Broad Distribution of the JC Virus Receptor Contrasts with a Marked Cellular Restriction of Virus Replication

Abstract

To investigate the early events of JC virus (JCV) infection, including attachment, penetration, transport to the nuclei, and replication of the virus, we analyzed the susceptibility of 15 different cell lines to infection using a semiquantitative polymerase chain reaction (PCR) assay, in situ hybridization, laser scanning confocal microscopy, and a viral replication assay. The cell lines examined were human permissive and nonpermissive cells as well as cells of monkey and mouse origin. JCV entry into the nuclei of the all cell lines was observed within 10 min after inoculation, demonstrating that the virus receptor is widely distributed among mammalian cells. Inhibition of viral entry by an anti-JCV VP1 antibody and sialidase treatment to remove sialic acid residues, which are considered a candidate for the JCV receptor, suggested that VP1 may interact with the cellular surface sialic acid. In addition, chlorpromazine, a clathrin-dependent pathway inhibitor, significantly suppressed entry of JCV into nuclei. In spite of the broad spectrum of cells susceptible to JCV entry, replication of the virus occurred exclusively in human neuroblastoma cell lines. These results suggest that whereas JCV can enter a wide variety of cell types and localize to the nuclei, cell-specific intranuclear mechanisms are required for virus replication.

Nuclear Entry Mechanism of the Human Polyomavirus JC Virus-like Particle

“…We had previously shown that wtVLPs are transported to the nucleus of various other cell types (19). These observations indicate that wtVLPs are able to enter the nucleus of both permissive and nonpermissive cells, and, together with the results of previous studies (4,19), they suggest that the neurotropism of JCV is attributable to intranuclear mechanisms such as DNA replication, transcription, or virus assembly.…”

“…The cells were then examined with a laser-scanning confocal microscope (Olympus, Tokyo, Japan). Preparation of VLPs-VLPs composed of wtVP1 or ⌬NLS-VP1 were prepared as previously described (19), with slight modifications. The expression plasmids for wtVP1 and ⌬NLS-VP1 were separately introduced into competent E. coli BL21(DE3)/pLys cells (Stratagene, La Jolla, CA) by transformation, and expression of the recombinant proteins was induced by incubation of the cells for 4 h at 30°C with 1 mM isopropyl-␤-D-thiogalactopyranoside.…”

“…This NLS of JCV VP1 was shown to be inefficient in mediating nuclear transport (16). However, JCV virus-like particles (VLPs) consisting exclusively of recombinant VP1 purified from either Escherichia coli or a baculovirus-insect cell expression system and assembled by the formation of disulfide bonds (17) were found to be transported to the nucleus of host cells (18,19). We have now investigated the mechanism of JCV nuclear entry with VLPs that exhibit both morphological characteristics and physiological functions, including hemagglutination activity, cell attachment, and cellular trafficking, similar to those of JCV virions (18 -20).…”

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

“…Hemagglutin (HA) Assay-Measurement of JCV titer was based on HA of human type O erythrocytes as described previously (35). Human blood was supplied from the Hokkaido Red Cross Blood Center.…”

Detection and characterization of a novel polyomavirus in wild rodents

“…Cells were fixed for 3 min in 100 % methanol at 220 uC. Non-specific binding sites were blocked with 1 % BSA and the cells were incubated with a mAb to SV40 TAg (PAb416; Calbiochem), which recognizes the epitope corresponding to aa 83-128, and a polyclonal antibody to JCV VP1 (Suzuki et al, 2001) overnight at 4 uC. Immune complexes were visualized by incubation with AlexaFluor 488-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature.…”