A JC virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway

Abstract

Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.

FIG. 1.

Genistein inhibits JCV and SV40 infection of glial cells. (A) SVG-A cells grown on coverslips were pretreated with 100 μM genistein for 1 h at 37°C. Cells were then infected with JCV or SV40 in the continued presence of genistein for 4 h at 37°C, and anti-JCV or anti-SV40 neutralizing antibodies were added at 4 h postinfection to neutralize any remaining extracellular virus. At 72 h postinfection the cells were washed and fixed, and infection was scored by an indirect immunofluorescence assay for the late viral protein VP1. (B) VP1-positive cells were scored by counting, and the results are graphed.

FIG. 2.

Sodium orthovanadate does not inhibit JCV infection of glial cells. (A) SVG-A cells grown on coverslips were pretreated with 50 or 100 μM sodium orthovanadate for 1 h at 37°C. Cells were then infected with JCV or SV40 in the continued presence of NaVO₃ for 4 h at 37°C, and anti-JCV or anti-SV40 neutralizing antibodies were added at 4 h postinfection to neutralize any remaining extracellular virus. At 72 h postinfection the cells were washed and fixed, and infection was scored by an indirect immunofluorescence assay for the late viral protein VP1. (B) VP1-positive cells were scored by counting, and the results are graphed.

FIG. 3.

Genistein and chlorpromazine inhibit entry of FITC-labeled JCV. SVG-A cells grown on coverslips were either untreated (control) (a and d) or pretreated with 100 μM genistein (b and e) or 25 μM chlorpromazine (c and f) for 1 h. The cells were then incubated with FITC-labeled JCV (a to c) for 4 h in the continued presence of drug or incubated with 35 μg of rhodamine-labeled transferrin/ml (d to f) for 30 min also in the continued presence of the drug. Cells were washed and fixed in 4% paraformaldehyde, and coverslips were mounted on slides with mounting medium containing propidium iodide (a to c) or DAPI (d to f). Slides were viewed using a laser scanning confocal microscope with a 63× objective.

FIG. 4.

Schematic representation of full-length eps15 and the dominant-negative mutant constructs used in these experiments. eps15 has an amino terminus containing three protein-protein interaction EH domains, a central coiled-coil domain thought to be involved in homodimerization, and a carboxyl terminus involved in association with the clathrin adaptor protein AP-2 during clathrin pit formation. The DIII (D3) and EH95 constructs are dominant-negative inhibitors of eps15 function and inhibit clathrin-dependent endocytosis, whereas the DIIIΔ2 (D3D2) construct serves as a negative control and has no effect on clathrin-dependent endocytosis. The FLAG constructs are either a control full-length eps15 construct or full-length eps15 with a point mutation (Tyr→Phe) in the major tyrosine phosphorylation site, Y850, which has been shown to block EGF internalization but not transferrin internalization.

FIG. 5.

Dominant-negative eps15 constructs inhibit JCV infection. SVG-A cells grown on coverslips were transfected with the indicated GFP- or FLAG-tagged dominant-negative eps15 constructs or control constructs for 24 h. Cells were then infected with JCV, and at 72 h postinfection the cells were fixed and stained with an anti-V antigen (VP1) monoclonal antibody (red). The GFP-expressing constructs were directly visualized, and the FLAG-tagged constructs were visualized using anti-FLAG antibody and an Alexa-Fluor 488-labeled secondary antibody. JCV was unable to infect any of the cells expressing a dominant-negative construct (EH95, D3, and eps15-Y850F). In contrast, JCV readily infected cells expressing control constructs (D3D2 and eps15wt).

FIG. 6.

Dominant-negative eps15 constructs block JCV entry. SVG-A cells grown on coverslips were transfected with either D3 (a to c) or EH95 (d and e) dominant-negative eps15-GFP constructs or the D3Δ2 control construct (f to h) for 24 h. Twenty-four hours posttransfection, cells were exposed to Alexa-Fluor 594-labeled JCV (red) for 4 h at 37°C and then fixed in 4% paraformaldehyde for 30 min. The cells were mounted using fluorescent mounting medium and viewed using a confocal microscope. Arrows indicate the presence of internalized virus (c) or membrane-bound virus (f and i).

FIG. 7.

JCV activates the MAPKs ERK1 and ERK2. SVG-A cells serum starved overnight were incubated with JCV or just medium (mock) for 30 min on ice, and then cells were shifted to 37°C to allow virus internalization. Lysates were collected at the indicated time points following the shift to 37°C, and samples were run on 4- to-15% gradient gels, transferred to nitrocellulose, and blotted with antibodies recognizing the phosphorylated or active form of ERK1 and -2 or with antibodies to tubulin as a loading control.