Β-annulus Peptide Research Articles

A nanoparticle vaccine based on the VP121–26 and VP2 structural proteins of Senecavirus A induces robust protective immune responses

Senecavirus A (SVA) is a causative agent that can cause vesicular disease in swine, which causes a great threat to the swine husbandry in the world. Therefore, it is necessary to develop a vaccine that can effectively prevent the spread of SVA. In this study, we developed a 24-polymeric nano-scaffold using β-annulus peptide from tomato bushy effect virus (TBSV) by coupling this antigen to SVA B cell epitope VP121–26 and VP2 proteins via linkers, respectively. The SVA-based nanoparticle protein of the VP1(B)-β-VP2 was expressed and purified by low-cost prokaryotic system to prepare a SVA nanoparticle vaccine. The immunological protective effect of SVA nanoparticle vaccine was evaluated in mouse and swine models, respectively. The results suggested that both mice and swine could induce high levels SVA neutralizing antibodies and IgG antibodies after two doses immunization. In addition, the swine challenge protection experiment showed that the protection rate of immune SVA nanoparticle vaccine and SVA inactivated vaccine both were 80 %, while the negative control had no protection effect. It demonstrated that SVA nanoparticle vaccine effectively prevented SVA infection in swine. In summary, the preparation of SVA vaccine by using β-annulus peptide is a promising candidate vaccine for prevent SVA transmission, and provides a new idea for the development of novel SVA vaccines.

Enveloped Viral Replica Equipped with Spike Protein Derived from SARS-CoV-2.

Synthetic viral nanostructures are useful as materials for analyzing the biological behavior of natural viruses and as vaccine materials. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus embedding a spike (S) protein involved in host cell infection. Although nanomaterials modified with an S protein without an envelope membrane have been developed, they are considered unsuitable for stability and functionality. We previously constructed an enveloped viral replica complexed with a cationic lipid bilayer and an anionic artificial viral capsid self-assembled from β-annulus peptides. In this study, we report the first example of an enveloped viral replica equipped with an S protein derived from SARS-CoV-2. Interestingly, even the S protein equipped on the enveloped viral replica bound strongly to the free angiotensin-converting enzyme 2 (ACE2) receptor as well as ACE2 localized on the cell membrane.

Alkyl anchor–modified artificial viral capsid budding outside-to-inside and inside-to-outside giant vesicles

ABSTRACT The budding of human immunodeficiency virus from an infected host cell is induced by the modification of structural proteins bearing long-chain fatty acids, followed by their anchoring to the cell membrane. Although many model budding systems using giant unilamellar vesicles (GUVs) induced by various stimuli have been developed, constructing an artificial viral budding system of GUVs using only synthesized molecules remains challenging. Herein, we report the construction of an artificial viral capsid budding system from a lipid bilayer of GUV. The C-terminus of the β-annulus peptide was modified using an octyl chain as an alkyl anchor via a disulfide bond. The self-assembly of the β-annulus peptide with an octyl chain formed an artificial viral capsid aggregate. The fluorescence imaging and transmission electron microscopy observations revealed that the addition of the tetramethylrhodamine (TMR)-labeled octyl chain–bearing β-annulus peptide to the outer aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle outside-to-inside the mother GUV. Conversely, the encapsulation of the TMR-labeled octyl chain–bearing β-annulus peptide in the inner aqueous phase of GUV induced the budding of the capsid-encapsulated daughter vesicle inside-to-outside the mother GUV. Contrarily, the addition of the TMR-labeled β-annulus peptide to GUV barely induced budding. It was demonstrated that the higher the membrane fluidity of GUV, the more likely budding would be induced by the addition of the alkyl anchor–modified artificial viral capsid. The simple virus-mimicking material developed in this study, which buds off through membrane anchoring, can provide physicochemical insights into the mechanisms of natural viral budding from cells.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified β-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

A supramolecular system mimicking the infection process of an enveloped virus through membrane fusion

Membrane fusion is an essential step for the entry of enveloped viruses, such as human immunodeficiency virus and influenza virus, into the host cell, often triggered by the binding of membrane proteins on the viral envelope to host cell membrane. Recently, external stimuli was shown to trigger membrane fusion in an artificial system. Direct observation of artificial membrane fusion using a giant unilamellar vesicle (GUV), which is similar in size to a cell, is useful as a biological model system. However, there are no model systems for studying membrane fusion of enveloped viruses with host cells. Here, we report a supramolecular model system for viral entry into a GUV or cell through membrane fusion. The system was constructed by complexing a cationic lipid bilayer on an anionic artificial viral capsid, self-assembled from viral β-annulus peptides. We demonstrate that the cationic enveloped artificial viral capsid electrostatically interacts with the anionic GUV or cell, and the capsid enters the GUV or cell through membrane fusion. The model system established in this study will be important for analyzing membrane fusion during infection of a natural virus.

Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled β-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

An artificial viral capsid decorated with a DNA aptamer internalizing into lymphoma cells.

Tumor-specific drug-delivering nanocarriers could be a promising modality for next-generation tumor therapy. Here we developed a Burkitt lymphoma-specific DNA aptamer-labeled nanocarrier using the β-Annulus peptide, which forms a spherical nanoassembly called artificial viral capsid. Dynamic light scattering and transmission electron microscopy of the DNA aptamer-decorated artificial viral capsid showed the formation of spherical assemblies with a diameter of approximately 50-150 nm. The artificial viral capsid was selectively internalized into the Burkitt lymphoma cell line, Daudi, and doxorubicin complexed with the capsid selectively killed Daudi cells.

Stabilization of Artificial Viral Capsid Using Surface Modification with BODIPY

This study shows that β-annulus peptide modified with boron-dipyrromethene (BODIPY) at the C-terminal self-assembled into stable artificial viral capsid even at 1 µM, in contrast to unmodified or N-terminally modified peptides, which cannot self-assemble. Even when sodium dodecyl sulfate was present, the artificial viral capsid, which was stabilized by presumably π–π stacking interactions between BODIPYs at the surface, nearly retained the apparent diameter and formation ratio.

Virus-like particles of Macrobrachium rosenbergii nodavirus: Particle size and capsid protein assembly domain

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in the giant freshwater prawn, which poses a major issue in the aquaculture sector. The capsid protein (CP) of MrNv expressed in Escherichia coli self-assembles into virus-like particles (VLPs) with a triangulation number, T = 3. However, the region in MrNv CP that involved in the viral capsid formation has yet to be identified. In the present study, a series of N- and C-terminal MrNv CP deletion mutants were constructed based on the viral subatomic structure. These mutants were then expressed in E. coli and purified with an immobilized metal affinity chromatography (IMAC). The formation of VLPs was determined using transmission electron microscopy (TEM). The results showed that, the first 29 amino acid residues at the N-terminal end of CP are dispensable for VLP formation, but deletion of these residues produced a smaller VLP. Disruption of the CP N-terminal arm (NTA) that contains a β-annulus motif resulted in no protein expression in E. coli. The β-annulus peptide was chemically synthesized, and analysed with TEM, dynamic light scattering (DLS), size-exclusion chromatography (SEC) and native agarose gel electrophoresis, but it did not assemble into VLPs. Truncation of the entire protruding (P) domain did not disrupt the VLP formation. However, deletion of the amino acid residues involved in dimeric interaction between subunits in the shell (S) domain impaired VLP assembly. In conclusion, the minimum contiguous sequence of the CP required for VLP formation was delineated in residues 30 and 252. This study provides mechanistic insights into the assembly of MrNv VLP, which will further broaden its applications in vaccine development and nucleic acid delivery.

Artificial Nanocage Formed via Self-Assembly of β-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the β-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

Embedding a membrane protein into an enveloped artificial viral replica.

Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from β-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection via the anti-Cx43 antibody, using fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Interestingly, many spherical structures connected to each other were observed in TEM images of the Cx43-embedded enveloped viral replica. In addition, it was shown that fluorescent dyes could be selectively transported from Cx43-embedded enveloped viral replicas into Cx43-expressing HepG2 cells. This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins.

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid.

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer β-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of β-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (Kd) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

A Photoresponsive Artificial Viral Capsid Self-Assembled from an Azobenzene-Containing β-Annulus Peptide.

Photoinduced structural changes in peptides can dynamically control the formation and dissociation of supramolecular peptide materials. However, the existence of photoresponsive viral capsids in nature remains unknown. In this study, we constructed an artificial viral capsid possessing a photochromic azobenzene moiety on the peptide backbone. An azobenzene-containing β-annulus peptide derived from the tomato bushy stunt virus was prepared through solid-phase synthesis using Fmoc-3-[(3-aminomethyl)-phenylazo]phenylacetic acid. The azobenzene-containing β-annulus (β-Annulus-Azo) peptide showed a reversible trans/cis isomerization property. The β-annulus-azo peptide self-assembled at 25 μM into capsids with the diameters of 30–50 nm before UV irradiation (trans-form rich), whereas micrometer-sized aggregates were formed after UV irradiation (cis-form rich). The artificial viral capsid possessing azobenzene facilitated the encapsulation of fluorescent-labeled dextrans and their photoinduced release from the capsid.

Horseradish Peroxidase-Decorated Artificial Viral Capsid Constructed from β-Annulus Peptide via Interaction between His-Tag and Ni-NTA

Artificial construction of spherical protein assemblies has attracted considerable attention due to its potential use in nanocontainers, nanocarriers, and nanoreactors. In this work, we demonstrate a novel strategy to construct peptide nanocapsules (artificial viral capsids) decorated with enzymes via interactions between His-tag and Ni-NTA. A β-annulus peptide derived from the tomato bushy stunt virus was modified with Ni-NTA at the C-terminus, which is directed toward the exterior surface of the artificial viral capsid. The β-annulus peptide bearing Ni-NTA at the C-terminus self-assembled into capsids of about 50 nm in diameter. The Ni-NTA-displayed capsids were complexed with recombinant horseradish peroxidase (HRP) with a C-terminal His-tag which was expressed in Escherichia coli. The β-annulus peptide-HRP complex formed spherical assemblies whose sizes were 30–90 nm, with the ζ-potential revealing that the HRP was decorated on the outer surface of the capsid.

Encapsulation of mRNA into Artificial Viral Capsids via Hybridization of a β-Annulus-dT20 Conjugate and the Poly(A) Tail of mRNA

Messenger RNA (mRNA) drugs have attracted considerable attention as promising tools with many therapeutic applications. The efficient delivery of mRNA drugs using non-viral materials is currently being explored. We demonstrate a novel concept where mCherry mRNA bearing a poly(A) tail is encapsulated into capsids co-assembled from viral β-annulus peptides bearing a 20-mer oligothymine (dT20) at the N-terminus and unmodified peptides via hybridization of dT20 and poly(A). Dynamic light scattering measurements and transmission electron microscopy images of the mRNA-encapsulated capsids show the formation of spherical assemblies of approximately 50 nm. The encapsulated mRNA shows remarkable ribonuclease resistance. Further, modification by a cell-penetrating peptide (His16) on the capsid enables the intracellular expression of mCherry of encapsulated mRNA.

Functional Peptide Nanocapsules Self-Assembled from β-Annulus Peptides.

Spherical viruses are unique nanocapsules formed by self-assembly of coat proteins (capsids). By mimicking natural spherical capsids, various artificial viral capsids are developed by using self-assembled proteins and peptides as building blocks. We developed an artificial viral capsid consisting of a β-annulus peptide designed from natural viruses. The "β-annulus capsid" can be functionalized by encapsulating guest molecules to the inside and decoration of exogenous molecules on the outside. Here, we describe the encapsulation and decoration on the β-annulus capsids by connecting additional sequences to the β-annulus peptide, conjugation with objective molecules, and subsequent self-assembly in aqueous solutions.

Dressing up artificial viral capsids self-assembled from C-terminal-modified β-annulus peptides

A variety of chemical approaches for the rational design of artificial proteins and peptides have been developed in recent years for the construction of self-assembled nanocapsules. It was previously found that a synthetic 24-mer β-annulus peptide, which participates in the formation of the dodecahedral internal skeleton of the tomato bushy stunt virus capsid, spontaneously self-assembled into artificial viral capsids with a size of 30–50 nm. These artificial viral capsids were established to encapsulate various guest molecules, such as anionic dyes, DNA, quantum dots, and His-tagged proteins. The artificial viral capsids could also be dressed up with gold nanoparticles, single-stranded DNA, coiled-coil spikes, and proteins by modifying with these molecules at the C-terminus of β-annulus peptides. The artificial viral capsids were notably stabilized by dressing up with human serum albumin and acquired enzymatic activity by dressing up with ribonuclease. It was found that a synthetic 24-mer β-annulus peptide, which participates in the formation of the dodecahedral internal skeleton of the tomato bushy stunt virus capsid, spontaneously self-assembled into artificial viral capsids with a size of 30–50 nm. The artificial viral capsids could be dressed up with gold nanoparticles, single-stranded DNA, coiled-coil spikes, and proteins by modifying with these molecules at the C-terminus of β-annulus peptides. The artificial viral capsids were notably stabilized by dressing up with human serum albumin and acquired enzymatic activity by dressing up with ribonuclease.

Enveloped artificial viral capsids self-assembled from anionic β-annulus peptide and cationic lipid bilayer.

Anionic artificial viral capsids were self-assembled from β-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.

Construction of Artificial Viral Capsids Encapsulating Short DNAs via Disulfide Bonds and Controlled Release of DNAs by Reduction

To construct an artificial viral capsid encapsulated short single-stranded DNA, a β-annulus peptide conjugated with ssDNA through a disulfide bond at the N-terminus (DNA-SS-β-Annulus) was synthesiz...