Fig 3. SR-PLP supported viral replication in the context of a recombinant, chimeric SARS-CoV (rSCV).

(a) A chimeric rSCV containing SR-PLP (SR-PLP-rSCV) was generated by reverse genetics to investigate SR-PLP functions in the context of a replicating virus. SR-PLP (purple) was inserted at the genomic position of SA-PLP (blue). The plaque morphology was analyzed in Vero E6 cells. Therefore, cells were infected with (b) rSCV and (c) SR-PLP-rSCV (MOI 0.01) and overlaid with a highly viscous medium. At 3 dpi, cells were fixed and stained with crystal violet. (d) To investigate structural integrity of the SR-PLP domain in the molecular context of the SARS-CoV nonstructural protein 3, cells were infected with either rSCV or SR-PLP-rSCV (MOI 0.0001) and treated with the SA-PLP protease inhibitor 3e (24-well format). After 1 h, cells were washed twice with PBS and either DMSO (0 μM) or in DMEM serially diluted compound 3e (3.125, 6.25, 12.5, 25 and 50 μM) was added. Supernatants were collected at 24 hpi. For virus quantification a real-time RT-PCR for genomic SARS-CoV RNA was performed. The experiment was done in triplicate and repeated twice. Error bars indicate the standard deviations of the means. Statistical significance between DMSO- and compound-treated cells was determined using one-way ANOVA and Sidak- or Games-Howell post hoc tests for rSCV and SR-PLP-rSCV, respectively. The 50% effective concentrations (EC₅₀) were rSCV: IC₅₀ = 2.36 μM and SR-PLP-rSCV: IC₅₀ = 11.02 μM.