Proteolytic activation of influenza virus hemagglutinin and SARS spike protein is conserved between TMPRSS2 of human, porcine, avian and murine origin.

<p>(A) Expression plasmids encoding the HA of the 1918 influenza virus and the indicated proteases or empty vector (pcDNA) were transiently cotransfected into 293T cells. The cells were then treated with PBS or trypsin, and HA cleavage was detected by Western blot analysis of cell lysates using a monoclonal antibody specific for HA. Detection of ß-actin served as loading control. (B) Lentiviral reporter viruses bearing 1918 HA and NA or the VSV-G glycoproteins were generated in 293T cells coexpressing the indicated proteases or empty vector (pcDNA), treated with PBS (black bars) or trypsin (white bars), and used for infection of 293T target cells. Viruses harboring no glycoprotein were generated in parallel as control. Luciferase activities in the cell lysates were determined at 72 h post infection. The results of a representative experiment performed in triplicates are shown. Error bars indicate standard deviation (SD). Comparable results were obtained in a separate experiment. (C) To detect SARS-S cleavage in cis, expression plasmids coding for SARS-S and the indicated proteases or empty vector (pcDNA) were transiently cotransfected into 293T cells, which were then treated with trypsin or PBS. Subsequently, S-protein cleavage was detected by Western blot analysis of cell lysates using a serum specific for the S1 subunit of SARS-S. SARS-S cleavage fragments produced by trypsin and TMPRSS2 are indicated by asterisks. Detection of ß-actin served as a loading control. (D) Effector 293T cells were cotransfected with a SARS-S expression plasmid and a plasmid encoding GAL4-VP16 and mixed with target cells cotransfected with a plasmid encoding a GAL4-VP16 responsive luciferase expression cassette and an ACE2 expression plasmid or protease expression plasmid or empty plasmid. The effector and target cells were mixed, treated with PBS (black bars) or trypsin (white bars) and the luciferase activities in cell lysates quantified at 48 h after cell mixing. The results of a representative experiment performed in triplicates are shown. Error bars indicate standard deviation (SD). Similar results were observed in two independent experiments.