Figure 3.

VP1 mutation confers resistance to the inhibitory effects of RA. (A) Growth curves of WT and N104K viruses. The data shown is from one of two independent experiments. (B) Attachment of wild-type and N104K variant viruses with RD cells. The amount of the attached virus was estimated using quantitative RT-PCR and was normalized the DMSO-treated control, set as 1. The data were expressed as mean ± SD from three independent experiments and analysed using the Student's t-test (*P < 0.05; **P < 0.01). (C-D) Effect of RA on the binding of EV-A71 variants to heparan sulfate and PSGL1-hFc. The EV-A71 variants were first pretreated with DMSO or RA at 4°C for 1 h and were then incubated with heparan sepharose beads (C) or PSGL1-hFc-conjugated protein G Mag Sepharose (D). The results are representative of at least three independent experiments. In (D), the ratio of EV-A71 binding to PSGL1 was defined by the levels of EV-A71 proteins over PSGL1. (E–F) Effect of RA (28 or 280 μM) on binding of non-heparan sulfate binding viruses to heparan sulfate beads or RD cells. (E) Equal copy numbers of viruses were subjected to heparan sulfate binding analysis. Bound viruses were assessed using RT-qPCR. (F) Viruses were incubated with RD cells in the presence of DMSO or RA on ice for 1 h. The medium was replaced with E2 containing RA and incubated for another 6 h. Total RNA was harvested using TRIzol for RT-qPCR. RNA copy number of each experiment was normalized to wild-type (left panel) or respective DMSO control (right panel), arbitrarily set as 1. The data are expressed as the mean ± SD of three independent experiments and were analysed by Student's t-test. *P < 0.05, **P < 0.01, and ns, no significance.