JC virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis

Abstract

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal alpha(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.

FIG. 1

Kinetics of JCV entry into human glial cells. JCV was preadsorbed to SVG cells for 30 min at 4°C, and then the cells were shifted to 37°C. At 0 min, 30 min, 1 h, 2 h, and 3 h following the shift to 37°C the cells were treated with either medium (+), preimmune antiserum (Pre), or anti-JCV antiserum (Imm). The percentage of infected cells were scored at 3 days postinfection by counting V antigen-positive cells by an indirect immunofluorescence assay. The error bars were derived by calculating the standard error of the mean of the average number of positive cells counted within one experiment. Experiments were repeated at least three times.

FIG. 2

Kinetics of SV40 entry into human glial cells. SV40 was preadsorbed to SVG cells for 30 min at 4°C, and then the cells were shifted to 37°C. At 0 min, 30 min, 1 h, 2 h, and 3 h following the shift to 37°C the cells were treated with either medium (+), preimmune antiserum (Pre), or anti-SV40 antiserum (Imm). The percentage of infected cells were scored at 3 days postinfection by counting V antigen-positive cells by an indirect immunofluorescence assay. The error bars were derived by calculating the standard error of the mean of the average number of positive cells counted within one experiment. Experiments were repeated at least three times.

FIG. 3

JCV infectious entry is inhibited by chlorpromazine but not by nystatin or PMA. SVG cells were either untreated or pretreated with PMA, nystatin, or chlorpromazine. The cells were then infected with JCV at 37°C for 4 h in the presence or absence of drug. JCV that remained bound to the cell surface was neutralized by the addition of anti-JCV antiserum. Preimmune antiserum was used as a negative control. The percentage of infected cells is indicated on the X axis. The error bars were derived by calculating the standard error of the mean of the average number of positive cells counted within one experiment. Experiments were repeated at least three times.

FIG. 4

SV40 infectious entry is inhibited by nystatin and PMA, but not by chlorpromazine. SVG cells were either untreated or pretreated with PMA, nystatin, or chlorpromazine. The cells were then infected with JCV at 37°C for 4 h in the presence or absence of the drug. SV40 that remained bound to the cell surface was neutralized by the addition of anti-SV40 antiserum. Preimmune antiserum was used as a negative control. The percentage of infected cells is indicated on the x axis. The error bars were derived by calculating the standard error of the mean of the average number of positive cells counted within one experiment. Experiments were repeated at least three times.

FIG. 5

Inhibition of the clathrin-dependent endocytic pathway by chlorpromazine. Clathrin-dependent receptor-mediated endocytosis of TRITC-transferrin was visualized in SVG cells that were either untreated or treated with PMA, nystatin, or chlorpromazine. (A) SVG cells alone; (B) SVG cells incubated with TRITC-transferrin in the absence of the drug; (C) SVG cells incubated with TRITC-transferrin in the presence of PMA; (D) SVG cells incubated with TRITC-transferrin in the presence of nystatin; (E) SVG cells incubated with TRITC-transferrin in the presence of chlorpromazine. Note the absence of endosomal staining (punctate cytoplasmic staining) in panel E compared to panels B to D.

FIG. 6

Colocalization of JCV and transferrin in endosomes. SVG cells were incubated with FITC-JCV and TRITC-transferrin for 20 min at 37°C. Cells were analyzed at a ×63 magnification by laser-scanning confocal microscopy. Images recorded under the FITC (green) (A) and TRITC (red) (B) channels, respectively, are shown. An overlay of the red and green channels to yield yellow where FITC-JCV and TRITC-transferrin colocalize is shown (C). An overlay of the red and green channels from cells that had been treated with chlorpromazine is shown (D).