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. 2012 Sep;86(18):10028-35.
doi: 10.1128/JVI.00051-12. Epub 2012 Jul 11.

Heparan sulfate is an attachment factor for foamy virus entry

Kathrin Plochmann  1 , Anne Horn, Eva Gschmack, Nicole Armbruster, Jennifer Krieg, Tatiana Wiktorowicz, Conrad Weber, Kristin Stirnnagel, Dirk Lindemann, Axel Rethwilm, Carsten Scheller
Affiliations

Heparan sulfate is an attachment factor for foamy virus entry

Kathrin Plochmann et al. J Virol. 2012 Sep.

Abstract

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

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Figures

Fig 1
Fig 1
PFV binds to heparin. Replication-incompetent PFV encoding an EGFP expression cassette was loaded on an FPLC column packed with Poros-heparin. The column was washed with 20 mM sodium phosphate buffer, pH 7.0, and the column was eluted with 500 mM NaCl, pH 7.0. FPLC fractions were tested for infectivity by transduction of HT1080 cells. The proportion of transduced cells was visualized by fluorescence microscopy and quantified by flow cytometry detecting EGFP fluorescence. (A) FPLC chromatogram displaying the affinity purification of PFV particles. The blue line represents absorbance at 280 nm detecting protein. The yellow line represents the applied NaCl gradient, and the brown line represents the corresponding conductivity. The x axis displays the fractions collected. (B) Fluorescence microscopy of HT1080 cells incubated with the indicated fractions. (C) Flow cytometric quantification of EGFP-expressing cells of HT1080 cultures transduced with the collected FPLC fractions.
Fig 2
Fig 2
PFV does not bind to Poros carrier. The experiment was performed as described in the legend to Fig. 1. Instead of using a Poros-heparin column, a column packed with Poros alone was used. (A) FPLC chromatogram. (B) Fluorescence microscopy of HT1080 cells incubated with the indicated FPLC fractions indicated below. (C) Flow cytometric quantification of EGFP-expressing cells of HT1080 cultures transduced with the collected FPLC fractions.
Fig 3
Fig 3
Heparin inhibits PFV and FFV infection. HT1080 cells were incubated with replication-incompetent PFV (MD9) or replication-competent FFV (Chatul-3) in the presence of different concentrations of heparin (He), a GAG closely related to HS. Virus-infected cells were monitored by EGFP expression (MD9) or by immunostaining for FV Gag (FFV) after 24 h by flow cytometry. Using a sigmoidal curve function, the EC50 (the concentration at which the half-maximal inhibitory effect was visible) of heparin was determined by nonlinear regression. (A) HT1080 cells transduced with PFV. (B) HT1080 cells infected with FFV. For both panels, data represent means ± standard deviations from triplicates.
Fig 4
Fig 4
Cells that lack HS are less susceptible to PFV infection. Mouse L cells and their HS-deficient subclone Sog9 were incubated with a dilution series of the same FV vector suspension, and transduction rate was determined by flow cytometry detecting EGFP expression. (A and B) Flow cytometric determination of surface HS expression. Green line, cells stained with anti-HS antibody; blue line, cells stained with isotype control antibody. (A) Mouse L cells. (B) Sog9 cells. (C) Mouse L and Sog9 cells were incubated with a dilution series of the same vector preparation. EGFP-positive cells were determined after 24 h by flow cytometry. Using a sigmoidal curve function, CCID50 (the vector dilution at which the half-maximal transduction rate was visible) was determined by nonlinear regression.
Fig 5
Fig 5
The amount of cell surface HS correlates with susceptibility to PFV infection. Relative surface HS expression of different cell lines (CRL-2242, HepG2, 293T, BHK, CRFK-LL, SK-N-SH, MRC, human mesenchymal stem cell line MSC-Tert, COS-7, CHO-K1, HT1080) was determined by the shift of mean fluorescence of cells stained with anti-HS antibody versus cells stained with isotype control antibody. The different cells were incubated with a dilution series of the same vector preparation. EGFP-positive cells were determined after 24 h by flow cytometry. Using a sigmoidal curve function, CCID50 was determined by nonlinear regression. (A) Correlation of relative HS surface expression versus cell-specific CCID50. Data represent means ± standard deviations from triplicates. (B) Histograms of HS expression of the cells from flow cytometric analysis. Cells were stained with anti-HS antibody (blue line) or an isotype-matched control antibody (red line). Numbers indicate geometric mean fluorescence intensity of cells stained with anti-HS (blue numbers) and isotype control (red numbers). (C) Correlation of relative CD138 (syndecan 1) surface expression with cell-specific CCID50. (D) Correlation of surface expression of HS with CD138 (syndecan 1).
Fig 6
Fig 6
Enzymatic removal of cell surface HS reduces susceptibility for PFV infection. HT1080 cells were treated with heparinase I or heparinase III for 1 h and subsequently incubated for 1 h with a dilution series from the same vector preparation. EGFP-positive cells were determined after 24 h by flow cytometry. Using a sigmoidal curve function, CCID50 was determined by nonlinear regression. (A) HT1080 cells treated with (red triangles) or without (green squares) heparinase I. (B) HT1080 cells treated with (red triangles) or without (green squares) heparinase III. (A and B) Data represent means ± standard deviations from triplicates. (C) Flow cytometric analysis of HT1080 cells treated with heparinase III in the presence or absence of a protease inhibitor (PI) cocktail. (Top) Histogram of HS expression (blue line) versus isotype control staining (red line). (Bottom) Histogram of CD138 (syndecan 1) expression (blue line) versus isotype control staining (red line). Numbers indicate geometric mean fluorescence intensity of cells stained with anti-HS or anti-CD138 (blue numbers) and isotype controls (red numbers).
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