Coxsackievirus A16 utilizes cell surface heparan sulfate glycosaminoglycans as its attachment receptor

Figures

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  Figure 1. Inhibitory effect of soluble heparin on CVA16 attachment and infectivity. (A, B) Inhibition of CVA16 infection by soluble heparin. One hundred TCID50 of (A) CVA16/SZ05 or (B) CVA16/G08 was pre-incubated with various concentrations of heparin sodium salt for 1 h at 37 °C before proceeding to infection of RD cells at 37 °C. Three days later, the infected cells were analyzed for viability by a MTT assay. The data are reported as the mean±sd of relative cell viability for triplicate samples. Statistical significance between the treated samples and the control (cell-only) is indicated as follows: NS, P≥0.05; *P<0.05; **P<0.01; ***P<0.001. (C, D) Inhibition of CVA16 attachment to RD cells by soluble heparin. Three hundred TCID50 of (C) CVA16/SZ05 or (D) CVA16/G08 was mixed with various concentrations of heparin sodium salt at 37 °C for 1 h. Then, the mixture was added to 1 × 105 RD cells pre-seeded 1 day ahead in a 24-well plate and incubated at 4 °C for 2 h. After the incubation, the cells were washed with serum-free medium three times. The cell-attached virus was quantified by qRT-PCR. The y axis shows the viral genomic RNA level of heparin-treated cells relative to that of the…

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  Figure 2. Heparinase I treatment impaired CVA16 attachment onto RD cells. RD cells were pretreated with heparinase I at 37 °C for 1 h and then inoculated with 8.6 × 103 TCID50 of (A) CVA16/SZ05 or (B) CVA16/G08. After washes, the cells were analyzed for bound virus by qRT-PCR. The data are expressed as the mean±sd for triplicate samples. Statistical significance between the treated samples and the control (without heparinase I treatment) was determined by Student’s two-tailed t-test and is indicated as follows: **P<0.01; ***P<0.001. Representative results from two independent experiments are shown.

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  Figure 3. Sodium chlorate pretreatment of RD cells inhibited CVA16 attachment and infection. RD cells (1 × 105/well) were cultured for 40 h in the presence of sodium chlorate at concentrations ranging from 0 to 50 mM. The treated cells were inoculated with 100 TCID50/well of CVA16/SZ05, followed by incubation (A) at 37 °C for 10 h or (B) at 4 °C for 2 h. The infected cells were analyzed for viral RNA by qRT-PCR. For each treatment, the viral RNA levels relative to those for the group infected with virus only (no sodium chlorate) are presented. The mean±sd for triplicate wells are shown. Statistical significance is indicated as follows: *P<0.05; **P<0.01; ***P<0.001.

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  Figure 4. CVA16 directly interacts with heparin in vitro. (A) CVA16 specifically binds heparin-immobilized agarose beads. CVA16/SZ05 or CVA16/G08 (3 × 106 TCID50 virus in a 500 μL final volume) was added to 20 μL of empty agarose beads or heparin-immobilized agarose beads, respectively, followed by incubation at 4 °C for 2 h to allow interaction. After separation by brief centrifugation, the precipitated agarose beads were washed with PBS, collected and analyzed for CVA16 protein by western blotting with an anti-VP1 polyclonal antibody. The virus input was also subjected to western blotting. (B) CVA16 binds heparin-immobilized agarose beads in a virus dose-dependent manner. Different amounts (5 × 105 or 3 × 106 TCID50) of CVA16/SZ05 or CVA16/G08 in a 500 μL final volume were mixed with 20 μL of heparin-immobilized agarose beads, followed by incubation at 4 °C for 2 h to allow interaction. Beads treated with medium-only served as a control in the assay. After incubation, the beads were processed and analyzed as described above.

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  Figure 5. Location of five positively charged residues of VP1 in the CVA16 capsid. The five positively charged residues of VP1 are located in a cluster at the five-fold vertex of the CVA16 capsid. The three-dimensional structure of a pentamer of the CVA16 mature virion (PDB code: 5C4W)14 is presented with sticks. The five VP1 residues, K141, R166, K241, K242 and H245, are highlighted in red, green, blue, light blue and purple, respectively. (A) Top view of the CVA16 pentamer. (B) Side view of the CVA16 pentamer.

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  Figure 6. Effect of alanine substitution of the five positively charged residues of VP1 on heparan sulfate binding and cellular attachment of CVA16. (A) Sequence of the designed CVA16 mutants. (B) Analysis of heparin-binding ability of the CVA16 mutants by pull-down assays. Equal amounts (equivalent to 4 × 106 copies of viral RNA genome) of CVA16 mutant or wild-type viruses were separately mixed with 20 μL heparin–agarose beads. The mixtures were incubated at 4 °C overnight. After three washes with PBS, the agarose beads were subjected to RNA extraction and qRT-PCR analysis as described in the ‘Materials and Methods’ section. The data are expressed as the mean±sd of the absolute viral RNA copies of triplicate samples. Representative results from two independent experiments are shown. (C) Ability of the mutant viruses to bind RD cells. The same amount of wild-type or mutant virus was added to RD cells and incubated at 4 °C for 1 h. Cell-bound virus was quantified by qRT-PCR. The data are reported as the mean±sd of the relative viral RNA genome numbers. Representative results from two independent experiments are shown. Statistical significance is indicated as follows: **P<0.01; ***P<0.001.