Figure 5.

S20 blocks HA-mediated membrane fusion in a pH-dependent manner. After mixing a suspension of chicken erythrocytes with (a, b) rWSN-WT virus or (c) rWSN-HA/M59I virus on ice, S20 was added at the indicated concentrations. The mixture was then acidified with a pH of either (a) 5 or (b, c) a range from 4.8 to 6. The suspension was then incubated at 37 °C for 30 min and assayed at 340 nM for NADH released from the erythrocytes, as a measurement of fusion. Data are expressed as percentage relative to the DMSO control and means of triplicates ± SD are shown. (d, e) Trypsin sensitivity assay showing S20 protection of purified (d) WT HA but not (e) HA-M59I from trypsin digestion in a pH-dependent manner. Purified A/WSN/33 HA was incubated with DMSO (D) or S20 at the CC₁₀ concentration for 15 min at 31 °C prior to acidification to the indicated pH. The mixture was neutralized to a final pH of 7.4 and treated with trypsin for 30 min at 37 °C. The extent of trypsin cleavage was analyzed on a gradient SDS-PAGE gel and visualized with Coomassie staining. Trypsin digestion of HA at neutral pH (pH 7.4) was used as a control and is shown in the first lane.