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. 2001 Feb;75(3):1565-70.
doi: 10.1128/JVI.75.3.1565-1570.2001.

Human papillomavirus infection requires cell surface heparan sulfate

T Giroglou  1 , L Florin, F Schäfer, R E Streeck, M Sapp
Affiliations

Human papillomavirus infection requires cell surface heparan sulfate

T Giroglou et al. J Virol. 2001 Feb.

Abstract

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.

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Figures

FIG. 1
FIG. 1
Heparin is an inhibitor of pseudoinfection. Pseudovirions were preincubated with glycosaminoglycans for 1 h at 4°C at the indicated concentrations, and 30 μl of this mixture was added to 270 μl of COS-7 cell suspension and incubated for 1 h at 4°C. Cells were washed with PBS, subsequently plated, and grown for 72 h in culture medium. A value of 100% infectivity corresponded to 36 fluorescent cells. The average of at least three independent experiments is shown.
FIG. 2
FIG. 2
Cell surface heparan sulfate is essential for pseudoinfection. (A) COS-7 cells were treated with the indicated amounts of heparinase I and subsequently subjected to infectivity assays. (B) COS-7 cells were grown for 40 h in the presence of the indicated concentrations of sodium chlorate dissolved in culture medium and subsequently subjected to infectivity assays. A value of 100% infectivity corresponded to 45 (A) or 53 (B) fluorescent cells.
FIG. 3
FIG. 3
VLPs of carboxy-terminally deleted L1 bind to heparin-BSA. Wild-type (wt) VLPs and VLPs lacking the carboxy-terminal seven (1/492) or 22 (1/477) amino acids of HPV-33 L1 were reacted with heparin-BSA or BSA, respectively, which had been immobilized to microtiter plates. Bound VLPs were visualized with VLP-specific antiserum and horseradish peroxidase-coupled secondary antibodies using tetramethylbenzidine as the substrate.
FIG. 4
FIG. 4
Postattachment neutralization of HPV-33 pseudovirions. Pseudovirions were bound to COS-7 cells for 1 h at 4°C. Cells were washed with PBS, supplied with culture medium, and grown at 37°C. VLP antiserum (1:500) or heparin (100 μM) was added at the indicated times. Fluorescent cells were counted 72 h after the temperature shift. The average of at least three independent experiments is shown.
FIG. 5
FIG. 5
α6 integrin-negative cell line DG75 is susceptible to HPV-33 pseudovirions. (A) Flow cytometry analysis of α6 integrin expression before (left panel) and after (right panel) treatment with heparinase. Fine lines represent autofluorescence, and bold lines show expression of α6 integrin stained with antibody GoH3. (B) Cells were subjected to HPV-33 pseudoinfection. RNA was isolated 72 h postinfection and subsequently reverse transcribed. RNA coding for dimeric GFP was amplified by nested PCR, and PCR products were analyzed by agarose gel electrophoresis. Tubulin mRNA amplification served as a control.
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