In vitro refolding of the structural protein VP1 of parvovirus B19 produces virus-like particles with functional VP1 unique region.

Virus-like particles (VLPs) from Parvovirus B19 (B19V) can be obtained by the self-assembly of the structural proteins VP1 and VP2. It is possible to produce B19V VLPs either from VP2 or a mixture of VP1 and VP2, through its heterologous expression in eukaryotic cells. The difference between VP1 and VP2 protein is a tract of 227 residues located at the N-terminal region of VP1, known as the VP1 unique region (VP1u). This region is critical for B19V infection, including tropism, cell internalization, and lysosomal scape through its phospholipase 2A activity. Herein, we report the in vitro self-assembly of VP1 to form VLPs. These species have phospholipase activity, suggesting that the phospholipase domain is correctly folded. Furthermore, VP1 and VP2 were co-assembled to produce hybrid VLPs which were able to bind and internalize in the non-permissive HepG2 cells, another evidence of the functionality of the in vitro refolded VP1u.

Decoration of virus-like particles with an enzymatic activity of biomedical interest.

The natural properties of virus-like particles (VLPs), like their nanometric size, polyvalence, monodispersity and biocompatibility, had called the attention of scientists from different fields. VLPs constitute an excellent platform for the development nanomaterials with a broad spectrum of applications, ranging from physics of soft matter to the development of vaccines and biological nanocarriers. To expand the repertoire of functions of VLPs, they can be decorated with different molecules. In this research, the α-glucosidase Ima1p of Saccharomyces cerevisiae was attached to the surface of in vitro assembled VLPs of parvovirus B19, by using the SpyTag/SpyCatcher system. The resulting particles were structurally characterized displaying a noticeable increase in size compared to the non-decorated VLPs. The study of the biochemical properties of the coupled enzyme indicate that it increased its Vmax by three-fold toward p-nitrophenyl-α-D-glucopyranoside (p-NPG) as substrate. In addition, the linked enzyme displayed a notorious 10 °C shift in its optimal temperature, from 35 °C for the non-attached enzyme, to 45 °C for the enzyme attached to VLPs. The decorated VLPs were also able to act on glycogen; therefore, these particles may be further developed as part of the therapy for treatment of lysosomal storage diseases derived from defects in the human acid α-glucosidase.

Thermal, conformational and rheological properties of κ-carrageenan-sodium stearoyl lactylate gels and solutions.

Polysaccharide-surfactant blends are widely used in foods. However, their possible mutual interactions have not been extensively studied. The purpose of this work was to examine how the anionic surfactant sodium stearoyl lactylate (SSL) affects different properties of κ-carrageenan solutions and gels. Rheometry, differential scanning calorimetry, asymmetrical flow field-flow fractionation coupled with multiangle laser light scattering, among others, were used to determine the flow and viscoelastic behavior, thermal transitions, and conformation changes, respectively. Interference caused by SSL is postulated as the primary factor to explain the variations in the conformation of κ-carrageenan in gels and solutions. However, electrostatic repulsions between κ-carrageenan and SSL can also be involved. These latter interactions are more important for high SSL concentrations (13 mmol dm-3) without addition of KCl, because of the higher net negative charge density of the system. SSL significantly modifies the properties of κ-carrageenan in aqueous media.

Production and characterization of a nanocomposite of highly crystalline nanowhiskers from biologically extracted chitin in enzymatic poly(ε-caprolactone).

A nano-composite from biologically obtained chitin nanofillers homogenously dispersed in a poly(ε-caprolactone) matrix was successfully achieved by an ultrasonication-assisted non-toxic and non-aqueous methodology. For this purpose, biological chitin was obtained from lactic acid fermentation of shrimp wastes and converted into chitin whiskers by acidic hydrolysis in a novel process at low temperature (4°C) that enhanced the distribution and yield. Additionally, the polyester matrix was enzymatically produced in a non-toxic compressed fluid (1,1,1,2-tetrafluoroethane at 25bar and 65°C) medium. The homogeneous distribution of the nanofiller in the matrix was corroborated by confocal and atomic force microscopies. Films of the nanocomposite were physicochemically characterized to assess its adequate properties. Additionally, the qualitative viability of human fibroblasts and osteoblasts cells was studied on the produced nanocomposite films showing good biocompatibility.

Construction of protein-functionalized virus-like particles of parvovirus B19.

Decoration of virus-like particles (VLPs) expands the repertory of functions these particles can display. In the last years, VLPs have successfully been used as scaffolds to present different molecules, frequently through the specific reaction of chemical groups on the surface of the particles, or by protein engineering when the presentation of peptides or proteins is the primary goal. VLPs of parvovirus B19 (B19V), have been previously produced in vitro and its stability and ability to assemble into hybrid particles composed of wild-type and chimeric proteins evidenced their potential as research tools. Herein, we report the presentation of functional proteins on the surface of B19V VLPs, through the fusion of the gene coding for the heterologous protein within the gene coding for the structural protein VP2. Two model proteins were used for the construction of chimeras, a lipase from Bacillus pumilus (BplA) and the enhanced green fluorescent protein (EGFP). Both chimeras were folded and successfully assembled in vitro into VLPs. While the BplA chimera exhibited esterase activity, the chimera of EGFP showed no fluorescence. We replaced the EGFP by its fast-folding derivative "super folder GFP" (sfGFP) flanked by larger linkers to increase its movement freedom, which resulted in fluorescent protein able to assemble fluorescent VLPs. These results expand the toolbox for VLP decoration as well as for the construction of new nanobiomaterials.

Peptide display on a surface loop of human parvovirus B19 VP2: Assembly and characterization of virus-like particles.

Virus-like particles (VLPs) are valuable tools for nanotechnology and nanomedicine. These particles are obtained by the self-assembly, either in vivo or in vitro, of structural proteins of viral capsids. VLPs are excellent scaffolds for surface display of molecules. The N-termini of the structural proteins of human parvovirus B19 (B19V) have been already modified to display peptides or proteins. However, other surface-exposed elements have not been studied as potential locations for peptide display. In this research, we tested the potential of surface loop 62-75 of VP2 protein for the presentation of a 64-residue heterologous peptide. The chimeric protein was able to self-assemble in vitro into VLPs. Improved colloidal stability was observed for these particles, indicating that the peptide is on the surface of the particle. AFM analysis of the chimeric particles shows no obvious difference between the surfaces of particles assembled with VP2 and those assembled with the chimeric VP2. Our results indicate that loop 62-75 is a good candidate for heterologous peptide presentation on the surface of B19V VLPs.