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 ␣1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on ␣2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal ␣2-3-or ␣2-6-linked sialic acid or the branched ␣2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal ␣2-6-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 ␣2-6-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.The human neurotopic polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). To date, the mortality rate from PML is high, and there is no appropriate therapy for treating PML. The viral route of infection and the mechanisms of viral spread have not been conclusively identified; however, as JCV is frequently detected in untreated urban sewage and in sewage-exposed shellfish, a fecal-oral route of transmission has been suggested (4). Recently, JCV DNA was detected in several organs, including the tonsils and upper and lower gastrointestinal tracts (13,22). Given that JCV ingested by the oral route may enter the intestinal wall and Peyer's patches and thus peripheral blood lymphocytes and various organs (4), the cell surface receptor for JCV is one of the potential therapeutic targets for controlling JCV infection.The JCV receptor has been described as a glycoprotein containing terminal ␣2-6-linked sialic acid, on the basis of the finding that sialidase but not ␣2-3-specific sialidase inhibited infection of glial cells by JCV. In addition, the N glycosylation inhibitor tunicamycin inhibited JCV infection (12). Certain viruses utilize the sialooligosaccharides of glycoproteins and glycolipids for their attachment to host cells an...