Optimizing the synthesis and purification of MS2 virus like particles.

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents' delivery.

Transfer Rate of Enveloped and Nonenveloped Viruses between Fingerpads and Surfaces.

Fomites can represent a reservoir for pathogens, which may be subsequently transferred from surfaces to skin. In this study, we aim to understand how different factors (including virus type, surface type, time since last hand wash, and direction of transfer) affect virus transfer rates, defined as the fraction of virus transferred, between fingerpads and fomites. To determine this, 360 transfer events were performed with 20 volunteers using Phi6 (a surrogate for enveloped viruses), MS2 (a surrogate for nonenveloped viruses), and three clean surfaces (stainless steel, painted wood, and plastic). Considering all transfer events (all surfaces and both transfer directions combined), the mean transfer rates of Phi6 and MS2 were 0.17 and 0.26, respectively. Transfer of MS2 was significantly higher than that of Phi6 (P < 0.05). Surface type was a significant factor that affected the transfer rate of Phi6: Phi6 is more easily transferred to and from stainless steel and plastic than to and from painted wood. Direction of transfer was a significant factor affecting MS2 transfer rates: MS2 is more easily transferred from surfaces to fingerpads than from fingerpads to surfaces. Data from these virus transfer events, and subsequent transfer rate distributions, provide information that can be used to refine quantitative microbial risk assessments. This study provides a large-scale data set of transfer events with a surrogate for enveloped viruses, which extends the reach of the study to the role of fomites in the transmission of human enveloped viruses like influenza and SARS-CoV-2. IMPORTANCE This study created a large-scale data set for the transfer of enveloped viruses between skin and surfaces. The data set produced by this study provides information on modeling the distribution of enveloped and nonenveloped virus transfer rates, which can aid in the implementation of risk assessment models in the future. Additionally, enveloped and nonenveloped viruses were applied to experimental surfaces in an equivalent matrix to avoid matrix effects, so results between different viral species can be directly compared without confounding effects of different matrices. Our results indicating how virus type, surface type, time since last hand wash, and direction of transfer affect virus transfer rates can be used in decision-making processes to lower the risk of viral infection from transmission through fomites.

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.

Structural basis for the adsorption of a single-stranded RNA bacteriophage.

Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion 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.

A Light-Activated Antimicrobial Surface Is Active Against Bacterial, Viral and Fungal Organisms.

Evidence has shown that environmental surfaces play an important role in the transmission of nosocomial pathogens. Deploying antimicrobial surfaces in hospital wards could reduce the role environmental surfaces play as reservoirs for pathogens. Herein we show a significant reduction in viable counts of Staphylococcus epidermidis, Saccharomyces cerevisiae, and MS2 Bacteriophage after light treatment of a medical grade silicone incorporating crystal violet, methylene blue and 2 nm gold nanoparticles. Furthermore, a migration assay demonstrated that in the presence of light, growth of the fungus-like organism Pythium ultimum and the filamentous fungus Botrytis cinerea was inhibited. Atomic Force Microscopy showed significant alterations to the surface of S. epidermidis, and electron microscopy showed cellular aggregates connected by discrete surface linkages. We have therefore demonstrated that the embedded surface has a broad antimicrobial activity under white light and that the surface treatment causes bacterial envelope damage and cell aggregation.

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded ß-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ.

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.

Mimicking filtration and transport of rotavirus and adenovirus in sand media using DNA-labeled, protein-coated silica nanoparticles.

Rotavirus (RoV) and adenovirus (AdV) are important viral pathogens for the risk analysis of drinking water. Despite this, little is known about their retention and transport behaviors in porous media due to a lack of representative surrogates. We developed RoV and AdV surrogates by covalently coupling 70-nm sized silica nanoparticles with specific proteins and a DNA marker for sensitive detection. Filtration experiments using beach sand columns demonstrated the similarity of the surrogates' concentrations, filtration efficiencies and attachment kinetics to those of the target viruses. The surrogates showed the same magnitude of concentration reduction as the viruses. Conversely, MS2 phage (a traditional virus model) over-predicted concentrations of AdV and RoV by 1- and 2-orders of magnitude respectively. The surrogates remained stable in size, surface charge and DNA concentration for at least one year. They can be easily and rapidly detected down to a single particle. Preliminary tests suggest that they were readily detectable in a number of environmental waters and treated effluent. With up-scaling validation in pilot trials, the surrogates developed here could be a cost-effective new tool for studying virus retention and transport in porous media. Examples include assessing filter efficacy in water and wastewater treatment, tracking virus migration in groundwater after effluent land disposal, and establishing safe setback distances for groundwater protection.

UVC Inactivation of dsDNA and ssRNA Viruses in Water: UV Fluences and a qPCR-Based Approach to Evaluate Decay on Viral Infectivity.

Disinfection by low-pressure monochromatic ultraviolet (UVC) radiation (253.7 nm) became an important technique to sanitize drinking water and also wastewater in tertiary treatments. In order to prevent the transmission of waterborne viral diseases, the analysis of the disinfection kinetics and the quantification of infectious viral pathogens and indicators are highly relevant and need to be addressed. The families Adenoviridae and Polyomaviridae comprise human and animal pathogenic viruses that have been also proposed as indicators of fecal contamination in water and as Microbial Source Tracking tools. While it has been previously suggested that dsDNA viruses may be highly resistant to UVC radiation compared to other viruses or bacteria, no information is available on the stability of polyomavirus toward UV irradiation. Here, the inactivation of dsDNA (HAdV2 and JCPyV) and ssRNA (MS2 bacteriophage) viruses was analyzed at increasing UVC fluences. A minor decay of 2-logs was achieved for both infectious JC polyomaviruses (JCPyV) and human adenoviruses 2 (HAdV2) exposed to a UVC fluence of 1,400 J/m(2), while a decay of 4-log was observed for MS2 bacteriophages (ssRNA). The present study reveals the high UVC resistance of dsDNA viruses, and the UV fluences needed to efficiently inactivate JCPyV and HAdV2 are predicted. Furthermore, we show that in conjunction with appropriate mathematical models, qPCR data may be used to accurately estimate virus infectivity.

The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.

Simple, spherical RNA viruses have well-understood, symmetric protein capsids, but little structural information is available for their asymmetric components, such as minor proteins and their genomes, which are vital for infection. Here, we report an asymmetric structure of bacteriophage MS2, attached to its receptor, the F-pilus. Cryo-electron tomography and subtomographic averaging of such complexes result in a structure containing clear density for the packaged genome, implying that the conformation of the genome is the same in each virus particle. The data also suggest that the single-copy viral maturation protein breaks the symmetry of the capsid, occupying a position that would be filled by a coat protein dimer in an icosahedral shell. This capsomere can thus fulfill its known biological roles in receptor and genome binding and suggests an exit route for the genome during infection.

The role of surface functionalization of colloidal alumina particles on their controlled interactions with viruses.

Materials that interact in a controlled manner with viruses attract increasing interest in biotechnology, medicine, and environmental technology. Here, we show that virus-material interactions can be guided by intrinsic material surface chemistries, introduced by tailored surface functionalizations. For this purpose, colloidal alumina particles are surface functionalized with amino, carboxyl, phosphate, chloropropyl, and sulfonate groups in different surface concentrations and characterized in terms of elemental composition, electrokinetic, hydrophobic properties, and morphology. The interaction of the functionalized particles with hepatitis A virus and phages MS2 and PhiX174 is assessed by virus titer reduction after incubation with particles, activity of viruses conjugated to particles, and imaged by electron microscopy. Type and surface density of particle functional groups control the virus titer reduction between 0 and 99.999% (5 log values). For instance, high sulfonate surface concentrations (4.7 groups/nm(2)) inhibit attractive virus-material interactions and lead to complete virus recovery. Low sulfonate surface concentrations (1.2 groups/nm(2)), native alumina, and chloropropyl-functionalized particles induce strong virus-particle adsorption. The virus conformation and capsid amino acid composition further influence the virus-material interaction. Fundamental interrelations between material properties, virus properties, and the complex virus-material interaction are discussed and a versatile pool of surface functionalization strategies controlling virus-material interactions is presented.

Evidence that viral RNAs have evolved for efficient, two-stage packaging.

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

Removal of bacteriophage MS2 from aqueous solution using Mg-Fe layered double hydroxides.

The objective of this study was to investigate the removal of bacteriophage MS2 from aqueous solution using Mg-Fe layered double hydroxides (LDHs). Batch experiments were performed under various experimental conditions to examine bacteriophage removal with LDHs. The bacteriophage was enumerated by the plaque assay method. Results showed that among the Mg-Fe LDHs calcined at different temperatures (105, 300, 500, 700 ° C), Mg-Fe-300 LDH had the highest removal capacity at (2.34 ± 0.01) × 10(8) pfu/g with a removal percent of 99.44 ± 0.48 %. This result could be attributed to the fact that calcination could alter chemical compositions and physical properties of Mg-Fe LDHs. Kinetic experiments indicated that the removal of MS2 by Mg-Fe-300 LDH was a fast process, reaching equilibrium within 60 min. Results also showed that the effect of solution pH on MS2 removal by Mg-Fe-300 LDH was minimal at pH 4.0-9.0. The influence of anions (NO(3)(-), SO(4)(2-), CO(3)(2-), HPO(4(2-); concentrations 1-100 mg/L) on the removal of bacteriophage was important. SO(4)(2-), CO(3)(2-), and HPO(4)(2-) influenced removal due to their competition with bacteriophage at the sorption sites, while the effect of NO(3)(-) was negligible. Generally, the impact of the anions was in the order of NO(3)(-) < SO(4)(2-) < CO(3) (2-) < HPO(4)(4) (2-). This study improves our knowledge of potential applications of LDHs as adsorbents for virus removal in water treatment.

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

BACKGROUND: Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. METHODOLOGY/PRINCIPAL FINDINGS: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. CONCLUSION/SIGNIFICANCE: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

Cationic phenylene ethynylene polymers and oligomers exhibit efficient antiviral activity.

The antiviral activities of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPE) and oligo-phenylene ethynylenes (OPE) were investigated using two model viruses, the T4 and MS2 bacteriophages. Under UV/visible light irradiation, significant antiviral activity was observed for all of the CPEs and OPEs; without irradiation, most of these compounds exhibited high inactivation activity against the MS2 phage and moderate inactivation ability against the T4 phage. Transmission electron microscopy (TEM) and SDS polyacrylamide gel electrophoresis (SDS-PAGE) reveal that the CPEs and OPEs exert their antiviral activity by partial disassembly of the phage particle structure in the dark and photochemical damage of the phage capsid protein under UV/visible light irradiation.

Visualising a viral RNA genome poised for release from its receptor complex.

We describe the cryo-electron microscopy structure of bacteriophage MS2 bound to its receptor, the bacterial F-pilus. The virus contacts the pilus at a capsid 5-fold vertex, thus locating the surface-accessible portion of the single copy of the pilin-binding maturation protein present in virions. This arrangement allows a 5-fold averaged map to be calculated, showing for the first time in any virus-receptor complex the nonuniform distribution of RNA within the capsid. Strikingly, at the vertex that contacts the pilus, a rod of density that may include contributions from both genome and maturation protein sits above a channel that goes through the capsid to the outside. This density is reminiscent of the DNA density observed in the exit channel of double-stranded DNA phages, suggesting that the RNA-maturation protein complex is poised to leave the capsid as the first step of the infection process.

Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.

Immobilization and one-dimensional arrangement of virus capsids with nanoscale precision using DNA origami.

DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (approximately 100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.