FIGURE 9.

The IRF-3 pathway regulates SARS-CoV replication in cell culture.A, Huh7 DN-18 cells with tet-regulated, conditional expression of a dominant negative form of IRF-3 (DN IRF-3) were cultured in the presence or absence of tet for 3 days, to repress or induce DN IRF-3 expression, respectively, and were subsequently mock-infected or challenged with SeV for 12 h. Expression of IRF-3, ISG56, SeV, and actin proteins were detected in cell lysates by immunoblot analysis. The endogenous IRF-3 (FL) and the DN IRF-3 (DN) bands were marked as filled and empty circles, respectively. The asterisk in the ISG56 panel denotes a nonspecific band. B, Huh7 DN-18 cells repressed or induced for DN IRF-3 expression were infected with SARS-CoV at an m.o.i. of 0.01 at 37 °C for 1 h. The virus inoculum was then removed, and cells were washed extensively and refed with complete medium. Cell-free culture supernatants were collected at the indicated times postinfection and stored at -80 °C until they were subjected to assessment of infectious viral titers using a standard TCID₅₀ assay in Vero E6 cells, as described under “Experimental Procedures.” The experiments were terminated at day 3 postinfection as CPE was observed in ∼30 and ∼70% of infected cells without and with DN IRF-3 expression, respectively. The growth curve of SARS-CoV shown was representative of three independently conducted experiments.