Fig. 4. Quantification of N-1 and C-1 inhibition of fusion mediated by SV5 W3A F. The conditions of the assay were similar to those in Figure 3 except that cleavage by TPCK–trypsin was not needed for W3A F and the samples were incubated at 15°C for 30 min between the 4°C binding of RBCs for 1 h and the 37°C incubation for 10 min. (A) Timeline of peptide order-of-addition experiment. Arrows denote the addition of peptide at either primary, secondary or tertiary stages. Samples were washed three times with PBS before each temperature change. (B) For cell–cell fusion by W3A F co-expressed with HN, N-1 inhibits fusion if present during either the 4, 15 or 37°C incubations, and is a more potent inhibitor as the incubation temperature is increased. (C) For cell–cell fusion by W3A F co-expressed with uncleaved influenza virus HA, N-1 does not inhibit fusion at 4°C, is a mild inhibitor of fusion at 15°C, and inhibits if present at 37°C. C-1 inhibits both HN+F and HA+F fusion only after thermal activation at 37°C. The peptide concentrations were 40 µM. The means and standard errors shown are for contents mixing from 3–5 fields.