Agglutination of Human Polyomaviruses by Using a Tetravalent Glycocluster as a Cross-Linker

Abstract

Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand–virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.

Middle-molecular-weight Glycoclusters for the Crosslinking of Multivalent Lectins

“…As a result, the virus particles were retained on the filtration membrane when the compound 10−MCPyV-LP aggregates were applied to a general sterilization membrane filter (0.20 µm) for microbial filtration. Furthermore, this membrane filtration assay showed effective virus trapping of infectious human polyomavirus JCPyV (60). In other words, these results suggest the possibility of virus aggregation using middlemolecular-weight glycoclusters as a new method for virus capture and/or purification method.…”

Virus-Like Particles With FLAG-Tagged Envelope Protein as A Tetravalent Dengue Vaccine Candidate

“…TEM analysis was performed as described previously 31 . In brief, VLP solutions were concentrated with Amicon ultra 0.5 centrifugal lter 10k (Merck, Darmstadt, Germany) and exchange solvent to ultra pure water, followed by placing onto a formvar-coated copper grid and negatively stained with 2% uranyl acetate solution.…”