FIG. 2.

TMPRSS2 and TMPRSS4, but not hepsin, activate influenza virus hemagglutinin by cleavage. (A) Cleavage activation of lentiviral pseudotypes bearing HA. (Left) Lentiviral reporter viruses bearing 1918 HA and NA were generated in 293T cells coexpressing the indicated protease, treated with PBS or trypsin, and used for the infection of Huh7 target cells. Luciferase activities in cell lysates were determined at 72 h postinfection. The results of a representative experiment performed in triplicate are shown; error bars indicate standard deviations. Comparable results were obtained in two independent experiments. (Right) Incorporation of HA into lentiviral pseudotypes. Lentiviral pseudotypes generated in 293T cells coexpressing the indicated protease were pelleted through a sucrose cushion, and the incorporation of influenza virus HA and HIV-1 capsid protein (p24) into virions was assessed by Western blotting. (B) Cleavage activation of PR8 (H1N1) and SCM35 (H7N7). The indicated proteases were transiently expressed in 293T cells, and the cells were infected at an MOI of 0.01 with PR8 (H1N1) or SCM35 (H7N7) produced in hens' eggs. Subsequently, the cells were treated with PBS or trypsin as indicated, and at 24 h postinfection, viral spread was quantified as the release of infectious particles into the culture supernatants, as measured by a focus formation assay. The results of a representative experiment performed in triplicate are shown; error bars indicate standard deviations. Comparable results were obtained in a separate experiment.