Herpes Simplex Virus Type-1 (HSV-1) Entry into Human Mesenchymal Stem Cells Is Heavily Dependent on Heparan Sulfate
Figure 5
3-OS HS but not Nectin-1 or HVEM serves as the critical gD receptor in mediating HSV-1 entry into hMSCs. (a) Expression of HSV-1 glycoprotein D (gD) receptors in hMSCs. RT-PCR analysis of nectin-1, 3-OST-3, and HVEM demonstrated that all 3 genes are normally expressed by hMSCs. No products were detected in the H2O or RNA lanes in which H2O or the same RNA that was used in generating cDNA templates was used as PCR template to control for potential genomic DNA contamination. The products were separated by electrophoresis on an agarose gel. (b) HSV-1 entry into hMSCs was not significantly affected by anti-nectin-1 or anti-HVEM antibody treatment. Cells were preincubated with anti-nectin-1 and anti-HVEM antibodies (1 : 10 dilution) followed by infection with reporter HSV-1 (KOS) gL86 virus. β-galactosidase activity was recorded 6 h later to determine HSV-1 entry. The experiment was repeated three times with similar results. (c) Visualization of heparinase treatment on HSV-1 binding into hMSCs. Cultured hMSCs were mock-treated with PBS (H−) or heparinase I (1.5 U mL−1) (H+), followed by exposure to GFP-tagged HSV-1 K26GFP virus at 50 pfu/cell for 30 min at 4°C. Cells were then stained with rhodamine phalloidin that recognizes F-actin (red). Only mock-treated but not heparinase-treated cells showed HSV-1 GFP binding, which appeared yellow when its green fluorescence overlaps with red fluorescence. Virus attachment was determined by using Olympus IX50 inverted fluorescence microscope. (d) Effect of heparinase treatment on HSV-1 entry into hMSCs. Cells were treated with heparinase I (1.5 U mL−1) (white bar) or mock-treated with PBS alone (black bar), followed by exposure to HSV-1 KOS gL86 at 50 pfu/cell. Viral entry was quantitated 6 hr later by ONPG assay. Viral entry was reduced more than 3 folds in cells pretreated with heparinase compared to mock-treated cells (**, t test). Data shown are the means of triplicate measures. (e) Effect of heparinase treatment on HSV-1 gD binding to hMSCs. The cells were either pretreated with a mixture of heparinase-I (treated at 1.5 U mL−1; white bar) or mock-treated (untreated; black bar), followed by exposure to a soluble recombinant form of HSV-1 gD (gD:Fc). Binding of gD:Fc was detected by using HRP-tagged antibody against IgG:Fc and subsequent HRP enzyme detection. Asterisks indicate significant difference from the control treatment (**, t test). Error bars represent standard deviation (SD).
