Artificial Protein Cage Delivers Active Protein Cargos to the Cell Interior.

Artificial protein cages have potential as programmable, protective carriers of fragile macromolecules to cells. While natural cages and VLPs have been extensively exploited, the use of artificial cages to deliver active proteins to cells has not yet been shown. TRAP-cage is an artificial protein cage with an unusual geometry and extremely high stability, which can be triggered to break apart in the presence of cellular reducing agents. Here, we demonstrate that TRAP-cage can be filled with a protein cargo and decorated with a cell-penetrating peptide, allowing it to enter cells. Tracking of both the TRAP-cage and the cargo shows that the protein of interest can be successfully delivered intracellularly in the active form. These results provide a valuable proof of concept for the further development of TRAP-cage as a delivery platform.

Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor.

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation.IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.

Virus Like Particles as Immunogens and Universal Nanocarriers.

Over the last two decades virus-like particles (VLPs) have become an important tool in biomedical research and medicine. VLPs are multiprotein structures that resemble viable virus particles in conformation but lack the viral genome. Consequently, they are non-infectious and non-replicative, but retain the ability to penetrate cells, making them useful for a vast spectrum of applications. Above all, VLPs mimicking genuine viruses in antigenic structure provide a safe alternative to attenuated and inactivated viruses in vaccine development. Moreover, due to their transducing proprieties, VLPs may efficiently deliver foreign nucleic acids, proteins, or conjugated compounds to the organism, or even to specific cell types. Additionally, VLPs are versatile nanovectors due to their flexibility in terms of composition and expression systems. In this review, different approaches for of virus-like particle synthesis and manipulation, as well as their potential applications, will be discussed.

Virus-like particles derived from bacteriophage MS2 as antigen scaffolds and RNA protective shells.

The versatile potential of bacteriophage MS2-derived virus-like particles (VLPs) in medical biotechnology has been extensively studied during the last 30 years. Since the first reports showing that MS2 VLPs can be produced at high yield and relatively easily engineered, numerous applications have been proposed. Particular effort has been spent in developing MS2 VLPs as protective capsules and delivery platforms for diverse molecules, such as chemical compounds, proteins and nucleic acids. Among these, two are particularly noteworthy: as scaffolds displaying heterologous epitopes for vaccine development and as capsids for encapsulation of foreign RNA. In this review, we summarize the progress in developing MS2 VLPs for these two areas.

Hollow, nanosized protein particles have many potential uses. If they can be appropriately engineered, they may for example be able to carry therapeutic cargoes to diseased cells or be used as a vaccine where appropriate antigens are mounted on their external surface. Many viruses offer a ready-made protein particle, the capsid, which can be made hollow by exclusion of the viral genetic material. MS2 is a virus that targets bacteria ­ a bacteriophage ­ which is well characterized and has been developed over many years for a number of applications. It has particular promise for development as a vaccine and for RNA delivery, both of which are reviewed here.

An artificial protein cage made from a 12-membered ring.

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.

Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron.

BACKGROUND: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. RESULTS: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. CONCLUSIONS: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

Low-volume label-free SARS-CoV-2 detection with the microcavity-based optical fiber sensor.

Accurate and fast detection of viruses is crucial for controlling outbreaks of many diseases; therefore, to date, numerous sensing systems for their detection have been studied. On top of the performance of these sensing systems, the availability of biorecognition elements specific to especially the new etiological agents is an additional fundamental challenge. Therefore, besides high sensitivity and selectivity, such advantages as the size of the sensor and possibly low volume of analyzed samples are also important, especially at the stage of evaluating the receptor-target interactions in the case of new etiological agents when typically, only tiny amounts of the receptor are available for testing. This work introduces a real-time, highly miniaturized sensing solution based on microcavity in-line Mach-Zehnder interferometer (µIMZI) induced in optical fiber for SARS-CoV-2 virus-like particles detection. The assay is designed to detect conserved regions of the SARS-CoV-2 viral particles in a sample with a volume as small as hundreds of picoliters, reaching the detection limit at the single ng per mL level.

Programmable polymorphism of a virus-like particle.

Virus-like particles (VLPs) have significant potential as artificial vaccines and drug delivery systems. The ability to control their size has wide ranging utility but achieving such controlled polymorphism using a single protein subunit is challenging as it requires altering VLP geometry. Here we achieve size control of MS2 bacteriophage VLPs via insertion of amino acid sequences in an external loop to shift morphology to significantly larger forms. The resulting VLP size and geometry is controlled by altering the length and type of the insert. Cryo electron microscopy structures of the new VLPs, in combination with a kinetic model of their assembly, show that the abundance of wild type (T = 3), T = 4, D3 and D5 symmetrical VLPs can be biased in this way. We propose a mechanism whereby the insert leads to a change in the dynamic behavior of the capsid protein dimer, affecting the interconversion between the symmetric and asymmetric conformers and thus determining VLP size and morphology.

Functional Severe Acute Respiratory Syndrome Coronavirus 2 Virus-Like Particles From Insect Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major epidemic threat since the beginning of 2020. Efforts to combat the virus and the associated coronavirus disease 2019 (COVID-19) disease are being undertaken worldwide. To facilitate the research on the virus itself, a number of surrogate systems have been developed. Here, we report the efficient production of SARS-CoV-2 virus-like particles (VLPs) in insect cells. Contrary to widely used pseudovirus particles, where only one coronaviral protein is displayed within a heterologous scaffold, developed VLPs are structurally similar to the native virus and allow for more throughput studies on the biology of the infection. On the other hand, being devoid of the viral genome, VLPs are unable to replicate and thus safe to work with. Importantly, this is the first report showing that SARS-CoV-2 VLPs can be efficiently produced in insect cells and purified using scalable affinity chromatography.

Kallikrein 13 serves as a priming protease during infection by the human coronavirus HKU1.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.

Novel coronavirus-like particles targeting cells lining the respiratory tract.

Virus like particles (VLPs) produced by the expression of viral structural proteins can serve as versatile nanovectors or potential vaccine candidates. In this study we describe for the first time the generation of HCoV-NL63 VLPs using baculovirus system. Major structural proteins of HCoV-NL63 have been expressed in tagged or native form, and their assembly to form VLPs was evaluated. Additionally, a novel procedure for chromatography purification of HCoV-NL63 VLPs was developed. Interestingly, we show that these nanoparticles may deliver cargo and selectively transduce cells expressing the ACE2 protein such as ciliated cells of the respiratory tract. Production of a specific delivery vector is a major challenge for research concerning targeting molecules. The obtained results show that HCoV-NL63 VLPs may be efficiently produced, purified, modified and serve as a delivery platform. This study constitutes an important basis for further development of a promising viral vector displaying narrow tissue tropism.

The inhibitory effect of secretory leukocyte protease inhibitor (SLPI) on formation of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs), web-like DNA structures, provide efficient means of eliminating invading microorganisms but can also present a potential threat to its host because it is a likely source of autoantigens or by promoting bystander tissue damage. Therefore, it is important to identify mechanisms that inhibit NET formation. Neutrophil elastase (NE)-dependent chromatin decondensation is a key event in the release of NETs release. We hypothesized that inhibitors of NE, secretory leukocyte protease inhibitor (SLPI) and α(1)-proteinase inhibitor (α(1)-PI), has a role in restricting NET generation. Here, we demonstrate that exogenous human SLPI, but not α(1)-PI markedly inhibited NET formation in human neutrophils. The ability of exogenous SLPI to attenuate NET formation correlated with an inhibition of a core histone, histone 4 (H4), cleavage, and partial dependence on SLPI-inhibitory activity against NE. Moreover, neutrophils from SLPI(-/-) mice were more efficient at generating NETs than were neutrophils from wild-type mice in vitro, and in experimental psoriasis in vivo. Finally, endogenous SLPI colocalized with NE in the nucleus of human neutrophils in vitro, as well as in vivo in inflamed skin of patients with psoriasis. Together, these findings support a controlling role for SLPI in NET generation, which is of potential relevance to infectious and autoinflammatory diseases.

The structural basis for the integrity of adenovirus Ad3 dodecahedron.

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Maternally inherited diabetes with deafness and obesity: body weight reduction response to treatment with insulin analogues.

Maternally inherited diabetes with deafness (MIDD) is a rare, monogenic form of diabetes mellitus caused by mutations in the mitochondrial genome, the most frequent being the A3243G substitution of the tRNA(Leu) gene. We screened 520 individuals with type 2 diabetes mellitus and 45 probands from families with a clinical picture of maturity onset diabetes of the young (MODY) using restriction fragment length polymorphism. One carrier of the mutation being investigated was found in a proband from a MODY family. The patient was a 20 year-old woman, diagnosed at the age of 16 years as having type 1 diabetes mellitus. On entry to the study, she was treated by a multiple daily injection regimen (MDI) with regular human insulin and human NPH insulin. Typical extra-pancreatic symptoms of MIDD were present, such as macular pattern dystrophy and mild bilateral sensory hearing loss. Additionally, the patient presented abdominal obesity (BMI 32.0), an uncommon feature in monogenic insulin secretion defects, including MIDD. To facilitate weight loss, the diabetes treatment was modified. Since metformin treatment is considered to be contraindicated in MIDD because of the increased risk of lactic acidosis, we used insulin analogues (aspart and detemir) in an MDI regimen and hypocaloric diet. This resulted in a 6.3 kg weight reduction (BMI 27.4) and normalization of HbA1c level (from 7.2 to 6.1 %) during a three-month follow-up. On the basis of this case, we suggest that an MDI regimen with insulin analogues may be a preferred therapeutic option in some rare clinical situations, such as MIDD associated with obesity.