Figure 4
(A) The inhibitory effect of peptides on viral adsorption into target cells. Hemagglutination inhibition (HI) assay was performed using 4 times the HA units (4HAU) of virus per well. 25 μL of peptides from a twofold serial dilution in saline was added into 25 μL virus (4HAU), following the addition of 50 μL of erythrocytes (0.5% v/v in saline) into each well. The hemagglutination reaction results were read after incubation for 1 h. PBS without virus was used as positive control, while virus only as negative control. (B) Hemolysis inhibition assay. Peptide (10 μM) in PBS was mixed with the influenza virus A/PR/8/34 (H1N1) strain. 200 μL of 2% chicken erythrocytes pre-warmed at 37 °C was then added. After incubation at 37 °C for another 30 min, 100 μL of sodium acetate (0.5 M; pH 4.6–6.0) was added and mixed with the erythrocyte suspension to trigger hemolysis. After incubation for 30 min, the mixture was centrifuged and the supernatants containing released hemoglobin were measured at OD535. (C) The interactions between C20-Jp-Hp and HA-FP-O analyzed with circular dichroism (CD) spectra. (i) C20-Jp-Hp interacted with HA-FP-O in PBS (pH 7.4) and (ii) acidic condition (pH 5.0); (iii) HA-FP-O mixed with HA-FP-1 in PBS (pH 7.4) and (iv) in acidic condition (pH 5.0). CD curves of individual or mixed peptides with equimolar concentrations were scanned from 195 to 260 nm with an average of four scans.
