Herpes Simplex Virus Type-1 (HSV-1) Entry into Human Mesenchymal Stem Cells Is Heavily Dependent on Heparan Sulfate
Figure 6
Effect of heparinase on quantitative HSV-1 glycoprotein-induced fusion with hMSCs. (a) In this experiment, the effector CHO-K1 cells were transfected with HSV-1 glycoproteins gB, gD, gH, and gL, and T7 RNA polymerase. The target hMSCs were transfected with plasmids expressing luciferase reporter gene. A luciferase reporter assay was performed 24 h after the two cell populations were mixed together. Cell fusion was measured in relative luciferase units (RLUs). Expression of gD receptors in hMSCs results in the fusion of CHO-K1 cells with HSV-1 glycoprotein expressing cells (black bar), while in a negative control coculture of target cells with effector cells devoid of all four glycoproteins-encoding genes, no luciferase activity was recorded (white bar). Coculture of heparinase (H+) pretreated-target cells with effector cells resulted in significant decrease in cell fusion (grey bar). (b). The effects of heparinase on multinucleated polykaryocytes formation were visualized. Multinucleated cells or polykaryocytes were microscopically observed with the hMSC target cells cocultured with effector CHO-K1 cells expressing HSV-1 glycoproteins (panel (B)) but not with CHO-K1 cells expressing control vector pDream2.1 (panel (A)) or with the pretreatment of target hMSCs with heparinase-I (1.5 U mL−1) before mixing with effector cells (panel (C)). Both type of cells were cocultured and stained with Giemsa at 24 h postmixing. Shown are photographs of representative cells (Olympus IX50 inverted microscope) after 24 h postmixing. (c) Quantitative determination of the number of fused polykaryocytes formed after coculture of target hMSCs cocultured with the effector CHO-K1 cells expressing HSV-1 glycoproteins in presence (grey bar) or absence (black bar) of heparinase. Effector CHO-K1 cells expressing empty vector (pCDNA3.1) cultured with target hMSCs was used as a negative control (white bar). The data shown are the means of triplicate measures with three independent experiments. Asterisks indicate significant difference from other treatments (**, t test), and error bars represent standard deviation (SD).
