and ionic strength 0.03 M. The values were found to be 30 nm and -29 or -34 mV (by Smoluchowski or Ohshima approximations), respectively. The MS2 bacteriophage surface was also investigated using a series of acid-base indicator dyes of various charge type, size, and structure. Their spectral and acid-base properties (pKa) are very sensitive to the microenvironment in aqueous solution, including containing nanoparticles. The electrostatic potential of the surface Ψ was estimated using the common formula: Ψ = 59 × (pKai - pKa) in mV at 25 °C. The Ψ values were -50 and +10 mV, respectively, which indicate the "mosaic" way of the charge distribution on the surface. These data are in good agreement with the obtained ζ-potential values and provide even more information about the virus surface. It was found that the surface of the MS2 virus is hydrophilic in solution in contrast to the commonly accepted hypothesis of the hydrophobicity of virus particles. No hydrophobic interactions between various molecular probes and the capsid were observed.

Genome-regulated Assembly of a ssRNA Virus May Also Prepare It for Infection.

Many single-stranded, positive-sense RNA viruses regulate assembly of their infectious virions by forming multiple, cognate coat protein (CP)-genome contacts at sites termed Packaging Signals (PSs). We have determined the secondary structures of the bacteriophage MS2 ssRNA genome (gRNA) frozen in defined states using constraints from X-ray synchrotron footprinting (XRF). Comparison of the footprints from phage and transcript confirms the presence of multiple PSs in contact with CP dimers in the former. This is also true for a virus-like particle (VLP) assembled around the gRNA in vitro in the absence of the single-copy Maturation Protein (MP) found in phage. Since PS folds are present at many sites across gRNA transcripts, it appears that this genome has evolved to facilitate this mechanism of assembly regulation. There are striking differences between the gRNA-CP contacts seen in phage and the VLP, suggesting that the latter are inappropriate surrogates for aspects of phage structure/function. Roughly 50% of potential PS sites in the gRNA are not in contact with the protein shell of phage. However, many of these sit adjacent to, albeit not in contact with, PS-binding sites on CP dimers. We hypothesize that these act as PSs transiently during assembly but subsequently dissociate. Combining the XRF data with PS locations from an asymmetric cryo-EM reconstruction suggests that the genome positions of such dissociations are non-random and may facilitate infection. The loss of many PS-CP interactions towards the 3' end of the gRNA would allow this part of the genome to transit more easily through the narrow basal body of the pilus extruding machinery. This is the known first step in phage infection. In addition, each PS-CP dissociation event leaves the protein partner trapped in a non-lowest free-energy conformation. This destabilizes the protein shell which must disassemble during infection, further facilitating this stage of the life-cycle.

Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria.

DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites' (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) in a variety of sizes (200-1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serve as an excellent protective shell. These engineered NCs pose critical properties that are yet unattainable by current fabrication methods.

Visualizing a viral genome with contrast variation small angle X-ray scattering.

Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryo-EM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

Colloidal Transformations in MS2 Virus Particles: Driven by pH, Influenced by Natural Organic Matter.

Enteric viruses, such as enterovirus, norovirus, and rotavirus, are among the leading causes of disease outbreaks due to contaminated drinking and recreational water. Viruses are difficult to remove from water through filtration based on physical size exclusion-for example by gravity-driven filters-due to their nanoscale size. To understand virus removal in drinking water treatment systems, the colloidal nanostructure of a model virus, the MS2 bacteriophage, has been investigated in relation to the effect of pH and natural organic matter in water. Dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy demonstrated that the water pH has a major influence on the colloidal structure of the virus: The bacteriophage MS2's structure in water in the range pH = 7.0 to 9.0 was found to be spherical with core-shell-type structure with a total diameter of 27 nm and a core radius of around 8 nm. Reversible transformations from 27 nm particles at pH = 7.0 to micrometer-sized aggregates at pH = 3.0 were observed. In addition, the presence of natural organic matter that simulates the organic components present in surface water was found to enhance repulsion between virus particles, reduce the size of aggregates, and promote disaggregation upon pH increase. These findings allow a better understanding of virus interactions in water and have implications for water treatment using filtration processes and coagulation. The results will further guide the comprehensive design of advanced virus filter materials.

Effect of polymer and glass physicochemical properties on MS2 recovery from food contact surfaces.

Viruses are transmissible via their interaction with contact surfaces of food containers or tools. This study evaluated the recoveries of MS2 coliphage, a virus surrogate, from polypropylene (PP), polyvinyl chloride (PVC), polyethylene (PE), and glass (borosilicate and soda lime), as influenced by the surface chemistry and topography. MS2 (5-6 logs) in PBS with 1% TSB was inoculated onto each of 9 different surfaces, 24-h cold-incubated, and recovery was quantified by infectivity. The order of MS2 recovery efficiency from smooth surfaces was PP > PE ≥ soda lime glass, which classified into 3 ANOVA groups, p = 0.05. The MS2 recovery ratios of smooth vs. rough surfaces were 1.4-1.5. Atomic force microscopy revealed 21-nm diam pinholes (<28-nm of MS2 size) in the borosilicate glass. The lowest and highest MS2 recoveries among the 9 surfaces were demonstrated by the hole-bearing borosilicate glass (34 ±â€¯8%) and smooth PP (69 ±â€¯14%) respectively. Generally greater MS2 recovery was obtained from smooth PP and PE surfaces compared to glass, but topographic alterations (pinholes or increased roughness) decreased recovery possibly by trapping the viruses.

Assessing Antibody Specificity in Human Serum Using Deep Sequence-Coupled Biopanning.

Affinity selection using phage-display technologies is a powerful tool for identifying the peptide epitopes of monoclonal antibodies. Coupling affinity selection with deep sequencing technologies allows for the broad assessment of selectant populations. Here, we describe a method for using a phage-display platform to assess antibody specificity in human serum. We describe the method with reference to the bacteriophage MS2 virus-like particle (VLP) platform, but it can be adapted to other phage-display technologies as well.

Determination of Antibody Population Distributions for Virus-Antibody Conjugates by Charge Detection Mass Spectrometry.

Virus-like particle (VLP) conjugates are being developed for biomedical applications; however, there is a lack of quantitative analytical methods to measure the extent of conjugation and modification of VLP based therapeutics. Charge detection mass spectrometry (CDMS) can measure mass distributions for large and heterogeneous complexes and is emerging as a valuable tool in the analysis of biologics. In this study, CDMS is used to characterize the stoichiometry and population distribution of antibodies covalently conjugated to the surface of a bacteriophage MS2 VLP. Initial CDMS analysis of the unconjugated MS2 particles suggested that they had packaged a broad distribution of exogenous genomic material. We developed procedures to remove the undesired genomic material from the VLP preparation and observed that, for the samples where the genomic fragments were removed, the antibody coupling reaction efficiency increased by almost a factor of 2. This meant there were (1) fewer VLPs with no antibodies bound, which is an important consideration for the efficacy of a targeted therapeutic and (2) fewer antibodies were wasted during the coupling reaction. CDMS could be employed in a similar manner as a tool to characterize coupling reaction product distributions and precursors and help inform the development of the next generation of conjugate-based therapies.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

Virus deposition onto polyelectrolyte-coated surfaces: A study with bacteriophage MS2.

HYPOTHESES: By selecting constituent polyelectrolytes and controlling conditions of their deposition, the resulting polyelectrolyte multilayers can be designed as surface coatings with controlled adhesive properties with respect to viruses. Charge and hydrophilicity of the polyelectrolyte multilayers govern virus adhesion. EXPERIMENTS: Four surfaces of different charges and hydrophobicities were designed using a layer-by-layer assembly of poly(styrene-4-sulfonate) and poly(dimethyl diallyl ammonium chloride). Contact angle measurements gave an estimate of MS2 hydrophilicity in terms of free energy of interfacial interaction in water. Experimental results on MS2 adhesion obtained using quartz crystal microbalance with dissipation monitoring were compared with predictions by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. FINDINGS: MS2 deposition onto polyelectrolyte multilayers occurred in two phases: an early phase defined by virus-surface interactions and a later phase with virus-virus interactions controlling deposition kinetics. Principal component analysis showed that the deposition rates in the two phases were independent one of another and that each was correlated to the depth of the secondary minimum of the corresponding XDLVO energy profile. Hydrophobic and electrostatic interactions governed the deposition process: short range hydrophilic repulsion prevented deposition into the primary minimum while electrostatic interactions defined the dependence of the deposition kinetics on the ionic strength. Different surfaces showed distinct kinetics of and capacities for MS2 deposition pointing to the potential of polyelectrolyte multilayers as easy-to-apply coatings for regulating virus adsorption, inactivating viruses via the virucidal action of cationic polyelectrolytes and reducing human exposure to viruses.

Encapsulation of Negatively Charged Cargo in MS2 Viral Capsids.

Encapsulation into virus-like particles is an efficient way of loading cargo of interest for delivery applications. Here, we describe the encapsulation of proteins with tags comprising anionic amino acids or DNA and gold nanoparticles with negative surface charges inside MS2 bacteriophage capsids to obtain homogeneous nanoparticles with a diameter of 27 nm.

Dual Surface Modification of Genome-Free MS2 Capsids for Delivery Applications.

One of the hallmarks of virus-like particles (VLPs) is the fact that they possess distinguishable interior and exterior surfaces. Taking advantage of our knowledge of the amino acid location from X-ray crystal structures, we have developed a series of synthetic modifications of the MS2 bacteriophage viral capsid, including small molecule and polymer attachment, as well as conjugation with peptides, DNA and other proteins. These constructs have found applications in nanomaterial fabrication and as delivery vehicles with therapeutic potential. Importantly, the dual-modification strategies described herein could be extended to other VLP systems.

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

MRI compatible MS2 nanoparticles designed to cross the blood-brain-barrier: providing a path towards tinnitus treatment.

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.

MS2 and Qß bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non-enveloped icosahedral viruses in SDS-based capillary zone electrophoresis.

SDS is commonly employed as BGE additive in CZE analysis of non-enveloped icosahedral viruses. But the way by which SDS interacts with the surface of such viruses remains to date poorly known, making complicate to understand their behavior during a run. In this article, two related bacteriophages, MS2 and Qß, are used as model to investigate the migration mechanism of non-enveloped icosahedral viruses in SDS-based CZE. Both phages are characterized by similar size and surface charge but significantly different surface hydrophobicity (Qß > MS2, where '>' means 'more hydrophobic than'). By comparing their electrophoretic mobility in the presence or not of SDS on both sides of the CMC, we show that surface hydrophobicity of phages is a key factor influencing their mobility and that SDS-virus association is driven by hydrophobic interactions at the surface of virions. The CZE analyses of heated MS2 particles, which over-express hydrophobic domains at their surface, confirm this finding. The correlations between the present results and others from the literature suggest that the proposed mechanism might not be exclusive to the bacteriophages examined here.

One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles.

MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.

Origins of the enhanced affinity of RNA-protein interactions triggered by RNA phosphorodithioate backbone modification.

The well-characterized interaction between the MS2 coat protein and its cognate RNA hairpin was used to evaluate changes in affinity as a result of phosphorodithioate (PS2) replacing phosphate by biolayer interferometry (BLI). A structure-based analysis of the data provides insights into the origins of the enhanced affinity of RNA-protein interactions triggered by the PS2 moiety.