Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61-392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin-streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

Adenovirus Structure: What Is New?

Adenoviruses are large (~950 Å) and complex non-enveloped, dsDNA icosahedral viruses. They have a pseudo-T = 25 triangulation number with at least 12 different proteins composing the virion. These include the major and minor capsid proteins, core proteins, maturation protease, terminal protein, and packaging machinery. Although adenoviruses have been studied for more than 60 years, deciphering their architecture has presented a challenge for structural biology techniques. An outstanding event was the first near-atomic resolution structure of human adenovirus type 5 (HAdV-C5), solved by cryo-electron microscopy (cryo-EM) in 2010. Discovery of new adenovirus types, together with methodological advances in structural biology techniques, in particular cryo-EM, has lately produced a considerable amount of new, high-resolution data on the organization of adenoviruses belonging to different species. In spite of these advances, the organization of the non-icosahedral core is still a great unknown. Nevertheless, alternative techniques such as atomic force microscopy (AFM) are providing interesting glimpses on the role of the core proteins in genome condensation and virion stability. Here we summarize the current knowledge on adenovirus structure, with an emphasis on high-resolution structures obtained since 2010.

Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2.

The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data.

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.

Superhemophobic and Antivirofouling Coating for Mechanically Durable and Wash-Stable Medical Textiles.

Medical textiles have a need for repellency to body fluids such as blood, urine, or sweat that may contain infectious vectors that contaminate surfaces and spread to other individuals. Similarly, viral repellency has yet to be demonstrated and long-term mechanical durability is a major challenge. In this work, we demonstrate a simple, durable, and scalable coating on nonwoven polypropylene textile that is both superhemophobic and antivirofouling. The treatment consists of polytetrafluoroethylene (PTFE) nanoparticles in a solvent thermally sintered to polypropylene (PP) microfibers, which creates a robust, low-surface-energy, multilayer, and multilength scale rough surface. The treated textiles demonstrate a static contact angle of 158.3 ± 2.6° and hysteresis of 4.7 ± 1.7° for fetal bovine serum and reduce serum protein adhesion by 89.7 ± 7.3% (0.99 log). The coated textiles reduce the attachment of adenovirus type 4 and 7a virions by 99.2 ± 0.2% and 97.6 ± 0.1% (2.10 and 1.62 log), respectively, compared to noncoated controls. The treated textiles provide these repellencies by maintaining a Cassie-Baxter state of wetting where the surface area in contact with liquids is reduced by an estimated 350 times (2.54 log) compared to control textiles. Moreover, the treated textiles exhibit unprecedented mechanical durability, maintaining their liquid, protein, and viral repellency after extensive and harsh abrasion and washing. The multilayer, multilength scale roughness provides for mechanical durability through self-similarity, and the samples have high-pressure stability with a breakthrough pressure of about 255 kPa. These properties highlight the potential of durable, repellent coatings for medical gowning, scrubs, or other hygiene textile applications.

Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus†5.

Amphiphilic surface groups play an important role in many biological processes. The synthesis of amphiphilic polyphenylene dendrimer branches (dendrons), providing alternating hydrophilic and lipophilic surface groups and one reactive ethynyl group at the core is reported. The amphiphilic surface groups serve as biorecognition units that bind to the surface of adenovirus 5 (Ad5), which is a common vector in gene therapy. The Ad5/dendron complexes showed high gene transduction efficiencies in coxsackie-adenovirus receptor (CAR)-negative cells. Moreover, the dendrons offer incorporation of new functions at the dendron core by in situ post-modifications, even when bound to the Ad5 surface. Surfaces coated with these dendrons were analyzed for their blood-protein binding capacity, which is essential to predict their performance in the blood stream. A new platform for introducing bioactive groups to the Ad5 surface without chemically modifying the virus particles is provided.

Nanoparticle Tracking of Adenovirus by Light Scattering and Fluorescence Detection.

The detailed characterization of biological nanoparticles is of paramount importance for various industrial sectors, as for production of viral therapeutics. More recently, technologies that allow real-time quantification with simultaneous sizing and determination of surface potentials of virus particles in solution have been developed. In this study, nanoparticle tracking analysis (NTA) was applied to determine the size and the zeta potential of human adenovirus type 5 (AdV5), one the most frequently used therapeutic/oncolytic agents and viral vectors. Virus aggregation was detected, and the kinetics of the dissolution of virus aggregates were studied in real time. In addition, advanced fluorescence detection of AdV5 was performed enabling the measurements in matrices and discrimination of viral subpopulations. It was shown that NTA is an efficient approach for investigating infectious viruses in a live viewing mode. Consequently, NTA provides a promising methodology for virus particle detection and analysis in real time beyond assays requiring nucleic acids or infectivity.

MnCaCs-Biomineralized Oncolytic Virus for Bimodal Imaging-Guided and Synergistically Enhanced Anticancer Therapy.

Oncolytic adenovirus (OA) is an ideal candidate for clinical anticancer treatment, because it can specifically replicate in tumor cells with high titer. However, its systemic administration is still hindered, because of severely compromised antitumor efficacy. Herein, an engineered OA was innovatively developed by enwrapping OA with calcium and manganese carbonates (MnCaCs) biomineral shell, which could protect the virus from removal of the host immune system and prolong its in vivo circulation. Upon accumulating in tumor sites, MnCaCs readily dissolved under the acidic microenvironment, releasing Mn2+ that could convert endogenous H2O2 into oxygen (O2) and then enhance the duplication ability of OA, thus significantly increased the antitumor efficacy. Meanwhile, Mn2+ and the increased O2 individually endowed the T1 modal magnetic resonance imaging (MRI) and photoacoustic imaging (PAI) feasibility, providing real-time monitoring information for the therapy. This versatile engineered OA demonstrated its promise for visible and efficient oncolytic virotherapy by systemic administration.

Rare missense variants in the human cytosolic antibody receptor preserve antiviral function.

The genetic basis of most human disease cannot be explained by common variants. One solution to this 'missing heritability problem' may be rare missense variants, which are individually scarce but collectively abundant. However, the phenotypic impact of rare variants is under-appreciated as gene function is normally studied in the context of a single 'wild-type' sequence. Here, we explore the impact of naturally occurring missense variants in the human population on the cytosolic antibody receptor TRIM21, using volunteer cells with variant haplotypes, CRISPR gene editing and functional reconstitution. In combination with data from a panel of computational predictors, the results suggest that protein robustness and purifying selection ensure that function is remarkably well-maintained despite coding variation.

Genetically Engineered Cell Membrane Nanovesicles for Oncolytic Adenovirus Delivery: A Versatile Platform for Cancer Virotherapy.

Currently, various oncolytic adenoviruses (OA) are being explored in both preclinical and clinical virotherapy. However, the pre-existing neutralizing antibodies (nAbs) and poor targeting delivery are major obstacles for systemically administered OA. Therefore, we designed bioengineered cell membrane nanovesicles (BCMNs) that harbor targeting ligands to achieve robust antiviral immune shielding and targeting capabilities for oncolytic virotherapy. We employed two distinct biomimetic synthetic approaches: the first is based on in vitro genetic membrane engineering to embed targeting ligands on the cell membrane, and the second is based on in vivo expression of CRISPR-engineered targeting ligands on red-blood-cell membranes. The results indicate that both bioengineering approaches preserve the infectivity and replication capacity of OA in the presence of nAbs, in vitro and in vivo. Notably, OA@BCMNs demonstrated a significant suppression of the induced innate and adaptive immune responses against OA. Enhanced targeting delivery, viral oncolysis, and survival benefits in multiple xenograft models were observed without overt toxicity. These findings reveal that OA@BCMNs may provide a clinical basis for improving oncolytic virotherapy by overcoming undesired antiviral immunity and enhancing cancer cell selectivity via biomimetic synthesis approaches.

Implementation of at-line capillary zone electrophoresis for fast and reliable determination of adenovirus concentrations in vaccine manufacturing.

A CZE method was validated and implemented for fast and accurate in-process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector-based vaccines. An analytical-quality-by-design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.

Structure-based Design of JOC-x, a Conjugatable Tumor Tight Junction Opener to Enhance Cancer Therapy.

Disorganized intercellular junctions are critical for maintaining the integrity of solid epithelial tumors and prevent the infiltration of oncological therapies into the bulk of the malignancy. We have developed small, recombinant proteins which bind a critical junction protein, desmoglein 2, triggering the transient and specific opening of tumor tight junctions allowing for infiltration of the tumor with immune cells, oncolytic viruses, drugs, and other therapeutics. Our new molecule, JOC-x, is a promising candidate for a new class of tumor-targeting agents that accumulate both around and within tumors and remodel the tumor microenvironment. Native cysteines were removed from the parental protein, JO-4, followed by addition of a single cysteine to allow for convenient attachment of various payloads that can be targeted directly to the tumor. Our tumor-targeting protein exhibits high avidity, minimal aggregation, and is easily purified at good yields from E. coli. For proof of concept, we demonstrate effective conjugation to biotin as a model for flexible co-targeting, addition of metal ion chelators as models for imaging and radiotherapy, and linkage of the TLR3 agonist poly(I:C) as a model immune-oncologic agent. This second-generation cancer co-therapeutic protein is optimized for activity and primed for cGMP manufacture in preparation for upcoming clinical studies.

Adenovirus 5 recovery using nanofiber ion-exchange adsorbents.

Viral vectors such as adenovirus have successful applications in vaccines and gene therapy but the manufacture of the high-quality virus remains a challenge. It is desirable to use the adsorption-based chromatographic separations that so effectively underpin the therapeutic protein manufacture. However fundamental differences in the size and stability of this class of product mean it is necessary to revisit the design of sorbent's morphology and surface chemistry. In this study, the behaviour of a cellulose nanofiber ion-exchange sorbent derivatised with quaternary amine ligands at defined densities is characterised to address this. This material was selected as it has a large accessible surface area for viral particles and rapid process times. Initially, the impact of surface chemistry on infective product recovery using low (440 µmol/g), medium (750 µmol/g), and high (1029 µmol/g) ligand densities is studied. At higher densities product stability is reduced, this effect increased with prolonged adsorption durations of 24 min with just ~10% loss at low ligand density versus ~50% at high. This could be mitigated by using a high flow rate to reduce the cycle time to ~1 min. Next, the impact of ligand density on the separation's resolution was evaluated. Key to understanding virus quality is the virus particle: infectious virus particle ratio. It was found this parameter could be manipulated using ligand density and elution strategy. Together this provides a basis for viral vector separations that allows for their typically low titres and labile nature by using high liquid velocity to minimise both load and on-column times while separating key product and process-related impurities.

Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Carbohydrate-Based Polymer Brushes Prevent Viral Adsorption on Electrostatically Heterogeneous Interfaces.

Chemical heterogeneity on biomaterial surfaces can transform its interfacial properties, rendering nanoscale heterogeneity profoundly consequential during bioadhesion. To examine the role played by chemical heterogeneity in the adsorption of viruses on synthetic surfaces, a range of novel coatings is developed wherein a tunable mixture of electrostatic tethers for viral binding, and carbohydrate brushes, bearing pendant α-mannose, ß-galactose, or ß-glucose groups, is incorporated. The effects of binding site density, brush composition, and brush architecture on viral adsorption, with the goal of formulating design specifications for virus-resistant coatings are experimentally evaluated. It is concluded that virus-coating interactions are shaped by the interplay between brush architecture and binding site density, after quantifying the adsorption of adenoviruses, influenza, and fibrinogen on a library of carbohydrate brushes co-immobilized with different ratios of binding sites. These insights will be of utility in guiding the design of polymer coatings in realistic settings where they will be populated with defects.

Systemic Administration and Targeted Delivery of Immunogenic Oncolytic Adenovirus Encapsulated in Extracellular Vesicles for Cancer Therapies.

Oncolytic viruses (OV) are engineered to infect, replicate in and kill cancer cells. Currently, the OV therapeutic approach is mainly restricted to neoplasia amenable to direct local administration of viral particles, while the possibility of a systemic delivery of cancer-tropic viruses would extend the OV application to the treatment of metastatic neoplasia. Herein, we applied in vivo/ex vivo imaging to demonstrate that cancer tropism is achieved when OV are encapsulated inside extracellular vesicles (EV) administered intravenously (i.v.), but not when injected intraperitoneally (i.p.). Moreover, we show that the therapeutic procedure adopted does not alter the immunomodulatory properties of the viruses.

Structural and functional annotation of hypothetical proteins of human adenovirus: prioritizing the novel drug targets.

OBJECTIVE: Human adenoviruses are small double stranded DNA viruses that provoke vast array of human diseases. Next generation sequencing techniques increase genomic data of HAdV rapidly, which increase their serotypes. The complete genome sequence of human adenovirus shows that it contains large amount of proteins with unknown cellular or biochemical function, known as hypothetical proteins. Hence, it is indispensable to functionally and structurally annotate these proteins to get better understanding of the novel drug targets. The purpose was the characterization of 38 randomly retrieved hypothetical proteins through determination of their physiochemical properties, subcellular localization, function, structure and ligand binding sites using various sequence and structure based bioinformatics tools. RESULTS: Function of six hypothetical proteins P03269, P03261, P03263, Q83127, Q1L4D7 and I6LEV1 were predicted confidently and then used further for structure analysis. We found that these proteins may act as DNA terminal protein, DNA polymerase, DNA binding protein, adenovirus E3 region protein CR1 and adenoviral protein L1. Functional and structural annotation leading to detection of binding sites by means of docking analysis can indicate potential target for therapeutics to defeat adenoviral infection.

Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

Galectin-9 (G9) is a tandem-repeat type ß-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.