Protein pVIII Research Articles

Mechanistic Insights Into an Ancient Adenovirus Precursor Protein VII Show Multiple Nuclear Import Receptor Pathways

ABSTRACTAdenoviral pVII proteins are multifunctional, highly basic, histone‐like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus (HAdV)2, the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre‐) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1), using a combination of structural, functional, and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)β1 and IMPα, respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting and intranuclear binding. Expression of wild‐type and NLS defective derivatives Pre‐pVII in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre‐pVII nuclear import is dependent on IMPα/β1 and IMPβ1, but not on transportin‐1 (IMPβ2). Clearly, AdVs evolved to maximize the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.

Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0×107 pfumL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy.

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Liquid Glycan Array.

The M13 phage platform is a stable and monodisperse nanoscale carrier, which can be modified with different molecules by chemical conjugation strategies. Here, we describe M13 phage acylated on pVIII protein with a dibenzocyclooctyne reacting with azido glycan to yield 30-1500 copy numbers of glycan per phage and monitored by MALDI-TOF spectrometry to generate multivalent glycoconjugates that contain desired densities of glycans. We prepared the liquid glycan arrays (LiGA) such that both the structure and density of glycans were encoded in the DNA of the bacteriophage. The LiGA can be used to validate the binding properties of glycans to purified lectins and explore the effect of glycan density on such binding. From a mixture of multivalent glycan probes, LiGAs can also identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and live animals in vivo.

Designing M13 Bacteriophage and Fe-Nanonest Self-Assembly System for Universal and Facile Preparation of Metal Single Atoms as Stable Mimicking Enzymes.

Metal single-atom catalysts (MSACs) possess multiple advantages in chemical synthesis; their efficient fabrication routes, however, remain a challenge to date. Here, an interdisciplinary design using M13 bacteriophage virus as a biotemplate to carry Fe nanoclusters, which we figuratively call "Fe-nanonests", is proposed to enable facile and versatile synthesis of MSACs. The feasibility and generality of this self-assembly method was demonstrated by the observation of six different metal single atoms (MSAs) including Ag, Pt, Pd, Zn, Cu, and Ni. With Pd as a representative, key factors dominating the fabrication were determined. The Pd single atoms exhibited excellent horseradish peroxidase (HRP)-like activity, which was further improved by 50% via genetic editing of the M13 pVIII protein terminals. Excellent stability was also observed in the quantification of acid phosphatase, a cancer predictor. X-ray absorption near-edge structure spectroscopy has been applied to the analysis of Pd single atoms as well, and the Pd-N4 coordination explained the mechanism of high HRP-like catalytic activity. The MSAs synthesized by the M13 phage and Fe-nanonest self-assembly method show promising prospects in non-cold-chain medical detection applications.

Bio-modification of M13 bacteriophage as signal amplification container for the highly sensitive detection of live E. Coli

Sensitive and rapid detection of E. coli is important for food safety. Existing methods are limited in detection time or discriminating viable/dead bacteria. In this work, we report a rapid and sensitive method based on M13K07 phage whose pVIII proteins (about 2700 copies) are biomodified by streptavidin binding peptides (SBP)-tag for live E. coli analysis. The M13K07-SBP was used to inoculate contaminated food. After specific infection and rapid propagation of M13K07-SBP in live E. coli, plentiful progeny phages carried SBP tags on pVIII proteins that can load numerous signal transducers were yielded for signal amplification output. Owing to the replication and the great loading capacity of M13K07-SBP, as low as 4 cfu/mL of E. coli was detected in less than 3.5 h. Moreover, specific analysis and practical application were successfully confirmed. In brief, this work shows great potential in providing sensitive, rapid, and specific detection method for viable E. coli.

Virus-Based Pyroelectricity.

The first observation of heat-induced electrical potential generation on a virus and its detection through pyroelectricity are presented. Specifically, the authors investigate the pyroelectric properties of the M13 phage, which possesses inherent dipole structures derived from the noncentrosymmetric arrangement of the major coat protein (pVIII) with an α-helical conformation. Unidirectional polarization of the phage is achieved through genetic engineering of the tail protein (pIII) and template-assisted self-assembly techniques. By modifying the pVIII proteins with varying numbers of glutamate residues, the structure-dependent tunable pyroelectric properties of the phage are explored. The most polarized phage exhibits a pyroelectric coefficient of 0.13 µC m-2 °C-1 . Computational modeling and circular dichroism (CD) spectroscopy analysis confirm that the unfolding of α-helices within the pVIII proteins leads to changes in phage polarization upon heating. Moreover, the phage is genetically modified to enable its pyroelectric function in diverse chemical environments. This phage-based approach not only provides valuable insights into bio-pyroelectricity but also opens up new opportunities for the detection of various viral particles. Furthermore, it holds great potential for the development of novel biomaterials for future applications in biosensors and bioelectric materials.

Cryo-EM structure of a bacteriophage M13 mini variant

Filamentous bacteriophages package their circular, single stranded DNA genome with the major coat protein pVIII and the minor coat proteins pIII, pVII, pVI, and pIX. Here, we report the cryo-EM structure of a ~500 Å long bacteriophage M13 mini variant. The distal ends of the mini phage are sealed by two cap-like complexes composed of the minor coat proteins. The top cap complex consists of pVII and pIX, both exhibiting a single helix structure. Arg33 of pVII and Glu29 of pIX, located on the inner surface of the cap, play a key role in recognizing the genome packaging signal. The bottom cap complex is formed by the hook-like structures of pIII and pVI, arranged in helix barrels. Most of the inner ssDNA genome adopts a double helix structure with a similar pitch to that of the A-form double-stranded DNA. These findings provide insights into the assembly of filamentous bacteriophages.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

Human adenovirus B7 (HAdV-B7) causes severe acute lower respiratory tract infections in children. However, neither the child-specific antivirals or vaccines are available, nor the pathogenesis is clear. Autophagy, as part of innate immunity, plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity. This study provided evidence that HAdV-B7 infection induced complete autophagic flux, and the pharmacological induction of autophagy decreased HAdV-B7 replication. In this process, the host protein Bcl2-associated athanogene 3 (BAG3) mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain. These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.

Engineered M13 bacteriophage-enhanced colorimetric detection of allergenic fungi

Fungal spores are an important component of bio-aerosols and ubiquitous in indoor environments, leading to adverse health problems. Because concerns about indoor air quality have increased, rapid and sensitive detection methods are needed for personal and public health. In this study, we developed a sensitive colorimetric biosensor for detecting allergenic fungal spores using engineered M13 bacteriophages. Aspergillus niger spore-binding phages, showing high binding affinity to A. niger spores, were identified by phage display screening. The phages were biotinylated on the major coat protein pVIII for binding with streptavidin–HRP conjugates, resulting in enhanced color change. This engineered phage exhibited higher sensitivity and specificity for A. niger spores than conventionally used antibody-based detection systems.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Efficient Pathogen Capture and Sensing Promoted by Dynamic Deformable Nanointerfaces.

The M13 bacteriophage (M13 phage)has emerged as an attractive bionanomaterial due to its chemistry/gene modifiable feature and unique structures. Herein, a dynamic deformable nanointerface is fabricated taking advantage of the unique feature of the M13 phage for ultrasensitive detection of pathogens. PIII proteins at the tip of the M13 phage are genetically modified to display 6His peptide for site-specific anchoring onto Ni-NTA microbeads, whereas pVIII proteins along the side of the M13 phage are orderly arranged with thousands of aptamers and their complementary strands (c-apt). The flexible M13 nanofibers with rich recognition sites act as octopus tentacles, resulting in a 19-fold improvement in the capture affinity toward the target. The competitive binding of the target pathogen releases c-apts and initiates rolling circle amplification (RCA). The sway motion of M13 nanofibers accelerates the diffusion of c-apts, thus promoting RCA efficiency. Benefiting from the strengthened capture ability toward the target and the accelerated RCA process, three-orders of magnitude improvement in the sensitivity is achieved, with a detection limit of 8 cfu mL-1 forStaphylococcus aureus. The promoted capture ability and assay performance highlights the essential role of the deformable feature of the engineered interface. This may provide inspiration for the construction of more efficient reaction interfaces.

Gold nanoparticles-decorated M13 phage SPR probe for dual detection of antigen biomarkers in serum

Label-free SPR is rarely used for biomarker detection in real samples due to its low sensitivity. Herein, we report a phage-based SPR (P-SPR) strategy for the detection of carcinoembryonic antigen (CEA) in undiluted serums. M13 phage is genetically engineered to display an anti-CEA single-chain variable region fragment on pIII protein for antigen targeting and to display the gold binding peptide (GBP) at the pVIII protein for gold nanoparticles (GNPs) binding to form the GNPs decorated M13 phage probe that can amplify the plasmon resonance signal. The proposed P-SPR method demonstrated ultrasensitive detection of CEA within 10 min. Under ideal conditions, P-SPR showed exceptional sensitivity with a limit of detection (LOD) of 0.83 fM, which is approximately 2000 times that of the conventional SPR and four orders of magnitude more sensitive than enzyme-linked immunosorbent assay. In particular, the proposed strategy can cross-validate the SPR results through naked-eye counting of the plaques generated by the P-SPR probes, enabling a reliable dual detection system. In addition, P-SPR scheme has excellent sensing performance for the detection of real samples at concentrations lower than LOD of ELISA. Therefore, the proposed P-SPR strategy holds potential for early detection of tumour biomarkers in liquid biopsy.

M13 bacteriophage as biometric component for orderly assembly of dynamic light scattering immunosensor

The ordered assembly of nanostructure is an effective strategy used to manipulate the hydrodynamic diameter (DH) of nanoparticles. Herein, a versatile dynamic light scattering (DLS) immunosensing platform is presented to sensitively detect small molecules and biomacromolecules by using the M13 phage as the building module to order the assembly of gold nanoflowers and gold-coated magnetic nanoparticles, respectively. After the directional assembly of M13 phage, the DH of the probes was significantly increased due to its larger filamentous structure, thus improving the detection sensitivity of the DLS immunosensor. The designed M13 assembled DLS immunosensor with competitive and sandwich formats showed high sensitivities for ochratoxin A and alpha-fetoprotein in real corn and undiluted serum samples, with the detection limits of 1.37 and 57 pg/mL, respectively. These values are approximately 15.8 and 164.9 times lower than those of traditional phage-based enzyme-linked immunosorbent assays. Collectively, this work provides a promising strategy to manipulate the DH of nanoparticles by highly evolved biomaterials such as engineered M13 phages and opens upon a new direction for developing DLS immunosensors to detect various targets by the fusion expression of special peptide or nanobody on the pIII or pVIII protein of M13 phage.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate‐based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido‐modified glycans. The display of glycans on each phage virion can be controlled from 30‐1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec‐2) and DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin), and in live mice (Nat. Chem. Bio. 17, 806–816, 2021). From a mixture of 50‐100 multivalent glycan probes, LiGA identifies the glycan‐phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo.Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec‐7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec‐1 using a cell‐based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans‐ glycan to overcome the cis‐ masking on the surface of immune cells.

Filamentous prophage capsid proteins contribute to superinfection exclusion and phage defence in Pseudomonas aeruginosa.

Filamentous prophages in Pseudomonas aeruginosa PAO1 are converted to superinfective phage virions during biofilm development. Superinfection exclusion is necessary for the development of resistance against superinfective phage virions in host cells. However, the molecular mechanisms underlying the exclusion of superinfective Pf phages are unknown. In this study, we found that filamentous prophage-encoded structural proteins allow exclusion of superinfective Pf phages by interfering with type IV pilus (T4P) function. Specifically, the phage minor capsid protein pVII inhibits Pf phage adsorption by interacting with PilC and PilJ of T4P, and overproduction of pVII completely abrogates twitching motility. The minor capsid protein pIII provides partial superinfection exclusion and interacts with the PilJ and TolR/TolA proteins. Furthermore, pVII provides full host protection against infection by pilus-dependent lytic phages, and pIII provides partial protection against infection by pilus-independent lytic phages. Considering that filamentous prophages are common in clinical Pseudomonas isolates and their induction is often activated during biofilm formation, this study suggests the need to rethink the strategy of using lytic phages to treat P. aeruginosa biofilm-related infections.

Binding Capabilities of Different Genetically Engineered pVIII Proteins of the Filamentous M13/Fd Virus and Single-Walled Carbon Nanotubes.

Binding functional biomolecules to non-biological materials, such as single-walled carbon nanotubes (SWNTs), is a challenging task with relevance for different applications. However, no one has yet undertaken a comparison of the binding of SWNTs to different recombinant filamentous viruses (phages) bioengineered to contain different binding peptides fused to the virus coat proteins. This is important due to the range of possible binding efficiencies and scenarios that may arise when the protein’s amino acid sequence is modified, since the peptides may alter the virus’s biological properties or they may behave differently when they are in the context of being displayed on the virus coat protein; in addition, non-engineered viruses may non-specifically adsorb to SWNTs. To test these possibilities, we used four recombinant phage templates and the wild type. In the first circumstance, we observed different binding capabilities and biological functional alterations; e.g., some peptides, in the context of viral templates, did not bind to SWNTs, although it was proven that the bare peptide did. The second circumstance was excluded, as the wild-type virus was found to hardly bind to the SWNTs. These results may be relevant to the possible use of the virus as a “SWNT shuttle” in nano-scale self-assembly, particularly since the pIII proteins are free to act as binding-directing agents. Therefore, knowledge of the differences between and efficiencies of SWNT binding templates may help in choosing better binding phages or peptides for possible future applications and industrial mass production.

Development of a Novel Cytokine Vehicle Using Filamentous Phage Display for Colorectal Cancer Treatment.

Due to its highly immunogenic nature and the great engineerability, filamentous phage has shown promising antitumor activities in preclinical studies. Previous designs of antitumor phage mainly focused on tumor targeting using a cancer-specific moiety displayed on the minor capsid protein, pIII. In this work, we developed a new therapeutic platform of filamentous phage, in which the major capsid protein pVIII was utilized for displaying an antitumor cytokine. We showcased that a 16.1-kD cytokine GM-CSF could be efficiently presented on the M13 phage particle using the 8 + 8 type display system through a highly tolerable pVIII variant P8(1a). We verified that the GM-CSF phage was a potent activator for STAT5 signaling in murine macrophage. The GM-CSF phage significantly reduced the tumor size by more than 50% as compared to the unmodified phage in a murine colorectal cancer model. Immunological profiling of the tumor-infiltrating leukocytes revealed that an increase of CD4+ lymphocytes in the GM-CSF phage treatment group. Furthermore, the combined therapy of the GM-CSF phage and radiation greatly improved the therapeutic potency with a 100% survival rate and a 25% complete remission rate. We observed that the IFN-γ expression was dramatically up-regulated by the combined therapy in multiple types of tumor-infiltrating immune cells. Overall, we created a novel vehicle for cytokine therapy using the pVIII filamentous phage display. This new platform can be multiplexed with other phage engineering approaches, such as displaying targeting ligands on pIII or encapsulating therapeutic genes inside phage capsids, to create multifunctional nanoparticles for cancer therapy.

Design and proof of concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage.

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.