Table 1.
A custom designed glycoarray containing hyaluronic acid, heparin, chondroitin sulfate and dermatan sulfate species.
The structure, molecular weight, number of sugar residues and sulfate groups per disaccharide for each glycosaminoglycan are listed.
Table 2.
A custom designed glycoarray containing different heparan sulfate species.
The structure, molecular weight, number of sugar residues and sulfate groups per disaccharide for each glycosaminoglycan are listed.
Fig 1.
Binding of human cytomegalovirus on glycosaminoglycan glycoarrays.
(A) Binding of purified extracellular CMV virions on a custom designed glycosaminoglycan glycoarray. Relative fluorescence units (RFU), which are directly proportional to the amount of virus binding, are plotted on the Y-axis in the graph. Ligand descriptions and chain structures are provided in Table 1. Six replicates for each GAG were used in the assay. NC1: Negative control 1 (print buffer), NC2: (Biotinylated Glycan), PC1: positive control; human IgG (0.1 mg/ml), PC2: mouse IgG (0.1 mg/ml), PC3: rabbit IgG (0.1 mg/ml), dp: degree of polymerization, triangles at the bottom of the graph represent an increasing degree of polymerization of GAGs from the left to the right. (B) Binding of purified extracellular CMV on a custom designed heparan sulfate glycoarray. Relative fluorescence units (RFU) are plotted on the Y-axis in the graph. Ligand descriptions and chain structures are provided in Table 2. Six replicates for each ligand were used. NC1: negative control 1 (print buffer), NC2: (biotinylated glycan), NC3: human IgG (0.1 mg/ml), PC1: mouse IgG (0.1 mg/ml), PC2: rabbit IgG (0.1 mg/ml). Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001). Standard deviation was plotted as error bars.
Fig 2.
Inhibition of HCMV growth by glycosaminoglycan derivatives.
Primary human foreskin fibroblasts (HFF) grown in 96 well plate were pretreated for one hour with 10 μM of 1) 6-O-desulfated Arixtra, 2) Unmodified Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) dp2, 6) dp4, 7) dp6, 8) dp8, 9) dp10, 10) dp12, 11) dp14, 12) dp16, 13) dp18, 14) dp20 15) > dp20 or control (dH2O). Cells were infected with GFP tagged HCMV (Towne strain) virus at an MOI of 3.0 in the presence of the test glycosaminoglycans, which were maintained in the cell culture medium. At 5 days post-infection, cells were fixed and number of foci (GFP) was counted in triplicate well for each sample under an epifluorescent microscope. An average of >200 foci were present in mock-treated infected cells. Percent of viral GFP was calculated compared to virus only infected control (100% GFP expression). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001).
Fig 3.
Effect of glycosaminoglycan derivatives on HCMV growth.
(A) Primary human foreskin fibroblasts (HFF) were pretreated for one hour with 10 μM of 1) 6-O-desulfated Arixtra, 2) Regular Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) dp2, 6) dp4, 7) dp6, 8) dp8, 9) dp10, 10) dp12, 11) dp14, 12) dp16, 13) dp18, 14) dp20 15) > dp20 or control (dH2O). Cells were infected with HCMV (Towne strain) virus at an MOI of 3.0 in the presence of test glycosaminoglycans. Cells and media were harvested at 5 days post-infection and titered for HCMV plaque forming units (pfu) on fresh fibroblasts in tissue culture dishes. Individual samples (3 replicates each) were quantified and displayed as total pfu/ml on Y-axis. (B) Virus titer is plotted (Y-axis) against degree of polymerization (X-axis). Data points ahead of the broken line is for a mixture of GAGs (dp>20). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. (C) Primary human foreskin fibroblasts (HFF) were pretreated for one hour with 0.05 g/L of 1) 6-O-desulfated Arixtra, 2) Regular Arixtra, 3) Heparin sodium salt from porcine intestinal mucosa (PIHSS), 4) Enoxaparin, or series of heparin oligosaccharide from enoxaparin: 5) dp2, 6) dp4, 7) dp6, 8) dp8, 9) dp10, 10) dp12, 11) dp14, 12) dp16, 13) dp18, 14) dp20 15) > dp20 or control (dH2O). Cells were infected with HCMV (Towne strain) virus at an MOI of 3.0 in the presence of test glycosaminoglycans. Cells and media were harvested at 5 days post-infection and titered for HCMV plaque forming units (pfu) on fresh fibroblasts in tissue culture dishes. Individual samples (3 replicates each) were quantified and displayed as total pfu/ml on Y-axis. (D) Virus titer is plotted (Y-axis) against degree of polymerization (X-axis). Data points ahead of the broken line is for a mixture of GAGs (dp>20). Results are representative of three independent replicates. Standard error of mean was plotted as error bars. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.0001).
Fig 4.
Mouse CMV replication in sulfotransferase knockout cell lines.
Cells were grown to 90% confluency and infected with wild-type MCMV (strain K181) at low (0.01, (A), (B) and high (3.0) (C), (D) MOI. Cells and the medium were harvested at 3- and 5-days post-infection, sonicated to release the virus and diluted for plating on to wild-type MEF in tissue culture dishes in order to enumerate total MCMV pfu/ml. Results are representative of three independent replicates. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test with control, and corrected using Dunnett’s post hoc test, showing significant differences among means (p <0.05). Standard error of mean was plotted as error bars. An asterisk (*) indicates significant inhibition compared to wild-type. Hs3st1 and Hs3st4: Glucosaminyl 3-O-sulfotransferase 1 and 4, respectively. WT: wild type; KO: knockout.
Fig 5.
Mouse CMV entry in sulfotransferase knockout cell lines.
(A) Cells were grown to 90% confluency and infected with wild-type MCMV (strain K181) at an MOI of 3.0. Cells were harvested at 2 hours post-infection, and whole cell lysates were loaded on a polyacrylamide gel for blotting. The blots were probed with anti IE1 antibody. Beta-actin was used as a loading control. (B) Bands from two independent experiments were quantified by densitometry and means were plotted. Standard error of mean was plotted as error bars. Hs6st1 and Hs6st4: Glucosaminyl 6-O-sulfotransferase 1 and 4, respectively. Hs3st1 and Hs3st4: Glucosaminyl 3-O-sulfotransferase 1 and 4, respectively. WT: wild type; dko: double knockout. Data was analyzed by ordinary one way ANOVA with multiple comparisons comparing the means of each test sample with control, and corrected using Dunnett’s post hoc test. Differences were considered significant (*) if p<0.05.
Fig 6.
(A) SPR sensorgrams of gB-heparin interaction. Concentrations of gB (from top to bottom): 1000, 500, 250, 125, and 63 nM, respectively. The black curves are the fitting curves generated using the T200 Evaluation software (version 3.2). (B) Sensorgrams of solution heparin or analogs /surface heparin competition. gB concentration was 250 nM, and concentrations of heparin/analogs in solution were 1000 nM. (C) Bar graphs (based on triplicate experiments with standard deviation) of normalized gB binding preference to surface heparin by competing with heparin or heparin analogs in solution. ES: Enoxaparin Sodium; ES-NS: Enoxaparin Sodium N-sulfated; ES-6 DeS: Enoxaparin Sodium 6-O-desulfated; Hep: Heparin.