Rika Komagome | PubMed Health

PubMed2005

The JC virus-like particle overlay assay.

JC virus (JCV) belongs to the family of double-stranded DNA polyomaviruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML). It has been reported that sialic acids play a pivotal role in hemagglutination of red blood cells and entry into host cells of JCV and that JCV can enter a wide variety of cell types and localize to the nuclei. The outer shell of the JCV virion comprises the major capsid protein VP1, and a virus-like particle (VLP) consisting of recombinant VP1 made from Escherichia coli exhibit a virion-like structure and physiological functions (cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. To examine the mechanism of cell attachment of JCV, an overlay assay using a VLP has been developed, revealing that sialoglycoproteins, including alpha1 acid-glycoprotein, fetuin, and transferrin receptor bind with VLP. In addition, VLPs bind to glycolipids, such as lactosylceramide and gangliosides including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and VLP weakly bind to GD1a. In this section, detailed procedures for the synthesis of VLP from E. coli and VLP overlay assay are described.

Electrophoresis, Polyacrylamide Gel↗

PubMed2002

Oligosaccharides as receptors for JC virus.

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including alpha1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on alpha2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal alpha2-3- or alpha2-6-linked sialic acid or the branched alpha2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal alpha2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal alpha2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.

Gangliosides↗