Skip to main content
. 2011 May;85(9):4122–4134. doi: 10.1128/JVI.02232-10

Fig. 4.

Fig. 4.

Shedding of SARS S by TMPRSS2 confers neutralization resistance. (A) The pseudoparticles indicated were produced in 293T cells in the presence or absence of TMPRSS2, concentrated by ultracentrifugation through a 20% sucrose cushion, preincubated with the indicated dilutions of the sera R1 and R1LI1, and then used for triplicate infections of 293T-ACE2 cells. Luciferase activities in cell lysates were determined after 72 h, and activities measured in the absence of serum were set as 100%. The results of a representative experiment are shown; error bars indicate standard deviations. The results were confirmed in two independent experiments. In the absence of serum, the following luciferase counts were measured: VSV-G, 509,961 ± 37,823 cps; SARS S plus pcDNA3, 497,873 ± 52,794 cps; SARS S plus TMPRSS2, 608,600 ± 97,835 cps. (B) The concentrated pseudotypes described in panel A were analyzed by Western blotting for the presence of SARS S (employing an S1-specific rabbit serum). (C) 293T cells were transiently cotransfected with expression plasmids for SARS S and TMPRSS2 or cotransfected with SARS S plasmid and empty vector (pcDNA3), and culture supernatants were harvested at 48 h after transfection. Subsequently, the supernatants were concentrated by ultrafiltration followed by ultracentrifugation through a 20% sucrose cushion. The presence of soluble SARS S protein in the supernatants of ultracentrifuged samples was analyzed by immunoblotting, as described in panel A. (D) Pseudoparticles bearing SARS S and purified by ultracentrifugation through a sucrose cushion were preincubated with the indicated dilutions of supernatants described in panel C and a 1:50 dilution of the sera R1 and R1LI1 for 60 min before addition to target cells (pcDNA3, supernatants from cells cotransfected with SARS-S and empty plasmid; TMPRSS2, supernatants from cells cotransfected with TMPRSS2 and SARS S expression plasmids). Luciferase activities in cell lysates were determined after 72 h. The results ± standard deviations of a representative experiment carried out in triplicate are shown; activities measured in the absence of serum, 1,307,409 ± 328118 cps, were set as 100%. The results were confirmed in two separate experiments.