FIG. 6.

TMPRSS2 and TMPRSS4 activate 1918 HA by cleavage. (A) 293T cells were transiently cotransfected with the 1918 HA (jointly with pcDNA3 or the 1918 NA) and TMPRSS2, TMPRSS4, or mouse matriptase-3 (HA and protease expression plasmids were transfected at a 3:1 ratio). Subsequently, the transfected cells were treated with trypsin or PBS, and proteolytic processing of HA was analyzed by Western blotting. (B) 293T cells were transfected with the 1918 HA alone or in combination with the indicated proteases (HA and protease expression plasmids were transfected at a 1:1 ratio); the cells were treated with PBS or trypsin, and proteolytic processing of HA was analyzed by Western blotting. (C) Pseudotypes were generated in 293T cells expressing the empty vector or the indicated proteases (HA and protease expression plasmids were transfected at a 3:1 ratio), normalized to p24 (150 pg/well), and employed for infection of Huh-7 cells. Three days after infection, luciferase activities in cellular lysates were determined. A representative experiment is shown, and similar results were obtained in two independent experiments. Error bars indicate standard deviations. (D) RNA was obtained from cells present in bronchoalveolar lavage fluids and reverse transcribed within reaction mixtures containing reverse transcriptase (RT) enzyme or PBS, and GAPDH and TMPRSS4 were amplified by PCR. M, molecular weight marker. cps, counts per second.