(a) Entry inhibitors are listed with their molecular weights, IC50 and IC90 values for neutralization potencies against SC4226618 and 6535 (and for T1249-Fc, also against 6535-ΔCT), and the fold above these values that they were used for fusion inhibitor imaging experiments in which the inhibitors were incubated at 130 µg/mL. N.N. = non neutralizing. N/A = not applicable. (b) Infectivity of SC4226618 pseudovirus after incubation with TZM-bl target cells for the indicated times at 37°C. Luciferase activity (measured in relative luminescence units, RLUs) of supernatants transferred to fresh TZM-bl cells after the following incubation times is presented as the mean and standard deviation for eight replicate measurements. ~ 20% of the input pseudovirus remained infectious after 48 hr at 37 °C. To address whether infectious pseudovirus was still present after a 48 hr incubation at 37 °C with T1249-Fc inhibitor and TZM-bl cells, we calculated how many molecules of T1249-Fc were added (1015 molecules) in the inhibition experiments, how many TZM-bl cells were cultured (~50,000 cells in each well, which contained one or two sapphire disks (3 mm each)), and how many SC4226618 pseudoviruses were added at the start of the incubation (2500 TCID50 = ~107 particles). Thus, there were ~2×106 infectious viruses, ~1015 molecules of T1249-Fc, and ~50,000 TZM-bl cells that were available for formation of spoke structures that could be captured by HPF and visualized by ET after the 48 hr incubation at 37°C.