Figure 4.

Relevance of Extracellular Carbohydrate-Rich Matrix in EHV-1 Cell-to-Cell Spread

(A and B) PBMC were infected with EHV-1 RFP + GFP (MOI = 0.1) for 5 min. The ECM were disrupted mechanically (extensive pipetting; EHV-1_5M_Cell_pip), chemically (heparin treatment; EHV-1_5M_Cell_hep), or both (EHV-1_5M_Cell_pip_hep). The disrupted cells (A) or the supernatant (EHV-1_5M_Sup_pip, EHV-1_5M_Sup_hep, EHV-1_5M_Sup_pip_hep) (B) were added to EC. As a control, the ECM was left undisrupted (EHV-1_5M_Cell or EHV-1_5M_Sup). The data represent the mean ± standard deviation of three independent and blinded experiments. Significant differences in plaque numbers on EC were seen between the different treatment procedures. n = 3; one-way ANOVA test and Dunnett's multiple comparisons test; ∗∗p < 0.01, ∗∗∗p < 0.001.

(C and D) Treatment with heparinase III or heparin fractionates and elution of ECM-viral assemblies. Confocal microscopy showing infected PBMC with EHV-1 RFP + GFP (red) treated with heparinase III (C) or heparin (D) and stained for cell surface carbohydrate-rich matrix with ConA (green). PBMC nucleus was stained with DAPI (blue). Data are representative of three independent experiments. Scale bar, 10 μm, and scale bar of magnification, 7 μm. Image stacks (number of stacks = 17 with 0.75 μm z stack step size) were photographed using VisiScope Confocal FRAP microscope. Presented here is a single optical section of the stacks.