FIG. 2.

Impact of spinoculation and protease inhibitors on 1918 influenza virus HA-driven viral entry. (A) 293T, Huh-7, and MDCK cells were seeded in 96-well plates and infected with p24-normalized pseudotypes bearing 1918 influenza virus HA, NA, and M2 or VSV-G. The virions were pretreated with trypsin or PBS and, subsequently, a trypsin inhibitor, before addition to target cells. Two plates were infected in parallel: one plate was incubated at 37°C immediately after addition of virus; the other plate was centrifuged for 2 h at 25°C and 1,200 rpm after infection and then incubated at 37°C. Three days after infection, luciferase activities in cellular lysates were determined. A representative experiment performed in triplicates is shown. Error bars indicate standard deviations. Similar results were obtained in two independent experiments. (B) Huh-7 cells were incubated with the indicated concentrations of the cysteine protease inhibitor E64d prior to infection with infectivity-normalized pseudotypes carrying the 1918 influenza virus HA, NA, M2, ZEBOV-GP, or VSV-G. Pseudotypes bearing influenza virus proteins were pretreated with PBS or trypsin for 10 min at room temperature, and soybean trypsin inhibitor was added. Three days after infection, luciferase activities in cellular lysates were determined. Infection of Huh-7 cells in the absence of protease inhibitors was set as 100%. The average of three independent experiments performed in triplicate is shown. Error bars indicate standard errors of the means. cps, counts per second.