Cysteine residues in the major capsid protein, Vp1, of the JC virus are important for protein stability and oligomer formation.

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.

Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells.

The efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is an important theme in the biomedical and pharmaceutical field. In this study, we synthesized virus-like particles (VLPs) coupled with cyclodextrins (CDs) as hydrophobic pockets through disulfide bonds inside the VLPs, where hydrophobic drugs can be incorporated. We report here the intracellular delivery of hydrophobic dyes or drugs encapsulated in VLPs through CDs with high efficiency and their subsequent release in cells in response to glutathione. As a model anticancer drug, paclitaxel (PTX)-CD complexes were encapsulated inside VLPs and the cytotoxic drug activity of PTX loaded VLPs against NIH3T3 cells was evaluated by CCK-8 assay. PTX-loaded VLPs exhibited a dose-dependent cytotoxic effect with a 20-fold smaller IC(50) than that of free PTX dissolved in DMSO. These results indicate that VLPs with removable CDs afford highly promising carriers of hydrophobic drugs without chemical modification of drugs.

Inspiration from chemical photography: accelerated photoconversion of AgCl to functional silver nanoparticles mediated by DNA.

We demonstrate a facile approach for converting AgCl to functional silver nanoparticles (AgNPs) via photoreduction in the presence of DNA. The resulting AgNPs are biofunctionalized, and exhibit photostable luminescence and DNA-specific Raman signatures, showing high potential for use in DNA-directed recognition and advanced bioimaging.

Gold nanoparticle arrangement on viral particles through carbohydrate recognition: a non-cross-linking approach to optical virus detection.

We propose a new approach to optical virus detection based on the spatial assembly of gold nanoparticles on the surface of viruses. Since JC virus-like particles (VLPs) comprise a repeating viral capsid protein that binds to sialic acid, the conjugation of sialic acid-linked Au particles with VLPs enables the spatial arrangement of Au particles on the VLP surface. This structure produced a red shift in the absorption spectrum due to plasmon coupling between adjacent Au particles, leading to the construction of an optical virus detection system. Our system depends not on the simple cross-linking of VLPs and Au particles, but on an ordered Au structure covering the entire surface of the VLPs and can be applied to various virus detection systems using the inherent ligand recognition of animal viruses.

Enhanced cellular uptake of virus-like particles through immobilization on a sialic acid-displaying solid surface.

Herein, we present the efficient cellular uptake of immobilized virus-like particles (VLPs) made of recombinant JC virus capsid proteins. VLPs expressed in Escherichia coli were labeled with fluorescein isothiocyanate (FITC). We compared two approaches for the cellular uptake of the FITC-VLPs. In the first approach, FITC-VLPs were immobilized on a polystyrene substrate, and then NIH3T3 cells were cultured on the same substrate. In the second approach, cells were cultured on a polystyrene substrate, and FITC-VLPs were then added to the cell culture medium. Flow cytometric analysis and confocal laser microscopic observation revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were the diffusive VLPs suspended in solution. The cellular uptake of VLPs on the substrate was increased in a VLP density-dependent manner. As a control, disassembling VLPs to form VP1 pentamers abolished incorporation into the cells. Displaying sialic acid on the substrate enhanced VLP density through the specific affinities between the VLPs and sialic acid, resulting in efficient incorporation into the seeded cells. These techniques can be applied to the development of novel drug delivery systems and cell microarrays not only of nucleic acids but also of small molecules and proteins through their encapsulation in VLPs.